<%BANNER%>

Bacterial Citrus Canker: Molecular Aspects of a Compatible Plant Microbe Interaction

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20110115_AAAACV INGEST_TIME 2011-01-15T18:07:46Z PACKAGE UFE0008379_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 51918 DFID F20110115_AABZHK ORIGIN DEPOSITOR PATH elyacoubi_b_Page_044.pro GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
015522b512449f69fa89e5e997de673d
SHA-1
0635b025b2614cb6a7db3cdd6ac608b1abd1ecf8
68707 F20110115_AABZGW elyacoubi_b_Page_083.jpg
3b6b818d8fc32cc0623bbf143ae30e39
4aa036b2881fdbfccf805b73b135e3c3dba2b9fe
1695 F20110115_AABZHL elyacoubi_b_Page_039.txt
f6f1047aeb532548396fb2a65d7daf00
ca461f528dd518182f4910bd48f73547ea196492
50183 F20110115_AABZGX elyacoubi_b_Page_038.pro
551610bba59fc40608d92b4b2f27a6a1
11c78a5825326dcfb8d9c993b35c1f3e2f5449f6
28936 F20110115_AABZHM elyacoubi_b_Page_025.jpg
7a85e46dbb40b1ab0e3aa99807501daf
cfbc43a78a4beab25eeff7404660a05ca72e7297
1053954 F20110115_AABZIA elyacoubi_b_Page_087.tif
5317e1daa6206edd390b75530ab8c508
2f0f3143277fd15e4fc30a43ecfacb21c0388545
3245 F20110115_AABZHN elyacoubi_b_Page_063thm.jpg
139067116529b0e2302f8389ff0f0419
f5474440958cfe6c2ec43eca30a8fa3eaecca7c8
25458 F20110115_AABZGY elyacoubi_b_Page_001.jp2
546cc595a327d562e95ddd47e093b5de
9b8fa95b59b2bcca9fcd75d81a6bf18848af75cb
25271604 F20110115_AABZIB elyacoubi_b_Page_034.tif
5c43ab1b31ac096c7cd5f73efd0ee4be
81c3c36a8b5af12a29c99c4484ab1cd8e7a41362
8669 F20110115_AABZHO elyacoubi_b_Page_074.pro
0baa1576e36fb54486c14a174fa2d89e
efd3ca976100686cceedec548d33a32457e5732a
62945 F20110115_AABZGZ elyacoubi_b_Page_031.jpg
a7d72250233db9ad93237f21ec9b50bf
4fac09df7eefaaa1cfb3cc90dcca4bf87b5c057f
2005 F20110115_AABZIC elyacoubi_b_Page_010.txt
441effdcea7fc25a8425bca677c0924c
298896987adeddbb9b7304443c21a704a6917e4b
57526 F20110115_AABZID elyacoubi_b_Page_063.jp2
17bdaceae14b8f3c279c87935177efee
68d0a986d9ed7c66229be24265446a00aa4f3643
112661 F20110115_AABZHP elyacoubi_b_Page_057.jp2
7d4ae27d738cb32d64bc2713bf25b3da
c948a88e193573e83091b1bb2c5968aa9638193c
42720 F20110115_AABZIE elyacoubi_b_Page_078.pro
5e0701421e0c77863965515c312b47e3
33f6b645fd0758100d8016acadddd332e14c9b3e
10963 F20110115_AABZHQ elyacoubi_b_Page_070.pro
f9eb363762d0f6ad3438f4cc9822b87d
eface57a4c509158961d4aa4e7dd37b19e89c644
1958 F20110115_AABZIF elyacoubi_b_Page_046.txt
79c8c90dbce93ddf642d617fbf645a23
a386d279832a3bb46921087d3019d56b73101471
6638 F20110115_AABZHR elyacoubi_b_Page_037thm.jpg
1ab7251a3bf0c4d82debdcb8c235ee30
545a91acd0c9b12431b75c3372a30a1f30fd5d87
2582 F20110115_AABZIG elyacoubi_b_Page_025thm.jpg
aee63ec2969db63f46964786729aa47c
5d1d8604ec47db639d6404235b401f2b39db01d6
73856 F20110115_AABZHS elyacoubi_b_Page_095.jp2
54b1194d1769bc66d9c59471ac4c2b2c
316c589c60566d80e30f5279f3b2bd0ceee8bc8b
74864 F20110115_AABZIH elyacoubi_b_Page_087.jpg
9689a9dcaca2cf661a651e34499e264c
029753e6d14e5245a55086b00a75392c9a34b677
6413 F20110115_AABZHT elyacoubi_b_Page_089.QC.jpg
81bbeb9d912d3791ff87472c95819266
b79017c14fcdb776a06c3f6cf896abc32ada4c52
4761 F20110115_AABZII elyacoubi_b_Page_012thm.jpg
ecf2c591b1ed3e210c6584cb857f82de
571411eafa29b8fb683c2c3f26bc1fc6094dac97
23787 F20110115_AABZHU elyacoubi_b_Page_018.QC.jpg
0c08a980e39b336b00c22f27556068d8
936994d10e610e5a5e3be5c92e6d0a99e8e5e0eb
111 F20110115_AABZIJ elyacoubi_b_Page_002.txt
ebbb9bf3710bc1ab634dab184ffa94e9
3e70bdaf7a6a6d8dda04eaaf1ace841c62ca0e16
24079 F20110115_AABZHV elyacoubi_b_Page_037.QC.jpg
ff0eb83d4a457b9395e49a7691ce9b3e
fdc7771f714a60b4404757f9a91a9ce72f2a5b7a
1912 F20110115_AABZIK elyacoubi_b_Page_013.txt
dd036ab099feefadb7201fb8d04a0c42
1f4a9f890da02858d6ee6c1e0178b290446e34e6
48492 F20110115_AABZHW elyacoubi_b_Page_086.pro
3d2ecd4d8aa83e9d11ade67f5ef04def
c5a67048eacacae135f14c525f03d62260c79e47
F20110115_AABZIL elyacoubi_b_Page_033.tif
49de29cc09387b4ebdb6640d78d80ea7
483d1d81de0cff12bf1f32c99c6e1185d2cd22f0
49222 F20110115_AABZHX elyacoubi_b_Page_080.pro
7e5b0608834bc30bb1bf2a1e80875739
74f4b9c2d9d64ad845ec5d382f7283d478c5dea7
100933 F20110115_AABZJA elyacoubi_b_Page_021.jp2
282089fe33cb80857c02c3e8c8b1cd02
18919766ba9d44c3c4f93cf76a741fd0d76705a2
24457 F20110115_AABZIM elyacoubi_b_Page_055.QC.jpg
9700f96c9cf50d7622b117fd0ca9a2a9
537f86467400a70415f6c386607ee538a269d68d
91177 F20110115_AABZHY elyacoubi_b_Page_101.jpg
b2ada201fab23b2e6cb9d017e6b45d7d
e0b3783f5de0d301035444902bd6eb8a7e24051b
102798 F20110115_AABZJB elyacoubi_b_Page_019.jp2
0f4d8b4111b1bf847072255b6a0e07eb
77191893c4f2de36e7f5594e3c0a046bf6a775ae
66959 F20110115_AABZIN elyacoubi_b_Page_061.jp2
3542dcaa42d22e814e92a09b9ba49f51
bf0d823cdcf64cc4c5aebc4a9625bd13fbbbb541
108929 F20110115_AABZJC elyacoubi_b_Page_060.jp2
3f9b7eced78258b3199defcd40dca691
2340263f1b45ebb68d4abe91704193f208307ada
68628 F20110115_AABZIO elyacoubi_b_Page_096.jp2
d0b21cb21aacca6c9fddd55762e4b950
fda7d22ed02a9b1df7825a1f87ac1ea632f9ed08
113687 F20110115_AABZHZ elyacoubi_b_Page_055.jp2
000c25de5a790b4a9371646535b34bb2
0960ce2896fa0f5c3cfd9fb811d78f73ec04de82
2599 F20110115_AABZJD elyacoubi_b_Page_100.txt
9b16b0528018b856b512e00aecc4dab9
387c4ff7bef53c0373af8abccc411e1f6b1ff8fc
3314 F20110115_AABZIP elyacoubi_b_Page_002.QC.jpg
d1dc38b31c369db82d3bc17d5c3dddcd
d2b0609895120e18e80f4ea25a825c0b66434f29
12850 F20110115_AABZJE elyacoubi_b_Page_091.QC.jpg
dff6f52f1eee4cdbd7cefe6f933294d4
7a4097f4327d589663fc805e3198567badc753d0
45476 F20110115_AABZIQ elyacoubi_b_Page_084.pro
15fb7aedeb27c502a8938b59d7d49e45
64d0adc63116a6be2223743f1ccf8ef6f79b0d1e
1723 F20110115_AABZJF elyacoubi_b_Page_031.txt
2bbfc04dd04a64a4e47ffaffd97d7a12
3340ac452ef45717a2579742a5443d156eefe515
17824 F20110115_AABZIR elyacoubi_b_Page_095.QC.jpg
c8251e492c039470e090e11551905eb3
82f89ed16f39fcfeadbfc87cd563d73b41321804
1809 F20110115_AABZJG elyacoubi_b_Page_083.txt
48fc849cf8fd68c26dbff271eb1154fb
67b3a8001b409e0c11477815d658207dca7d7144
686 F20110115_AABZIS elyacoubi_b_Page_025.txt
d66f407892f81a0afa9187b0b6e0484a
4a2d6801dd781dc2572553e4cca1974669ede7bb
5372 F20110115_AABZJH elyacoubi_b_Page_069.pro
e1ec824f13167d15261e7be420327f55
925819865dd5055c33965dda435ad4e54adad67c
2325 F20110115_AABZIT elyacoubi_b_Page_024thm.jpg
39f4d5a2b426dc1bbdacd3a89236e976
54566f3937a9e1fd3af983cbe101b51067c6dd78
111661 F20110115_AABZJI elyacoubi_b_Page_014.jp2
72e714b47b183c00c0e9f9fd1296d8ea
becdb03e12c9bb625998758ef4e5e83631ceaa34
20903 F20110115_AABZIU elyacoubi_b_Page_005.QC.jpg
79b7573e6416ed8a8ab1eccb14bc0211
4b51cd8dea7fd8e4434aca11fa03f48dc7e14990
F20110115_AABZJJ elyacoubi_b_Page_066.tif
f332d36fccbf9c804eec6c1c1e350acc
b193dfc9ab44ec4660d535a3a25cf165a9eebff0
66669 F20110115_AABZIV elyacoubi_b_Page_032.jpg
94da189411e1f3f2df816e0bda3634a8
c8c2dad9b3d9bc0b66a2aec4d399d62446f940db
F20110115_AABZJK elyacoubi_b_Page_082.tif
43e987ab4d8571dba3b232e3b163869a
93ff2ed8277022df444746dbd6bf160a4ba9cb03
437 F20110115_AABZIW elyacoubi_b_Page_074.txt
b42517c5ef760039c40d40310013e952
2cc97fdb5681825eab9db115bfde469e52118a33
4898 F20110115_AABZJL elyacoubi_b_Page_096thm.jpg
7168c0a4d146c8654ac3f4403d011e60
bc48799865ef027e7cf5c8218c5e13eb9100cc9c
107429 F20110115_AABZIX elyacoubi_b_Page_085.jp2
c1c9b266792c6ad1cb4eefdb2841b1cd
1f0cd4fa678b02586dbc91777bf2962a68c1372f
F20110115_AABZJM elyacoubi_b_Page_012.tif
8fe0c364dbb54cd298b31058b5205137
78344480a9d5f62048a4f784443966adb6a5c7e0
16495 F20110115_AABZIY elyacoubi_b_Page_097.pro
971ed9ab2450940344a52397432e1fcd
f17241e4d845f0cd1391a584f6be9686fb8d148a
157264 F20110115_AABZKA UFE0008379_00001.xml FULL
44b8c2d6661d6e2092da2c9c6c8288f7
38166792996d094a928a24e02dcf8bd0a5d563e4
2018 F20110115_AABZJN elyacoubi_b_Page_007thm.jpg
1a06e415975494da0542d442a268ca88
27c90c874ec2732c15774e59f79332aad0aeb5b1
45189 F20110115_AABZIZ elyacoubi_b_Page_061.jpg
b7baf38ce3e2a9e1edf5f687a6b09cb6
c51c3c13dd0c4144a5cd6bab4e142f506ac7f751
114092 F20110115_AABZJO elyacoubi_b_Page_020.jp2
dd8ab767fa8648371f47f91308386037
ecf0260167e7c57b96c9f24e928d19b14fd441d4
22932 F20110115_AABZJP elyacoubi_b_Page_023.QC.jpg
bff32e4f08eeb32c3d0231a78fc47f3d
4efe40d36c2db6bd4a4d0d2d4e5f7a1b82b8878a
10386 F20110115_AABZKD elyacoubi_b_Page_002.jpg
d82d50fb0c15f3e7ec1340d00e609eae
d18c25448e1c12da77a3b828b78e7ff173b21c8c
106860 F20110115_AABZJQ elyacoubi_b_Page_018.jp2
3a2dd00ca613860c7dd6ad43e402beb6
6135648b688286743fed1ff06f8bd26327020e1c
11525 F20110115_AABZKE elyacoubi_b_Page_003.jpg
544e1f3b29f6faf65b0a561602eeed49
b492bebf404a1e0a971e8738bb62374d1109a65e
1342 F20110115_AABZJR elyacoubi_b_Page_012.txt
9c14001391b8ad9059770de42028d283
f46272d097cd2040287e8f40dab4a5f1970d9fb2
36679 F20110115_AABZKF elyacoubi_b_Page_004.jpg
0cc70c2d29438af3c0a288653bd4636d
20f7bbdcbbb6b1663e2e5939bd454968af74868f
75231 F20110115_AABZJS elyacoubi_b_Page_056.jpg
2069615571d28613218c6392f74ae147
9be0f984a00dbb92ddc97db8efce23589b8e93b4
80132 F20110115_AABZKG elyacoubi_b_Page_005.jpg
5608b47f22dd5372723cb05fac8f112d
d0f0837955cd38569079d21a4d910022dee634fe
6872 F20110115_AABZJT elyacoubi_b_Page_069.QC.jpg
968868b0bd3754b1d4b468c1f1766653
e9d0c1dbaf0c45c0e74610761f185bcf5559e9b9
18406 F20110115_AABZKH elyacoubi_b_Page_007.jpg
f733db4d5f971eecb4cb091aa58ea165
37f118820ed07a3410ae9cf4d7943726073bdc25
63233 F20110115_AABZJU elyacoubi_b_Page_078.jpg
c113530879cd2484871e18bf985bc373
7c0276c77e0d7af1caba0fb33a4495d80208e14a
68355 F20110115_AABZKI elyacoubi_b_Page_010.jpg
1d5504303b6c045d7f91ff06fd39b77d
62039f78e7c08ca9afb2f7c06572848b6dec8b68
1908 F20110115_AABZJV elyacoubi_b_Page_085.txt
8b1317ff443b0c768e71d5e56709b430
898938026fba2e23b01257146748899c1e686803
59864 F20110115_AABZKJ elyacoubi_b_Page_011.jpg
1dc9fc7d4b44f83e00e234915f37df2e
3fad0deac3d077ff8487873bbda544c1d1511ffb
39196 F20110115_AABZJW elyacoubi_b_Page_063.jpg
2c7e6f696de0d732b0bc936fd82c2f92
6fe492321f5c9a7961c91e66c7462c0e24c7f101
51459 F20110115_AABZKK elyacoubi_b_Page_012.jpg
1ae57189e1bf967050059191c5c9a570
af5a957503a9a574eb731d0612c71d5ea2f63b52
F20110115_AABZJX elyacoubi_b_Page_093.tif
ed4acc69504f187e0c3c2449f27be2a1
acbb07e416f7961d552c5d9591bbbd4f0c9b9280
69305 F20110115_AABZKL elyacoubi_b_Page_013.jpg
519251a8001007c6be72b77d588450a7
394a971671aab3c1f017f02e04f9b6106f656eda
773 F20110115_AABZJY elyacoubi_b_Page_103.txt
ec2d17dc98d0211a699a2445afb11d8c
d17b0bf46ba46dc0dcddc71883d04c599c0a7693
61668 F20110115_AABZLA elyacoubi_b_Page_042.jpg
db57034d2f200c084f78358c78086e7e
226a45f52548bd303263f875a3257fd7c2f22b01
72658 F20110115_AABZKM elyacoubi_b_Page_014.jpg
9dbd06b5ecafd4295732b563afa8bdd3
ae621c9ecc08db1115940c71d1d2bc7bf7437a19
6487 F20110115_AABZJZ elyacoubi_b_Page_059thm.jpg
dabedb4c713ce31ecb0fce2dab123394
ec0be301a55f50e3cb77b1bda162560196adc3e5
76509 F20110115_AABZLB elyacoubi_b_Page_044.jpg
d9a0556210a05bb521a33735fceaac5e
5290e5817be2ac9ec8a37a8e890b76ac41cd8e12
72060 F20110115_AABZKN elyacoubi_b_Page_018.jpg
271271c410c43429b3661932e729e3a3
ba75aac3ef0ed8c5c18f384b611cafeefe3ec5a7
73789 F20110115_AABZLC elyacoubi_b_Page_046.jpg
cd00d109e1ef3fefc59cd5c96a386db9
67a39581a4c45227852a1eae0f73cc8f117757a2
75852 F20110115_AABZKO elyacoubi_b_Page_020.jpg
67a275afffc42308781c0e4e316827cb
288d2a6b587d71d34e9c02747f9398e17915cfba
66522 F20110115_AABZLD elyacoubi_b_Page_047.jpg
cdddeef45617c257730ca0b83aad2144
63660029c233b8b6931f1adc48ecd204fedd39aa
68379 F20110115_AABZKP elyacoubi_b_Page_021.jpg
b6caf908c2039ce16255e7286d84ad6f
4e3b5a3f50c1c140e2aebd7b540ab8991c5f1087
71957 F20110115_AABZLE elyacoubi_b_Page_048.jpg
44ca1876cd117516eb646b3a8aa2874d
b563dee5a6a1c72338a6f3926cef1b716f22d7fc
69599 F20110115_AABZKQ elyacoubi_b_Page_022.jpg
b657f77c52a9d9f78601a2f2e0dfe60b
f32b70718d37dcb1a2e083745fac535bdbe37eba
70857 F20110115_AABZLF elyacoubi_b_Page_049.jpg
ccb4cefd059622b97f3c917456425760
e37900c1e0cf6a4e9e9f186b143323b5cd19fd52
70350 F20110115_AABZKR elyacoubi_b_Page_023.jpg
3af7d3d08815019ae5aed65d15e0ddb9
b2863a9c6be62597ba5d2a038133b261cbf39518
72965 F20110115_AABZLG elyacoubi_b_Page_050.jpg
f7cad019f0170efaa53cded5fda4495d
bcf495bd9c4faa0d032e005cd51b0c5f75769002
20714 F20110115_AABZKS elyacoubi_b_Page_024.jpg
d0417918042a7c616e090268bec99a77
32d28002364dbfd581310d15589e51a246c50eae
71031 F20110115_AABZLH elyacoubi_b_Page_052.jpg
44e80865e13ad3ba62beb0558b167fe9
0402a6219f381f1389437fac5e826f0691c45eff
42682 F20110115_AABZKT elyacoubi_b_Page_028.jpg
2b6cea487be2a33212fc88c4bd4d7e14
8b81ca0f7ff29edf2fb3b69e02aef324edcbd3e1
69745 F20110115_AABZLI elyacoubi_b_Page_053.jpg
7cd4ba36552befdac88a52a62276f884
c79d16930f0567ec85ad637d7366e0d55e6e176d
56593 F20110115_AABZKU elyacoubi_b_Page_029.jpg
1b1daeddb301a390e7a82a3b6f04f0ad
7fcfaad7cc48188adab40ec00eea578bfa38284a
78075 F20110115_AABZLJ elyacoubi_b_Page_054.jpg
8ba1fd1dad34d8f856e698161923635a
cc70f2fb96a825916214a9a379c1f914e8e01430
53315 F20110115_AABZKV elyacoubi_b_Page_030.jpg
43d2c49634e957fec17a407381ba2a24
89bf984dd758a7a18818824c7889a75b52377653
72037 F20110115_AABZLK elyacoubi_b_Page_058.jpg
0ae1722d60ef4285331d6e8e66ef113c
34aefeceaa9f592b849911e20481255228cece8a
50804 F20110115_AABZKW elyacoubi_b_Page_034.jpg
edffc5249aa8985ff61fed06e1a27b85
882a469e5b0a1d064c114c66bd9bc499e41716ce
24414 F20110115_AABZLL elyacoubi_b_Page_065.jpg
c6faf4e4d7289682037c7083dada85b5
f519036a29a24145481393a3a23e9a2ef68cc539
72115 F20110115_AABZKX elyacoubi_b_Page_036.jpg
74ead00e5cab45c98cb30e01519b3c58
4f4ab6ff7eab6bf504e26893c6e8cffc28276720
41136 F20110115_AABZMA elyacoubi_b_Page_091.jpg
fe4ab27216f01aa0c627793edfec0723
630e932d00bae7d715fcdccf312a57553d43db31
49101 F20110115_AABZLM elyacoubi_b_Page_067.jpg
7a21c9331dd3eff59a090dfd254916f1
0e6468d5481c6fc38e32b038fd5377b8efe78fc5
74547 F20110115_AABZKY elyacoubi_b_Page_037.jpg
670983806340f25765cf69c8c19ef1e6
7b4ecbc255be8f919090c5bcdc09070a200a65f1
33467 F20110115_AABZMB elyacoubi_b_Page_092.jpg
4b7762594567905e96376efb75909486
c4a13f1bf91686c0dcd7bbc9c2b321f7a9c4da82
27026 F20110115_AABZLN elyacoubi_b_Page_071.jpg
07554c4535569ebdd889ca80291b0675
889a86368252f57c8fc3e50966e2e14546c2f46f
63571 F20110115_AABZKZ elyacoubi_b_Page_039.jpg
345942e65ae17a5a8d11a23075bcc7b9
fc285fec8ad8a63c7a594563f6c31d2d0751c298
88957 F20110115_AABZMC elyacoubi_b_Page_100.jpg
48065118b0b0bb6596efcbdebb8c960f
ef509ba91b106bd13e11b5b8a3bc26b190b08dac
58111 F20110115_AABZLO elyacoubi_b_Page_073.jpg
2380f959a91a49767d133ccde21ae165
a8e86fdc1241e21111de3dbabb88b414217a350e
5691 F20110115_AABZMD elyacoubi_b_Page_002.jp2
d067585d153caa51c6533e70527e3ace
b10a69a7b7a685b35177fa8858c73ec15278f6fc
49286 F20110115_AABZLP elyacoubi_b_Page_074.jpg
f4c1e24fe2c5fe0ddfd6fc3db2e60bad
f7025834003eb4f9076a7b2b9bf9f9deb6ae6a65
7469 F20110115_AABZME elyacoubi_b_Page_003.jp2
09ef1bd4a011422daeecdf55d2844e98
8d3275515054f33c373d7caecdf07684a2fd1941
40778 F20110115_AABZLQ elyacoubi_b_Page_075.jpg
be01413b753bf2e118be7fbe30f69aaf
433c5869326473dcc30f9dbc377315b8a6a01ebc
49928 F20110115_AABZMF elyacoubi_b_Page_004.jp2
8eac41eb1aa6becb00de989c7fd0b099
4ebd3167c7e30591bdcf05e1f8b2cb8000ac9914
61019 F20110115_AABZLR elyacoubi_b_Page_076.jpg
b2a7a6c2c7a1687dcef32fc620c190dd
b2f47a3bbeb93d5bfe8235ec3a207414aa208170
1051982 F20110115_AABZMG elyacoubi_b_Page_005.jp2
8f7f1c9c189fbc49564cf230e889711f
d4f736085dda3bc9d4e27b1839f10e2652b798e5
36821 F20110115_AABZLS elyacoubi_b_Page_077.jpg
7757373927385d648000479ea89c8d41
7e21d9584915ab1722209f53d4a171b12b165599
312307 F20110115_AABZMH elyacoubi_b_Page_008.jp2
708fb18f5d93c03e6e122fc66ad39747
f820f82a60a3641ad39358e513f440684746603f
74483 F20110115_AABZLT elyacoubi_b_Page_079.jpg
ed0b2fd538aed302aa331ed922efa28b
cfdbfd3d74233ef903a5caa77cb9755f7a27b619
1051958 F20110115_AABZMI elyacoubi_b_Page_010.jp2
7b5069815b2858ab663193238070d8ba
46218d7329d442ec5949be46df052175905a0869
71376 F20110115_AABZLU elyacoubi_b_Page_080.jpg
855a38826c7d20a81628724143349a9d
aa5fd5e8a56fa27ac6d0567501c45807dc47ed52
106657 F20110115_AABZMJ elyacoubi_b_Page_016.jp2
82235d357df9a5a822d4b34a1e6f01e3
db38522254a97534cc9683bf4e0e289dfad691c2
66710 F20110115_AABZLV elyacoubi_b_Page_082.jpg
ac838fdd37aae8178d00543b527534b4
9489afc309778a6da9d97d779cede7bbf06f2cb5
102399 F20110115_AABZMK elyacoubi_b_Page_017.jp2
afdf4f77b27f163899a9629a1bd60489
fe2686c5043ef73008aa3907cc34c9ebd1ab1e13
71907 F20110115_AABZLW elyacoubi_b_Page_085.jpg
944ab9705c21a386046e6fbc91799b69
671c388c4fe54931c75e6c4a8672bc43e52f82b4
108720 F20110115_AABZML elyacoubi_b_Page_023.jp2
4256ac56752b11b7dfea07bb17f4fc65
e0bd52ed0d14061abd64f4651cb72162274571f9
72943 F20110115_AABZLX elyacoubi_b_Page_086.jpg
5fd7e1e06bd21b9a39460fee81c65f24
ea5c527e6b4857b3441604fdcc80487471286279
24148 F20110115_AABZLY elyacoubi_b_Page_089.jpg
923fc4d779c21078d8f3b826496b3507
197436b3807036bfed7d82b7d7927ce0876e1751
106115 F20110115_AABZNA elyacoubi_b_Page_053.jp2
f791822913f8c15c37a64678aa962e2c
650034bda1ff741d05cf29652d5cec909b13dc4a
296833 F20110115_AABZMM elyacoubi_b_Page_025.jp2
e2ff033234a861c91e8423f8976fd165
14be4ab1b04917f61e3b146053f352ffb89abba5
36423 F20110115_AABZLZ elyacoubi_b_Page_090.jpg
426f10d18d7eb362aa2ebd4b80aa1998
e7120909c6e1406be3046e8f3c28c265695d25fe
116030 F20110115_AABZNB elyacoubi_b_Page_054.jp2
b183acce2d0a9738dc40521120b00ef7
e43dee88b1cadce9421ccd38b58891d77df529ce
355664 F20110115_AABZMN elyacoubi_b_Page_027.jp2
ba24c2a81075986cbd9837d754f3c716
b6a32989bedc67986142ca561a68a669866f5fb0
112988 F20110115_AABZNC elyacoubi_b_Page_056.jp2
19d2834ba61ab4f27892819270865b69
ee0f6972e5297603c734f11c7eacdaa50cf9b86a
816350 F20110115_AABZMO elyacoubi_b_Page_029.jp2
24c6427cf75bc2ccd9333d026c1f5cbf
5aacca0336c4c5f8372a3ace9f3f988ed6433b10
111180 F20110115_AABZND elyacoubi_b_Page_059.jp2
30c81c4558584901c5a7ad1f4579cdde
e0581e177a9c0c486728ddd8f8210b8cb90f7d55
861791 F20110115_AABZMP elyacoubi_b_Page_031.jp2
854793efaf6ef1dd841d344a86a4717a
5ad3423633d62d33e67b677664e243d4986a3e10
992161 F20110115_AABZNE elyacoubi_b_Page_064.jp2
cd1dec0d69301f60c311835be45deb4d
3961a4f4b06e8c50bc91ae4a228aa9ea551d36d4
1051977 F20110115_AABZMQ elyacoubi_b_Page_034.jp2
1823779f010acb6c92952b2e76ecbdbf
d8fdd95ef6999b3232b2655e78e19ca258b0bded
409830 F20110115_AABZNF elyacoubi_b_Page_065.jp2
7282c498da7b723ee7d4e8a08d25289b
53f3a8dfac6a3f49d229a08a87975914b5c2b08e
97480 F20110115_AABZMR elyacoubi_b_Page_035.jp2
bc934332e4e92e680105365dc48ae3c7
f4a03d4c88062e28e9a68734fd1bc94fa2a18e80
308934 F20110115_AABZNG elyacoubi_b_Page_066.jp2
214fe41040b87852a84575921ed6282a
b93259e1871261caa0f821a9828bc6dd6b83f5f0
111649 F20110115_AABZMS elyacoubi_b_Page_036.jp2
8db933bd4faf0067d4b3d3d54689b63b
33a4352c0bc95763db3f17947e55968ccd2ef53a
725179 F20110115_AABZNH elyacoubi_b_Page_067.jp2
6bec2c8e9856fc2d3a5e5fb2cf165cc4
04b94c94571e5415463465e75ead7ceca5c2b61f
113227 F20110115_AABZMT elyacoubi_b_Page_037.jp2
ef87da48f5281c33932048bf2149799f
c62c1b96073b62251888408cd17686cc5f1c5d3b
753676 F20110115_AABZNI elyacoubi_b_Page_068.jp2
4951ea1b9c9c779db142f530698681a7
05c8493b6fa9a3132e9f0ccbea28e72fdbfde141
102409 F20110115_AABZMU elyacoubi_b_Page_041.jp2
10130a8db28d655df53acc43f5b4073e
c339e5474b93923144a13694b17fb3322907240d
240963 F20110115_AABZNJ elyacoubi_b_Page_069.jp2
2f00ec28c58c075c91ce0a798facd149
a8f0c9c636cc13300102b336b43aefb16ff6009c
111941 F20110115_AABZMV elyacoubi_b_Page_043.jp2
07eb08ac90372b009130c46f9cc1862d
0cb801482db9cd92deb5217c003401242678dd6f
1034990 F20110115_AABZNK elyacoubi_b_Page_073.jp2
7a16afe262f21a3d1c7c9b6ebf09d282
69222117394a29a7bd007d30f5312ee7cee208cf
114995 F20110115_AABZMW elyacoubi_b_Page_044.jp2
1d99543ef5defc13f174386e2d2fe56d
3dd0b936ecf9839093c13d7707bac93e6d3bbfcf
562886 F20110115_AABZNL elyacoubi_b_Page_075.jp2
c183b04e0bdf6a607a6080baea6125fc
97cdf537558dbf4bd6a4de89b15fbfcd87c45b8a
106486 F20110115_AABZMX elyacoubi_b_Page_049.jp2
ab94ebe2de09478ee081e166ec3a1381
ef330b2d92e32b7b4d4ee87a695ffa23be43ff03
F20110115_AABZOA elyacoubi_b_Page_009.tif
842b32a8c096b9b8ded0dec8f113fe88
f69084de63f6c11827a7d39727979ed927366546
1051979 F20110115_AABZNM elyacoubi_b_Page_076.jp2
436197fd03f817dc6e3e7a46bf8970c5
41ea2ba0f765a67e2cccf5811d7365e03d356ebd
110901 F20110115_AABZMY elyacoubi_b_Page_050.jp2
83c03558233599d5d2d097e8156c9970
724d12ea6955fa3f487b937ebf81641caab361f1
F20110115_AABZOB elyacoubi_b_Page_014.tif
334f7464aade0912557c849764162614
aabfc7136f6a9c7d144f85795bd485855b867c27
40525 F20110115_AABZNN elyacoubi_b_Page_077.jp2
64eec78f9d237ae8ed4b52dbb74f9792
43eb8eeed55e26712d0cff7ac6040fa754cc8f75
113287 F20110115_AABZMZ elyacoubi_b_Page_051.jp2
1a75cc307defd80b4826e4021dbbdfc3
199612f2624ba5f7b29f8edf8ae88d68980c7fbf
F20110115_AABZOC elyacoubi_b_Page_015.tif
093d992f6bd25a53472a71fd6a445cf6
70dbef50f7b89468565aa47eb089fd01275d342a
108716 F20110115_AABZNO elyacoubi_b_Page_080.jp2
c57c5bcbe5eff065cb4921c47cb20edb
09e25ef55637a1a58dc03dca9b00ea7e5f7efdf9
F20110115_AABZOD elyacoubi_b_Page_016.tif
d8925818eeebb2f3e3338684f85877b6
9a01e944babec4fb22c54358ef2ca9414e528742
103372 F20110115_AABZNP elyacoubi_b_Page_083.jp2
21b98858cf0267ad1fde0149b28b40e0
c6389cc8231787add51dba04937827cb69383f4f
F20110115_AABZOE elyacoubi_b_Page_018.tif
b80379feb5c68225500ea27b7dfa2772
9ada5d6b778a1d2a621c3e18dea97093e1af69da
107704 F20110115_AABZNQ elyacoubi_b_Page_086.jp2
412982948b699834d5d611628c52bb4c
88dfe87180df1d415d30f71fccb57bac3927dc35
F20110115_AABZOF elyacoubi_b_Page_019.tif
66e3e76221c5b6789ab22663114b96cd
8c4dd5ab032f85acae4ec481124c47bcdab05b5b
100453 F20110115_AABZNR elyacoubi_b_Page_088.jp2
3031f3dedb8ec8e36af41c16a4cde2ee
befc6e4846a7b831d01ea181b8129753870e5cb6
F20110115_AABZOG elyacoubi_b_Page_020.tif
6aaa919dc6ab5bb8f5ffb15cd737227c
19143d1b4ee175e88290cfa0acabd2a0ce85db71
310708 F20110115_AABZNS elyacoubi_b_Page_089.jp2
503c597ac8bf2961f6bbbaab5452b66d
4fa68126cd8104ff566f0d7ba3446e61994ae1c0
F20110115_AABZOH elyacoubi_b_Page_023.tif
577ae8f66f82be212e8871726eb39c55
9a9db6abb226401f222f2b6c97e465753460a2ba
538528 F20110115_AABZNT elyacoubi_b_Page_091.jp2
5988bbc3164f8216f207537681e4f2e2
3ae5bba4178a35f40e3846f41dbdc3ab72cf27e4
F20110115_AABZOI elyacoubi_b_Page_025.tif
979f89d79ff7b4e70ecf2e28e2435995
774f692c2eca92749a81a8376263d33532d15af1
128389 F20110115_AABZNU elyacoubi_b_Page_099.jp2
b65e966de04a4ec551a8c73d018488b9
fbdd3ad0964f11607b0c7a02e57610d57a3c4089
F20110115_AABZOJ elyacoubi_b_Page_028.tif
648bef7e4176fd89bb359737986f5ff8
e52c7194c1d25be84574618ba1ab2f7c2a23c059
F20110115_AABZNV elyacoubi_b_Page_001.tif
f916900291e9d2af8c26249f14fa0dac
d391560e53b3607ef880f18b6088c5f631dcea75
F20110115_AABZOK elyacoubi_b_Page_029.tif
db846da0ad42d99dc4844be9eabd2266
b10c1f1a111b60ec713825ebd267822e2ffa7c3e
F20110115_AABZNW elyacoubi_b_Page_002.tif
736c5d5cb9fbf055f7db7dfa7673829a
e0a5bf902f5fd52d80c45dd9af9d2637ced6092e
F20110115_AABZOL elyacoubi_b_Page_030.tif
ebd57893d9540f8a632da926aceb6640
0e4ddf171d4b472887e0db2bf77ae5ff9ebcb977
F20110115_AABZNX elyacoubi_b_Page_005.tif
e35c79e0d25c1f1e946a37e38fcbb4a5
84691255f7192adb487736e859f9ff7bca0aa1d3
F20110115_AABZPA elyacoubi_b_Page_068.tif
91bc142b35a7935f23c05b341d0be3d9
6f74fc53014e9ad4d4065df467271af80659b01b
F20110115_AABZOM elyacoubi_b_Page_032.tif
b9fb22ada8337301ae8cc0f8bbd22194
25ec68bd35fbd5960000f855ea4f1d5acea50c6e
F20110115_AABZNY elyacoubi_b_Page_006.tif
928d2f518a53ae66fac0aa2ca3358741
ef3ea9aafab38a9a654fa861d4492d7e3af0724d
F20110115_AABZON elyacoubi_b_Page_039.tif
5e3b6506f51ca15d856d97d485ec7f19
10ec6f3aa232d056020440d0c10d96975b2d60e1
F20110115_AABZNZ elyacoubi_b_Page_008.tif
05965d0db2dbaafa01d35e4e73080521
e96ef8895986ee9cf4bb0e356a2a55acf313e467
F20110115_AABZPB elyacoubi_b_Page_069.tif
9375cec95185be71ff831dcb51b183fc
f9cb8032fd71c5d173f459ab0f31ebd212dbd2e8
F20110115_AABZOO elyacoubi_b_Page_044.tif
39f3f9e5cfaa57b7a4c62f74f687042e
80d8b84822c44e0038ae936ccdcc39e2142681e2
F20110115_AABZPC elyacoubi_b_Page_070.tif
52e6e791cc44e7ee00dc7268b5b36287
fbe3ffc4416892c7bccfd9e4267e865b390212f5
F20110115_AABZOP elyacoubi_b_Page_045.tif
f5db1e524d8f4954b0fea3d9da8422d8
1dacff14b0a4149b2f23969b8c56c6540f61d8e6
F20110115_AABZPD elyacoubi_b_Page_071.tif
bf075816ba6073b59b9171d8a9330094
e916c844d1a530e1cb329ab15e8a75c64b4a73ae
F20110115_AABZOQ elyacoubi_b_Page_048.tif
a48363a0bc433b97b04eb207b328dc3c
2c0f22b353ad648e25cb48487290dc117354d57e
F20110115_AABZPE elyacoubi_b_Page_072.tif
526c9a0f39dd24cb321a47b16bd2f3b9
3d16a0b46f8d045bb64571a3e121fee28a4095e2
F20110115_AABZOR elyacoubi_b_Page_050.tif
010d1168cb854370d9377faae38d6922
9fab2fbae0649e667c45aaafe1bd58434c3e6157
F20110115_AABZPF elyacoubi_b_Page_073.tif
af39d98e6dd00de8cdf48d7cdd25938c
31ed486ea112c319ab21b1a5e8cdf161dc8c0927
F20110115_AABZOS elyacoubi_b_Page_052.tif
07376daa33764ec0fdc902cc00a132e4
3003f7a0ca28369f4e94915a7791fee2529de239
F20110115_AABZPG elyacoubi_b_Page_077.tif
824290ddf3e9524e1664b12e6777da0e
7bacdb3f49ccbe7d2df3b1dc170a094cda254c38
F20110115_AABZOT elyacoubi_b_Page_053.tif
0524d093fe8a0d470eaeb69d4524d585
6d8c73d5e5b1810b6896a933c7f142c81e885177
F20110115_AABZPH elyacoubi_b_Page_078.tif
594e467ac0f5dc8b206375af6ab89d71
f7fe8ad2fea250bac84df80d8962bb6499691788
F20110115_AABZOU elyacoubi_b_Page_055.tif
67b2880b7f3deb2d5aed15b72fa18b4e
393c1855af7f6de29e061accb653f7a80c83178d
F20110115_AABZPI elyacoubi_b_Page_083.tif
50180b3b2eb696b9ee5246b1d198c766
cd965ad98f52b15f54f166682924e988545e035e
F20110115_AABZOV elyacoubi_b_Page_056.tif
bc8adda18e148d127c03b07125ed6b5c
f46ba3b64723497fc2fccba0080e8c2867484879
F20110115_AABZPJ elyacoubi_b_Page_084.tif
d5a7aab4a2110292a7c285fb5aca692a
02e36aeddfa48faa6688ba6ef061de41fc891021
F20110115_AABZOW elyacoubi_b_Page_058.tif
5a99273e4d1a1d4bd125a7c7fe100196
ba608caf6c2d5911a948b17e79b61fc7d747015b
F20110115_AABZPK elyacoubi_b_Page_091.tif
32d5ffbcfefce41e04b53cb4ed54c902
7f341ef9bb23a3c1974f7f04ad90930aa0025707
F20110115_AABZOX elyacoubi_b_Page_059.tif
9b31a0f6244a77d3349fc267a9736fcd
04abfaa45719539c6ab22c0ea94ad078edd61790
51109 F20110115_AABZQA elyacoubi_b_Page_020.pro
6ae92ca0708d892cb8013d43ae491f0a
4d256436633b56e1c5f14574fdc41c385e64ca71
F20110115_AABZPL elyacoubi_b_Page_094.tif
9196b86e3fb5cfe99620ed9a76363c32
19dd1757a142681a7d49b45d57a3588c66d62edc
F20110115_AABZOY elyacoubi_b_Page_060.tif
e789def714d95a9cf72faad51728b528
38e029ed56026b98042af09e029e4c54bd8413ff
47287 F20110115_AABZQB elyacoubi_b_Page_022.pro
9e497011483dcfca27521606cac352c5
d9d0b0ba44126a799624b2840e916d0880538247
F20110115_AABZPM elyacoubi_b_Page_097.tif
9aef206d2ca9778fa483858925fecdd2
e714aded7da518e05a8f0fbe3c0e9f8d6a15f16e
F20110115_AABZOZ elyacoubi_b_Page_063.tif
5240c2d702c5d92b9b1ea5060ccb79bc
89a87a44a174137bdfe30c2dabdb6fb9766a064f
F20110115_AABZPN elyacoubi_b_Page_098.tif
7a303014452ec113dfc28540b2ce6cdd
c7825351e1c6cd60d55398bbc3325fc2b44b2b28
47696 F20110115_AABZQC elyacoubi_b_Page_023.pro
ad871999d544a0cac0d286dcdbd60229
bed49f44eb87ff0b263b430615fae5ce48f6e2cb
F20110115_AABZPO elyacoubi_b_Page_099.tif
f8b668e917e0770d6124a26923115dff
9e35ddbe00d7850ad415b06857e8e45da07e6b4f
9754 F20110115_AABZQD elyacoubi_b_Page_024.pro
0cfbb4546671b477f24140a1094a3365
21e6578c42698c6158354a7bc620c736358714f3
F20110115_AABZPP elyacoubi_b_Page_100.tif
b4744a3b7f539d3a564a001862c2f1a9
ab6a328f677ecf634d4160b66d51adf4cdf16924
13454 F20110115_AABZQE elyacoubi_b_Page_025.pro
695524973d39d2115617ee85ad2e7a4f
261dc06af5434d7e74683b938b0be401f89cdc1e
F20110115_AABZPQ elyacoubi_b_Page_102.tif
42e6c683a465ead1d279e204f2c00af8
ec9a4dd75d287153f9e25c4341c4024b23505954
27267 F20110115_AABZQF elyacoubi_b_Page_026.pro
55651ba578fa300e2b9d9a74fdaa460c
54d5529cae22b447bc4ed4bafe270392cc02cdbd
8474 F20110115_AABZPR elyacoubi_b_Page_001.pro
35641f9bbedca8aca0062009ad86bbda
865754107f827f3c6ff951cbfe65261b73ff29ca
17700 F20110115_AABZQG elyacoubi_b_Page_028.pro
eb1ccc55fb6125b4b131a79f18d539c4
51b27cb479a76643da6c8f4cecceb797cf51a656
1203 F20110115_AABZPS elyacoubi_b_Page_002.pro
788e231084d454873b2e6ac2ba6a6657
d888e9ab3e5dd94ceefa367e373a9604b1771dc1
28853 F20110115_AABZQH elyacoubi_b_Page_029.pro
f4aaf229c98d2d99adf95046b5407897
85d7644803cb29ef2786d1312c4100aaf58e6089
1927 F20110115_AABZPT elyacoubi_b_Page_003.pro
943187aad0e7e54b9d2ddda04544f4cb
d784cf3e5f8b9dbeebba2718b4e4450541a4233d
29900 F20110115_AABZQI elyacoubi_b_Page_030.pro
8a6833250b9007cf9bb69e5429688b6f
30aa70e06d4732f26840558d1f3f0a89d3e59f7d
21489 F20110115_AABZPU elyacoubi_b_Page_004.pro
7bff488160ecc326dd919157398432bc
83188e460789a7be63970d486a4c081241155e87
34780 F20110115_AABZQJ elyacoubi_b_Page_031.pro
f01e82057329980d2d5c7bc76855fb2c
75cb56fde07ebc5eefdf060376f764f7d6792bac
94351 F20110115_AABZPV elyacoubi_b_Page_006.pro
1dbd92fa17a4dccb0ff627c25a3ce7bf
77db32f5f17eeb507a29a0e914e62fc1084610fd
39978 F20110115_AABZQK elyacoubi_b_Page_032.pro
35b387ee34de69b692416d89633f32ab
f5107c3afa8a76eb89ce8aae97cabe45298f37ce
9612 F20110115_AABZPW elyacoubi_b_Page_008.pro
0a78acd4208edaea3efcf268ec8a7e57
f2c75c853536f1e96b2e60b6983fcefb634b6935
8156 F20110115_AABZRA elyacoubi_b_Page_066.pro
032ea45779990b90313f8d4519f60650
7ad1773e1aaa94f9e94888bc7d93f69a78dba9a1
44384 F20110115_AABZQL elyacoubi_b_Page_035.pro
f3898d80d90ea7493302569d4f1212f4
3df8b12497a5087662bd05d5de880d3de16c6a6e
60461 F20110115_AABZPX elyacoubi_b_Page_009.pro
8112f9ca1623cae435911128ffd25498
71293f7a713abc6e1d5bb1b22ee2317980f067e3
35556 F20110115_AABZRB elyacoubi_b_Page_067.pro
8581b1d172a9e55e877a6bb9714b2c3f
ffc0933f4a6ab2e23c6a04962cab8360ca187989
50418 F20110115_AABZQM elyacoubi_b_Page_036.pro
22432e4f0ff6378e66abc436869325ab
871d46258813f20baff03aa3652a30b1d654962a
50348 F20110115_AABZPY elyacoubi_b_Page_015.pro
cc6490fff189fe36a2221ccd4f4dbc98
c2eeb40c5eb18a65cfcff56df31afd05cbabef56
10634 F20110115_AABZRC elyacoubi_b_Page_072.pro
6420614aa457efa1ea325a86fea969e3
60fb6a7012243596e1d429edd82238e15d9ab55f
F20110115_AABZQN elyacoubi_b_Page_039.pro
7551cf736287a5969833d62bd06fec40
631e611852acdbd426403f036756efedcafd3146
47398 F20110115_AABZPZ elyacoubi_b_Page_018.pro
2a6d3461ee4f120d4fd43cf7084e1cc0
a1edb95e057bfb428ac54935eb2f9f4bd79c4c5c
44522 F20110115_AABZQO elyacoubi_b_Page_041.pro
729f57061cb842a5cc26b4b5420dc301
59759ac44e077699192e27600f92be062f93af39
7120 F20110115_AABZRD elyacoubi_b_Page_076.pro
6a308af0d85028db0b0784c17432c945
dd54e355cd70e2a51a7e5866a458886f81620793
50878 F20110115_AABZQP elyacoubi_b_Page_043.pro
f0d03e59820e44f27f76d54753f01c27
3f84fe4ef57e5b2766b317deb3a66553f56ecf55
13274 F20110115_AABZRE elyacoubi_b_Page_077.pro
6b470da99abbdfb6cde97712b8c078e1
08339a4489bf6443cbbbc44adfdb42dabde35742
48797 F20110115_AABZQQ elyacoubi_b_Page_048.pro
d5815b46fd3dccab4871ae362035084c
a86ffdc619ae6468cb75b4eea3f7758eefa6c67a
45034 F20110115_AABZRF elyacoubi_b_Page_082.pro
874f08cdd036b3c012e4cd5cf85e7fe1
a3176398802e16a6201668df0a617b9ee3840ee8
48766 F20110115_AABZQR elyacoubi_b_Page_052.pro
8700d55f726f1b22930336ac18c98c6d
af3dc6dbe4c75de8241a80067b36126380952a65
48117 F20110115_AABZRG elyacoubi_b_Page_085.pro
b07e40240f5ae62a1001b2016d3e2d6a
963867335c5282a7a513df3d8aaafd7904912688
48217 F20110115_AABZQS elyacoubi_b_Page_053.pro
f8bfe799c03720759a7dfba26b1173cf
6e08c3665a231336b45cc07acc62b037b740b1a3
52275 F20110115_AABZRH elyacoubi_b_Page_087.pro
d422dd0d8058e97380ac7f23817fe3f3
786757d93e8d197ec8e46d23feb7e4ff154a5f52
53398 F20110115_AABZQT elyacoubi_b_Page_054.pro
5fd076b1dab3fbfc3f15c6b15e67ed11
a434324a21ca896b8fc17f26efae4dd28127c47f
13683 F20110115_AABZRI elyacoubi_b_Page_089.pro
43efed3c5113c5385c79185c79462e3b
5a60250a5d56a815c48a34e31c080973d8746512
52388 F20110115_AABZQU elyacoubi_b_Page_056.pro
663aae66a9a8bed7f7fdfa3752efe2cb
2314b715a47ec280241a60c82285d1cda386c45f
50707 F20110115_AABZQV elyacoubi_b_Page_057.pro
90b9d0b89fe620b6e0cee1e0c0cf4fec
19ea214b2943b23c4176a75de113dea7b63d393e
15946 F20110115_AABZRJ elyacoubi_b_Page_090.pro
bdc295711d4c9bd95082f66d1461c9f9
2fb874ff3dfc75a73aa668fb42a99a4462a98627
51734 F20110115_AABZQW elyacoubi_b_Page_059.pro
4228c9e16ebf719154bacdf05e5e181c
a685a46b60b94ff0121ae21d8f636155c09b63c8
12743 F20110115_AABZRK elyacoubi_b_Page_092.pro
1c39317ecdb009e187c2b04a28281f3e
e8f48b8f16c2be387dd18d6cedfb2e69a7777ef3
51072 F20110115_AABZQX elyacoubi_b_Page_060.pro
438a553f024dacaab58ed81176e2a39f
15986972c69b189a4086efe28c03c054dc79d09b
1902 F20110115_AABZSA elyacoubi_b_Page_022.txt
f7fbbe09df2a9b3037913c01de1936b9
43131fd1ce7f0dbb1567687936acbbfa30505024
16152 F20110115_AABZRL elyacoubi_b_Page_094.pro
629dbb8c4cef7ccc4f6127140f32e69b
faa44a596bc129d6238d16702215445c233f0f0c
39592 F20110115_AABZQY elyacoubi_b_Page_061.pro
066fa07b31bf2700f2e3c90bd2909857
22f1f9d7f34ed403c0b533d30f929f145bb8fee6
429 F20110115_AABZSB elyacoubi_b_Page_024.txt
e59bcf7462ed32849541c03321666ce7
74a8c3dd80ecbd6c2fff50a7df7b934a0fcf8895
33329 F20110115_AABZRM elyacoubi_b_Page_095.pro
800e4f394a91d4ba36105389f0c87350
209f96d1428a9ad5522f4a0b420ee59b0e2d98a8
5117 F20110115_AABZQZ elyacoubi_b_Page_065.pro
f902bdd49bef8eb2a7efaa0b90e7293a
fc3cc50e5ba31954492bf4f846c6d3f47ef7a471
323 F20110115_AABZSC elyacoubi_b_Page_027.txt
a2b968b0ded9e22572de7acd8c39c35f
a3d35f6eb0f38c92ad039492628967b1cc48fc65
31794 F20110115_AABZRN elyacoubi_b_Page_096.pro
d6468e695f267ad71396e2d59ff38ba8
fd814715148132e13f38969e86aff3d8729d305d
F20110115_AABZSD elyacoubi_b_Page_028.txt
4ee2ba57144e686e0bc8b442bdd3843c
6638c29c606fc51e75a94c1220a05eb64cf4c16a
64173 F20110115_AABZRO elyacoubi_b_Page_100.pro
bc254151d6908daffcdff4912f53b43b
c83ff344f0c6c38459d4c53a94b9a684ccffe3ba
62966 F20110115_AABZRP elyacoubi_b_Page_101.pro
37e28822b607c3ed4d9cbfe9d455ef45
49a89e986db4cee0781dbd13c0f1c7177f3a7211
1344 F20110115_AABZSE elyacoubi_b_Page_029.txt
c6b737e1c69f34174484c605f0d56703
ed7cf170cb8ba78ca226073f4c30d6e23471fec8
21248 F20110115_AABZRQ elyacoubi_b_Page_102.pro
e9c0ea6b6ad6d076dcf5fffa10bffdf0
514d9c390a447b235efb15dd78a3689bd6b91e60
1763 F20110115_AABZSF elyacoubi_b_Page_030.txt
d21ca4372671b1eeeca221fc0a15cb73
5deda357ea514f0e95e91b65b43b279ab1f0d65f
466 F20110115_AABZRR elyacoubi_b_Page_001.txt
eb62adfec9751f23452820dadcbbd6a2
fa234394b6fa1e9d5bca96c8ad263d9a4b6aac8d
1876 F20110115_AABZSG elyacoubi_b_Page_032.txt
de3f8626451c43ccfbe1021e9bcd1b05
11f830d4f383852676b7c82b3eb5b02fd78b3231
128 F20110115_AABZRS elyacoubi_b_Page_003.txt
eeaa922b2a5ce74705bb81c6a9cb9024
8d142ef0215c9cf129d1b6c75e4401613bbc72ba
1802 F20110115_AABZSH elyacoubi_b_Page_033.txt
942109ec6f6329678e255a551ee21ae4
39b002642005250127e9ada4f7e4003a8d0cd23e
908 F20110115_AABZRT elyacoubi_b_Page_004.txt
fff89a21d5d1b9eff9d20fbfc31406af
79048eba2c2af186241666f827e883d9ffb374d6
2061 F20110115_AABZSI elyacoubi_b_Page_037.txt
f29a0262263050f04d951b6b0252ede0
f0283b11abd97f8b6f5f209659696742e250f580
3933 F20110115_AABZRU elyacoubi_b_Page_006.txt
30d80aa868a4679fce4bbda8e7ec9c60
7ac198d07a0c9f02a40d253d03add895f08cab56
2007 F20110115_AABZSJ elyacoubi_b_Page_038.txt
cb0d1e4724b7ef41ff1c3d3a9f9f7ac6
5c879a4e7e8ec1751081928cb1e1dd601f4b7b16
2446 F20110115_AABZRV elyacoubi_b_Page_009.txt
85f7153bf69144d8a7b893392cf7333e
5f51c8714a2acdaf1ad2324412901f92e106ad8b
1796 F20110115_AABZSK elyacoubi_b_Page_040.txt
db8b017c1d730d3053a983895654a804
be5d3568a1d6d0677d948b7d572f17c848ec98f2
1974 F20110115_AABZRW elyacoubi_b_Page_015.txt
c29d73a9201ef852ac93650eebfb0ec5
1cf33135b9124b5080d840b1fd2f854f1f5b1fc5
539 F20110115_AABZTA elyacoubi_b_Page_072.txt
c1dcf23cead58c301b36f406c526d00f
815e1e2d28ae28578763b60bfaaea08a324f4489
2037 F20110115_AABZSL elyacoubi_b_Page_044.txt
e40213e8fd9ad3a27d7b86a0d48568f5
f316b072eec96e490aaedce0f0115b09bae33bd4
1856 F20110115_AABZRX elyacoubi_b_Page_017.txt
443751bf56595b5489e6c918621410a9
009ec80c1f6984892e4356fbd102438e80f8fb4f
1898 F20110115_AABZTB elyacoubi_b_Page_081.txt
47d6dcc3192ba6db5affd9a77a1f5d00
623d5ee5237d1eb3a2f4e6d85c09e38d91f4b00f
1731 F20110115_AABZSM elyacoubi_b_Page_047.txt
8f32d5ec273a23a2c61a28e72d248ec7
7e96d9cbcc6e196cbe9120b240d9325bce9fa7f3
1892 F20110115_AABZRY elyacoubi_b_Page_019.txt
ec010bf8893d9fbe40c576f122a1e121
05507f009a78558a2253467d5b2f16389a2814db
1783 F20110115_AABZTC elyacoubi_b_Page_082.txt
ede354c8b0805a3c86a3ea56ec083e17
bcfee88078d9e6f7751d931bfd91901000b55025
1941 F20110115_AABZSN elyacoubi_b_Page_049.txt
b876c19ba5d28b9a599610fb3e3296d0
91dd04a0c2c8694dee523665807855beccc63c52
F20110115_AABZRZ elyacoubi_b_Page_020.txt
27c5085d103e6f84a04c74594c112334
551308d1d5532d112b5d78e31cd8e6352b81f74f
1959 F20110115_AABZTD elyacoubi_b_Page_086.txt
c73871a46907c091ab239f9d7bb6e2ef
900453e9611c87ee8f8acc4b8dd3d6c00f9442f4
2050 F20110115_AABZSO elyacoubi_b_Page_050.txt
bdf484ec06d911998e0d97ae2b7d72be
e17a86d083586b9998d56ee2a57def2192e4c133
759 F20110115_AABZTE elyacoubi_b_Page_094.txt
dd2b38830a5154ed6873aa8218861dbe
22a8e34bfb3afde3e36fc9a4b59c1d82c6ef2080
2076 F20110115_AABZSP elyacoubi_b_Page_051.txt
b93ead326b7e0f548954b2539259d86d
228c62162f3c86c871404bf228bbe639c4647610
2100 F20110115_AABZSQ elyacoubi_b_Page_054.txt
270e35560ed535edb52e96688386fb7d
ac8d0da823e740976343b1569282a29588a20844
1928 F20110115_AABZTF elyacoubi_b_Page_095.txt
457432b30e81eed84791a506f33da248
cf3521daa0a1e8f7a8e6a55bebb32905cfd1739c
2062 F20110115_AABZSR elyacoubi_b_Page_056.txt
bd2871fbae795773977cf3a698e62545
dfa713aa2da9f246bfbfd73b9059a6e0e7d7dfdc
2484 F20110115_AABZTG elyacoubi_b_Page_099.txt
d8d7c79e5c1a368b4044f6254a84298d
ca7ebb0e0991d444ee532999ed43da2760cbdcd4
2001 F20110115_AABZSS elyacoubi_b_Page_057.txt
912f596b1ab09ff7dda552322ef7b79c
6166151e0aa580d18a59af198202619bd21e60e1
3216043 F20110115_AABZTH elyacoubi_b.pdf
8f8a449f76b17585cd53b409cc67d815
200acc0c03e5ebc381beaf6ca4641f375d810b50
2015 F20110115_AABZST elyacoubi_b_Page_060.txt
feb67633b245fb516676c0a2505588b7
d16e7ac92a4797bda7ed314e04449d40528db1c9
121421 F20110115_AABZTI UFE0008379_00001.mets
9e4c4f3f5b1ef7fda52d20499ced7dd5
910e5569899d9cc11785283ac6ed60d7e409737e
596 F20110115_AABZSU elyacoubi_b_Page_064.txt
29b634f9097b71102f90d45a13cdaa11
ef2d2f9e5f6375e21e83c9e79d5402874558e0d1
6891 F20110115_AABZTJ elyacoubi_b_Page_001.QC.jpg
8079ad863786ceceb4c1bf39a9a9f8de
f117203a3e95ebf57a5f92abbad135546e54901a
285 F20110115_AABZSV elyacoubi_b_Page_065.txt
3b1adf53c5cf3483ba30d9b99d3af090
d617083ba07da2175ff65c7466c5865225e5e8f8
1384 F20110115_AABZTK elyacoubi_b_Page_002thm.jpg
ede928e3b8a94561eb8852ce7bfda878
0f11be1d254d90d35f734be136ff7ee16682f11f
363 F20110115_AABZSW elyacoubi_b_Page_066.txt
80fb299f8dfb5f94c327b5266a08b271
2b3c79c95df3f98dde6a07d0f58df4d9124d4675
3439 F20110115_AABZTL elyacoubi_b_Page_003.QC.jpg
fb629dbc574f2749c85e46c4793ab3e0
e5bb359bca13ee42560f4238346cc5d8e683fc03
2483 F20110115_AABZSX elyacoubi_b_Page_067.txt
6d5ec5cfbec97e80d7855a21eaf42be1
cf0df56050ab97bd377741755348ccfe22d1ee2f
7071 F20110115_AABZUA elyacoubi_b_Page_020thm.jpg
1f6e45c0da31a5cbac7830cf1d55688c
7347e8c6c6db9a91f0dc2137db3da4fa64ad4d65
1453 F20110115_AABZTM elyacoubi_b_Page_003thm.jpg
495d931ea8aecaeed17da627be78f7c6
27283c86b7d4c8bff57f7384893eef227bce1cdb
412 F20110115_AABZSY elyacoubi_b_Page_069.txt
c3ab44c068d3cc089df79c13527d2550
c59246e2a4dd54d77103b9946d02d4ba610effc7
6523 F20110115_AABZUB elyacoubi_b_Page_021thm.jpg
70648645e58b4387fdd1456650e700d0
d965427c908e36e242c877adf69b2b68802d861d
3677 F20110115_AABZTN elyacoubi_b_Page_004thm.jpg
35e1383d2b3670385b32bd632df00a33
5517053b84f40dad3b3725235db8e2639bedeccc
439 F20110115_AABZSZ elyacoubi_b_Page_071.txt
f360b1329301eaa4063c16c8caabe9bb
992b65dad891828eb56a4cd1f69a279304e41d9c
22604 F20110115_AABZUC elyacoubi_b_Page_022.QC.jpg
845f50c615c64fc874b8cf22903fe2a5
57130936182c0f29030970e309acfb2cd709b631
6237 F20110115_AABZTO elyacoubi_b_Page_009thm.jpg
389aba198ce20159c7d16e45bfc9eff4
5a0b18a36510054d63d267af25142c5348b2c800
6222 F20110115_AABZUD elyacoubi_b_Page_023thm.jpg
2ac14b93c1eb9466024ae66001c3fdcf
7aeeacc0eed04b8481c4bb1d6477f8a413902179
19847 F20110115_AABZTP elyacoubi_b_Page_010.QC.jpg
0cb36c32311be1f9c7431ed3606e1abc
73a2e50d82c0e40666763fb825a60661b73facef
8285 F20110115_AABZUE elyacoubi_b_Page_025.QC.jpg
4cee8db1877a6b6c29f6797909b3cefa
0be1ea322a1daf72f32f06b6a54b6869324f4ac7
5232 F20110115_AABZTQ elyacoubi_b_Page_010thm.jpg
007994aef9dadaf3e737b33ddaa81431
b962408654bd6b46cd475ce63b3618d4c88abaf8
13441 F20110115_AABZUF elyacoubi_b_Page_026.QC.jpg
5f139ab7e51960988196332d5c7ab4ac
5857e3c4a5b2f19eee249c4dcee49bb75e065119
22199 F20110115_AABZTR elyacoubi_b_Page_013.QC.jpg
5c2b8dfaed2cf2f0eb97d25b66f3b82a
7555ce6237b9fe62a032958a33e7d999803557fb
6011 F20110115_AABZTS elyacoubi_b_Page_013thm.jpg
b6c467495358bf7a19f9f26f42a6f01f
55606ee66075387b04693593eb451068fef3d745
7432 F20110115_AABZUG elyacoubi_b_Page_027.QC.jpg
b988ebfe32991e51e7a76b1ad818d4cd
2b67a9cdf273ec464211523a3a3e130fc0f3a61b
6628 F20110115_AABZTT elyacoubi_b_Page_014thm.jpg
b8873b3629a58721b9c6ec42d0d090d0
1576973c9e063e0f35034b555c70199935901f53
2536 F20110115_AABZUH elyacoubi_b_Page_027thm.jpg
00f59cd7ee87bd1e4e3794cea092cbb0
74f70e4aea306a09f9173a50bdaadde9bdafa780
6672 F20110115_AABZTU elyacoubi_b_Page_015thm.jpg
9fde5eb62d16c6cca528d717d5f9ebdc
ffd4f7d9001ad71f9306845a394d840638616819
15211 F20110115_AABZUI elyacoubi_b_Page_029.QC.jpg
f16492e68d000483881f8f28220894d0
1a7e841f6b88a54b7903c960f5880cef0f8a3307
6238 F20110115_AABZTV elyacoubi_b_Page_016thm.jpg
c557a61a34c7dc83c7bbd8731be54010
22d1bbcaadf8465758dd26e591f3627b7c5055f9
4287 F20110115_AABZUJ elyacoubi_b_Page_029thm.jpg
9122a9535a03365c2b19728c91f81f44
750ec53699eaf03589c3eccbe3b212d2a6f1ea70
22755 F20110115_AABZTW elyacoubi_b_Page_017.QC.jpg
baa58bb0542f83b6192ce7116b1b0ac5
9382c61386c0ab48bd30f29353dc66ef6e34b6d5
14451 F20110115_AABZUK elyacoubi_b_Page_030.QC.jpg
30be16c9aee8b181a295896d872ed7fd
60ba550b88df1a672e686236591609fc9ce294a3
22868 F20110115_AABZTX elyacoubi_b_Page_019.QC.jpg
e2189d0096d47efd79a715457c82d0b6
d91605f21b5e5524463ae58dc87e2c6a79e55581
23266 F20110115_AABZVA elyacoubi_b_Page_045.QC.jpg
0415f4e48b121b75d84a765802787e0f
f8b692aac2eec928f42e7b68ba0d164bb0d34490
4075 F20110115_AABZUL elyacoubi_b_Page_030thm.jpg
e8cfcd0cae9e77205ac1b2c01b157ebd
8db561f8f8559c24ad029a4b1e4f7d89a3de4423
6439 F20110115_AABZTY elyacoubi_b_Page_019thm.jpg
c442690d834bf6cea39510c9ecd03028
57c9f48c8113b37e21d59efcc1f5de23fb283d75
6450 F20110115_AABZVB elyacoubi_b_Page_045thm.jpg
eac47afaebb70b57f039a81230e8a24f
1d3902acd8943e47295aa1a959e926be75a91fd8
16314 F20110115_AABZUM elyacoubi_b_Page_031.QC.jpg
0cd6cd99aef8586f6b85dbeff8cff19a
e91a5424be8023a0f7b9ab329c4aaab935f3ab9c
24958 F20110115_AABZTZ elyacoubi_b_Page_020.QC.jpg
09c822c750b1ad979461f7ce1946a620
7450dfa20c44eed4ac6b78c52cdf8e500b8a98b7
23898 F20110115_AABZVC elyacoubi_b_Page_046.QC.jpg
8cc53397b51a7b9c29ea27cf24000364
1661e697e84703695caf38d7bdaa19294fab3bcc
17781 F20110115_AABZUN elyacoubi_b_Page_032.QC.jpg
c9f874c882282ba1907a7440b49e0b37
a249051857a3fd7ccfa5ad541cc129b2ddcada65
6640 F20110115_AABZVD elyacoubi_b_Page_046thm.jpg
eefbc4eeeed4cf190fd9aea9f151cd84
5d81169cd3eb95f9c05d3a42801890b0b6442585
17626 F20110115_AABZUO elyacoubi_b_Page_033.QC.jpg
48e5a4dea0b5a62d0c935c274fe4298f
fd33cf2fdc98880e64f4dbdc66f9636774fd1fdf
22156 F20110115_AABZVE elyacoubi_b_Page_047.QC.jpg
2320d21a4fee31a94d60c6daba123975
f9672743b12982c9090582655939a18d8ae56fd4
14916 F20110115_AABZUP elyacoubi_b_Page_034.QC.jpg
65e9371a45c3d002f240088555378e9f
97966ead1b69d996f533ae3e8a826736c4e70f9f
6529 F20110115_AABZVF elyacoubi_b_Page_048thm.jpg
9e0bd124d4df1ad461731130dadcb16f
30382798924d3a20c1e22120adf01cd9632bb962
6125 F20110115_AABZUQ elyacoubi_b_Page_035thm.jpg
e2b7e40bdc50506d57d9d6dd0768d95f
7be967a886d929270769c40414647d8805e71399
6502 F20110115_AABZVG elyacoubi_b_Page_049thm.jpg
c4f80b47603bae9ac32fa5e5be65d7a8
9ba54447581bd709b6c76a4b2582a4e3a7e59a7d
23762 F20110115_AABZUR elyacoubi_b_Page_036.QC.jpg
43c1011d2b9a72c7f9d9858c090ab884
eb3f06f9e0d12665a2b876e001426e28a8a59b49
6592 F20110115_AABZUS elyacoubi_b_Page_038thm.jpg
acf43d861557e97cadbd5885242b4c64
bc8ce1b0e575eecfa57c203718058c497dc73de5
6637 F20110115_AABZVH elyacoubi_b_Page_050thm.jpg
57456d1cdf7248b32262ff1e3f7d3306
777e0164c7b3349dd595a238fcb78961965e91f6
6122 F20110115_AABZUT elyacoubi_b_Page_039thm.jpg
3b2d4ec87b3b45509b40728bf2aed82c
aed03e35b89319d6a1f4aaf97f780e851f880a51
6654 F20110115_AABZVI elyacoubi_b_Page_051thm.jpg
01ad7224ef861b09a5a0447e0da4b9a2
88dc5b508ccab4c2d878b827552f0c9e1bb05b16
22064 F20110115_AABZUU elyacoubi_b_Page_040.QC.jpg
9c3ed8e95e8ca05e49ea8960e36f6ac8
a9cdb19d4d8e14e80d1c60092c24d9a60f077316
23761 F20110115_AABZVJ elyacoubi_b_Page_052.QC.jpg
a6c80b4d9aea51987e7e91dbc34043a4
abfe8342a65ae82f4c7ab8999399dbdf95e2f180
6196 F20110115_AABZUV elyacoubi_b_Page_041thm.jpg
c862ea5763a89ef29994b35879e079f9
18de9095535c7184c29efe9843c7cf76a54907e1
6601 F20110115_AABZVK elyacoubi_b_Page_052thm.jpg
f1d944584f813aa6ecab9a176a3f4ad2
327af5a67680641d228ee9376c7bc8fcbacd6f39
19734 F20110115_AABZUW elyacoubi_b_Page_042.QC.jpg
12015691f4076fc9043bf66fc3a9d971
bd25eaa71544774d916cfc74d6dba3e9a3efe871
5181 F20110115_AABZWA elyacoubi_b_Page_073thm.jpg
fd167a41ebf130effc853b2fabf654e9
a19172ea61e0b2bc44203127486dc659c66ff93e
6578 F20110115_AABZVL elyacoubi_b_Page_053thm.jpg
467b935c2955547d8795e282cae2be7c
71a5d689b675826878635ca0386da5fceadac979
6683 F20110115_AABZUX elyacoubi_b_Page_043thm.jpg
84252584f6445d762a33a86c03bc6e9e
eec818e352a80db787fafd84756579f65ff8eb20
15967 F20110115_AABZWB elyacoubi_b_Page_074.QC.jpg
a45528b4c74593bb5ccf79a0efb161be
ca5b5d69f89ca58bf6eb2d365ed69653aff467d7
6917 F20110115_AABZVM elyacoubi_b_Page_054thm.jpg
cb99e527075b1cdecbf06078b886357d
bc7c4e5ae065f22293c2d395d1f406f580af5734
24860 F20110115_AABZUY elyacoubi_b_Page_044.QC.jpg
d1ff41eeadfd004913b67d3b98000edc
ab9929e564d8deb07878d7ef95f2df05a986629a
4885 F20110115_AABZWC elyacoubi_b_Page_074thm.jpg
a1b91721504d77916d6b4f08eaf38ff4
51ce56585d04e33a6bf965c00625634f4d359322
24758 F20110115_AABZVN elyacoubi_b_Page_056.QC.jpg
da85d06606411ae027db5ec5a1695e07
9a8dd06e17c5bacdb1013cda7a6a9537f11f477b
6667 F20110115_AABZUZ elyacoubi_b_Page_044thm.jpg
09ba16b592edad3bf0fe3c3e6833d97b
d7b1e285e695ed659d5932ed0355fd2c19da16b1
12008 F20110115_AABZWD elyacoubi_b_Page_075.QC.jpg
adb24df396ce173c9e0cf81d0591f2c7
478a84821860ffcac7a3219ad6532c01e72e860c
24412 F20110115_AABZVO elyacoubi_b_Page_057.QC.jpg
f97d2524c586c2984dcd9da0aa70a56b
0e3158cefbb0abba0a4ede8db6505a5357df0f1d
17108 F20110115_AABZWE elyacoubi_b_Page_076.QC.jpg
8a3388e7b614d0f12d40a926c0672981
5d91e60a6c94a9e0a16fb990d3df5d3c9d20072e
23995 F20110115_AABZVP elyacoubi_b_Page_059.QC.jpg
8e8274493252000a8555a065fe600a86
56f023b2002a9d4cfd93a46332787ba00df61bfd
12957 F20110115_AABZWF elyacoubi_b_Page_077.QC.jpg
c2feeda999e7535289e547e02f78f59e
ba0bb75cee67c39765a8d8c415e3466af3c63c26
6573 F20110115_AABZVQ elyacoubi_b_Page_060thm.jpg
04280afd0c38998f193c02b3b5305ec8
cbf36bffae3211b895c367404566522a48bd852b
5718 F20110115_AABZWG elyacoubi_b_Page_078thm.jpg
2ba2396b22a3afa8a63307bc271a1a0f
8492f7efeb9752c54385b4c331fb762ddf8ad5db
12260 F20110115_AABZVR elyacoubi_b_Page_061.QC.jpg
a25ce454cf1dc7e2616c0cdacfd7c990
81ae117f73c73310b6d6f8c5a6895592e3902213
3469 F20110115_AABZVS elyacoubi_b_Page_061thm.jpg
b1b2b51a6203e587f228b9441fc91a84
b0044996b77a6a75a9570eb30d6ac551c9ed0636
24032 F20110115_AABZWH elyacoubi_b_Page_079.QC.jpg
33a53bd0f428b03060c6ab168f162b2d
2721ea88c785690d177fd10e655244e55c5ba469
18966 F20110115_AABZVT elyacoubi_b_Page_062.QC.jpg
99aabe20f5829acdddb6cf9922027d0f
4ad9960282379710437d24d29bc49c94112fe868
9031 F20110115_AABZVU elyacoubi_b_Page_066.QC.jpg
932911d38df085d41ab6d0a2cdbae4de
a24ab5acc05d27f56936874931af3fe85d32ae5c
6415 F20110115_AABZWI elyacoubi_b_Page_080thm.jpg
2cf6b72ebc46d887e21a60a3d7521919
c8bbb7ebf3afc1eb970f000517d60c5d4a3008c0
3153 F20110115_AABZVV elyacoubi_b_Page_066thm.jpg
003e89821fa33c38eba2b1f3e3591c64
ead7b57703af3083324a77dcb50fc03e724d9751
22357 F20110115_AABZWJ elyacoubi_b_Page_081.QC.jpg
33054bfa5fae737485003a04134d0eb9
e74b755f9325ca97a6fcb24c81f17eda5aa2462c
5101 F20110115_AABZVW elyacoubi_b_Page_068thm.jpg
0066981ee7d7acc840beed11b183d174
eb094e0bb3078833fbb9b9d9993b8424dd418404
21922 F20110115_AABZWK elyacoubi_b_Page_082.QC.jpg
f43338b9b7cde68e625d25b369404fe4
d12d50d82577e7c5aa84ecafbad34fd0c0c768b4
7751 F20110115_AABZVX elyacoubi_b_Page_070.QC.jpg
d0c2fa66c537300324251ab0a64fa480
525b6c78f6256e3c563f51113e4278f0bbc133e7
11984 F20110115_AABZXA elyacoubi_b_Page_097.QC.jpg
ac7c36052edf2d35cda4ddf6475be49e
8b743bc801b0424ba635d3c735a5e26877fbc65f
6251 F20110115_AABZWL elyacoubi_b_Page_082thm.jpg
09a1db85456e5b34d0d9dfbe92415ef5
8e9c1466f5019b6abbe4e87b82dd9a610d26af9d
2597 F20110115_AABZVY elyacoubi_b_Page_070thm.jpg
dc42a3ae3ea08280d75bb92dce523412
e44eb4f392960c17464c91870a93f62c2459f25a
4145 F20110115_AABZXB elyacoubi_b_Page_097thm.jpg
cad64f6b933f4e48e23b7070927e06a1
df78197ebbdb4f6bc154c935b698fc98c541bb26
22976 F20110115_AABZWM elyacoubi_b_Page_083.QC.jpg
aa4f18c99d092e76aee8dd08dbfd51e5
52b5888c3c96a8f5b218792b42b7ba1bde37d480
17472 F20110115_AABZVZ elyacoubi_b_Page_073.QC.jpg
b0fb1219517eb51288be8c24b6d6345b
cb79016f4517aa7fb87af7630f6a74732d5b402f
6892 F20110115_AABZXC elyacoubi_b_Page_101thm.jpg
1e91185da0ba6b132b56010e4e76edda
516661819ae0fc8436171ba490416a713f5c4c21
6394 F20110115_AABZWN elyacoubi_b_Page_083thm.jpg
6bb13999cde6bca8adbdfe7c72988d84
2dc68f871a382f163b209693d10f82664e5e7645
10670 F20110115_AABZXD elyacoubi_b_Page_102.QC.jpg
4ebd04cd4d43c2815e2e6dd6b843a2b9
a2d4130243bf31880e97cfe983aaa6e1d3d29036
6236 F20110115_AABZWO elyacoubi_b_Page_084thm.jpg
d2f55cf633b678336cf0519ddb04ba63
280c4bc60236f96ebbf6760774bd43f61f240d7e
3056 F20110115_AABZXE elyacoubi_b_Page_102thm.jpg
4b7cddd8d9e92bba4d9ecdb44edf3311
5244168ab1c85b5a887344ae9b22bfa15f6d1075
23625 F20110115_AABZWP elyacoubi_b_Page_085.QC.jpg
2a7b9913c1c8787b3c8cc03a17266e66
a4d71f3d43bbab4e24e2551a9d856bb2147e5b87
6385 F20110115_AABZWQ elyacoubi_b_Page_085thm.jpg
4368d3c79dba51e82cbbe67f1cab297c
69482dfe9ee167d047ad1973860e14303d0b8169
23523 F20110115_AABZWR elyacoubi_b_Page_086.QC.jpg
7204d43c9f8320a5d4b9f96ce624dddb
cabf3080520d56a0fb8dc4133d7fff8b43ddc3e0
6506 F20110115_AABZWS elyacoubi_b_Page_086thm.jpg
e2b0a1e286559374642a4428b9dd11a4
de23928de8d72111b029321a9cbf7d11ae309ae9
21956 F20110115_AABZWT elyacoubi_b_Page_088.QC.jpg
b08c72c9fda48d8a2a9aa38470a91b05
cc1384232b00185cfc7d260becf5f83bc7ee320e
22660 F20110115_AABZAA elyacoubi_b_Page_049.QC.jpg
908b1b9c3fafbd51bde09286002a4d96
d090619d257b8c4cf7b7df7f1a72c517f1987aa3
6112 F20110115_AABZWU elyacoubi_b_Page_088thm.jpg
037649a0620135806695c067eae12132
62348b7754e163b612006e2f8a5829995869ee06
4848 F20110115_AABZAB elyacoubi_b_Page_033thm.jpg
2b3923fed0d86f5d8f4b322e70754d7e
1d1041e9a1306c77c61a28cdfcc935e9b25a4c5e
3220 F20110115_AABZWV elyacoubi_b_Page_090thm.jpg
5930f3f05d5d7624bc9e0d4871e2a11c
45ce861f5ab99b912bdfb08357e7bca7c77a539c
45045 F20110115_AABZAC elyacoubi_b_Page_021.pro
98ebcac14c24144921c7dc5b179aae6c
2d7d70959a7e590197493e6117b2d3e3e5fef680
9581 F20110115_AABZWW elyacoubi_b_Page_093.QC.jpg
b1912ed5cff7e15bf4a057b1118b3714
96caedc1b874efdb2f2efc912c39da162a813210
24052 F20110115_AABZAD elyacoubi_b_Page_043.QC.jpg
ff3e248c88f08ad0a4e313bd280e2992
2ed77116ca76105f49c51903c637becb8e045559
3922 F20110115_AABZWX elyacoubi_b_Page_094thm.jpg
f68407b988949319a88bfca2a6d05d65
1eb69a55b5866ef91fd0ee4e2f8f998244809a8a
851 F20110115_AABZAE elyacoubi_b_Page_091.txt
e1366f576f175f4413d1c08db150eaa0
34ec8a6a99f0dc9dc75e0483d8dcd19b7b7e45ea
5131 F20110115_AABZWY elyacoubi_b_Page_095thm.jpg
134f7dcde3bd5ae273f4f223a23b356d
a9cfeebb42dd7a841ac18a1ba8b6a5a5fd76e511
F20110115_AABZAF elyacoubi_b_Page_092.tif
f06bbaaf9584abc2ad79c2688c325ff2
49d8e7978a4f514b94752fa08295dd8e42207012
17545 F20110115_AABZWZ elyacoubi_b_Page_096.QC.jpg
4313969ff7406a7217886e184978b73e
484dbe4e5ffd67482e77b4ec83ec2a2e5b88e05f
6538 F20110115_AABZAG elyacoubi_b_Page_079thm.jpg
6216348cf68a125b81b8d86accc075bf
15282974ebd5623cef3101ace625b34f9641f768
16983 F20110115_AABZAH elyacoubi_b_Page_012.QC.jpg
de1d4ee68d7db2ff7223d8840d3e7521
772f3dd237ff0dc890841e82d39189ba9c4b2ab3
38555 F20110115_AABZAI elyacoubi_b_Page_011.pro
6adfdd3b3e8b30cbb228dbf9f5120615
b602366e67af569151483c697614d0dd36b17e44
F20110115_AABZAJ elyacoubi_b_Page_037.tif
8eabeecaa9a0049c1e838fe5b420d38b
c73064e3aeb301ddc0c5182d1582655cd9f630e6
F20110115_AABZAK elyacoubi_b_Page_007.tif
2998d5bf794ba7bcfe88c3691f2c4347
77387669ec85ccec883e3e8612bce8bfa5a1f313
F20110115_AABZAL elyacoubi_b_Page_075.tif
503c8ff0e96d360c2b51a8b7caeeef6e
c63984dbf9bf40d5db09997d2a056726c2a4a182
F20110115_AABZBA elyacoubi_b_Page_038.tif
336ab43477a9eff21295ce3401b6956b
b788e47113655d40c6a4db5b119b262ac77ae429
52390 F20110115_AABZAM elyacoubi_b_Page_037.pro
0d60976f3fb402bb107f3c81f8c166dd
cca0b5838b2e7e14fe89c3456d921ee01643487d
113540 F20110115_AABYVH elyacoubi_b_Page_087.jp2
d7a9ace47ef3b8845f0ea6c4552d9910
7b77a64072e9a2a069137c532fc32b14eb03f638
73787 F20110115_AABZBB elyacoubi_b_Page_057.jpg
3833330701105c574b18024122d6a020
f781f6f90b6f06e6b449e641d94b7686b4c3d9e6
68319 F20110115_AABZAN elyacoubi_b_Page_040.jpg
7af343bf95cea20b7f7eccec1b73fd8f
105dde0585844556e15b73937981a22f012358e7
F20110115_AABZBC elyacoubi_b_Page_057.tif
e14e254296d1ad7a16a73b69de2f18eb
71039811788f13de6a7448b0a09e365d3bb8f80d
47318 F20110115_AABZAO elyacoubi_b_Page_016.pro
c70966f41c83b3ed907cc2dcaab28c0d
201efe26e17144373b42b7702bc9a58377d9accc
93860 F20110115_AABYVI elyacoubi_b_Page_078.jp2
a92955d1ad7f70b8d77eaa7cbb80a3cb
ace59f9a61561cb5bfb4bf59d43e50d797fee984
21628 F20110115_AABZBD elyacoubi_b_Page_041.QC.jpg
a4e523b6ba1bedc0e56f2553ec20015c
2d3262bf86837504849cd0b66c2c659dd16adef3
61176 F20110115_AABZAP elyacoubi_b_Page_062.pro
e16b6f9585414e3330b58884578f2411
d0826bcae4e71b1d8adf61e10faf8830e43661fa
1585 F20110115_AABYVJ elyacoubi_b_Page_042.txt
24a246884a85b0bd0322ec48165096d4
760c5088740e9b1e53a1e04c74e0f208ca98cf0f
3572 F20110115_AABZBE elyacoubi_b_Page_075thm.jpg
e5ba4381bf6e94df4535b136e12e5b4f
10043cd652cd2feb927faee9931f8282db4c3a20
905 F20110115_AABZAQ elyacoubi_b_Page_102.txt
397da1f15ec570a08f96b8c55da11654
2377af1e959b09ffa7ddadfaa5289639c852c882
F20110115_AABYVK elyacoubi_b_Page_095.tif
1dfcc0e59ff96a8845e6f1f9e82d03e4
c6e2db690632a648a49c3384c1c4a6e6e25a489d
F20110115_AABZBF elyacoubi_b_Page_081.tif
5f4a3b95b0aa6d2f45698e9dce5ce740
9c5607fee598c7ab7296f6039cd5e2cbce8a89a3
1882 F20110115_AABZAR elyacoubi_b_Page_018.txt
f09448a105ee4e58d182662d8f729e29
343cfa4e3291f2da8f7434ddf793e9446be36865
4727 F20110115_AABYVL elyacoubi_b_Page_034thm.jpg
7134c19f9d2f72357fb1527f06006760
aaeae87b5889fa068ec991bd1e82afe964917b32
45557 F20110115_AABZBG elyacoubi_b_Page_088.pro
8ba07028b6477cb2f39e004533b85510
10848f36f3e6a8985726903ed9ad2cab3dad136f
79167 F20110115_AABYWA elyacoubi_b_Page_009.jpg
4e6a8098ebf07a63762540dd559aee06
d10bd2b4400d49228924bcdba22df03a15ce3c80
49528 F20110115_AABYVM elyacoubi_b_Page_058.pro
342c1b8417891395468360c6e76a45b8
7d41cc1e08564a80d3e056b6218b21405b1ecaac
F20110115_AABZBH elyacoubi_b_Page_040.tif
53cf1a5122ec54be63f4f44a6a942a6c
ccefd22a02884ead28c4796e5deb152b3e73fd3f
F20110115_AABYWB elyacoubi_b_Page_088.tif
3fdd33c57b811b191befafdec03fd321
762c16f28a6ec5b6345fed112c7312b26ab725cf
107842 F20110115_AABZAS elyacoubi_b_Page_052.jp2
257c74fbc6fca673e633f9b38119d1f0
7177f315871d0c5be907be3399e0abee1268a84c
7166 F20110115_AABYVN elyacoubi_b_Page_065.QC.jpg
b9dd5aabae11bb42439373fbe4a77c3c
60ab4c2796390b97458208a8314bc4ec88d8cd30
F20110115_AABZBI elyacoubi_b_Page_087.txt
0a7061b6df976ec586c4d201ebb23575
2a243c217efcf47f74f1385f5b4454c9b4cf22da
F20110115_AABYWC elyacoubi_b_Page_047.tif
74e7fb267c2912ee5b9c2ce52ebbb711
5f8c5e7ba97b2addbb2bf452fe699573e61fbf1f
14356 F20110115_AABZAT elyacoubi_b_Page_068.QC.jpg
249ca879a3e95796c15690a0fd591669
2b2130ae0bde941d8388d6baab9e5250bc285f6d
1887 F20110115_AABYVO elyacoubi_b_Page_045.txt
65946975cb53e56524585a672002bf4d
d2c36d5d5cec1aac6c7fda415b0fa496ce621e2a
F20110115_AABZBJ elyacoubi_b_Page_024.tif
9924791f63b7f898a92d82c205d299c7
f74410e069239a7f7a6ad7586c7242133579952c
11798 F20110115_AABYWD elyacoubi_b_Page_093.pro
eb29af1bd2e079a5b064844bed6f697b
478758149cd28f85bedb0ca8f5a888028d91d886
3382 F20110115_AABZAU elyacoubi_b_Page_071thm.jpg
0c8c6802d7abbf9b7d63dae71996c44b
abe5b0edd86f93a457edc7f054daa1eeb8d982e9
F20110115_AABYVP elyacoubi_b_Page_086.tif
1fc11d2c25ec89a42f29f178bf9654a7
55c6e81f649fee13c6cd90ae44d5ef18dbc2591c
2155 F20110115_AABZBK elyacoubi_b_Page_008thm.jpg
f46150b5ddc5b83381e84856aafd98b4
5c179015d93ae8bbe6da47653f9c628446318939
5838 F20110115_AABYWE elyacoubi_b_Page_072thm.jpg
b6c72384bfb96df8c92cbd5caf2b9d51
a8b88d1760aae959b59d026858908cc3cbf07e52
F20110115_AABZAV elyacoubi_b_Page_080.tif
deb7c5506cf6aecd4c9a186c36202152
6381f5aca155b1607f7786c828e38ae8a6899371
552850 F20110115_AABYVQ elyacoubi_b_Page_092.jp2
f6b1bdf1dbcb6b3db37b0077173d6100
f1c59e82f40726b92a7657b927786625de0dc487
11954 F20110115_AABZBL elyacoubi_b_Page_094.QC.jpg
d6b70a6186ad45fe17139337e608d8c2
d450fe3a29a0c6c30ec3efd015f451dc4cafc904
18297 F20110115_AABYWF elyacoubi_b_Page_103.pro
54cd0c3543d6b8a4dc701e34fdb627b0
c6d724d0d4976ee4ffe689c04a324de17f53b057
22523 F20110115_AABZAW elyacoubi_b_Page_053.QC.jpg
ebcb033250312ece581c0e2e32ea28ca
48048f47fed5864c4e7c036180b033eecf92c1ee
F20110115_AABYVR elyacoubi_b_Page_054.tif
ee597c6d62f6a4cd1fb1ce38e5c39875
45ad4d11b8af1665a70a284b56fc440f9ad26f53
72995 F20110115_AABZCA elyacoubi_b_Page_059.jpg
da3f788b510580410cee87dfdefb82c3
8317f04957db214e4f4c3a1652747c64aa40faf6
23811 F20110115_AABZBM elyacoubi_b_Page_024.jp2
1b72217863e95bfe6b1aff21aabb6c2a
03391911e767b91001d287e52c46b9467bdf9056
7133 F20110115_AABYWG elyacoubi_b_Page_100thm.jpg
b6cf0c9726b7cae3ad56c50fec657525
0f5f108e5f44dcbad680835e52b321e7371be199
2392 F20110115_AABZAX elyacoubi_b_Page_001thm.jpg
cd0d1259f289429e010259f4204fc43d
5b2fd4ec1b5ca47ab2819644e17d1f6a7f7bec48
9379 F20110115_AABYVS elyacoubi_b_Page_071.pro
36d9e74de50aae41dab67999c254099c
5e33a434b556e264a80cadcc0a12b530f8547f03
1940 F20110115_AABZCB elyacoubi_b_Page_048.txt
5c811565f45b55d62a69b3588a46f4cd
f587ee84fc332eea47f66de08339c1dc99064686
37362 F20110115_AABZBN elyacoubi_b_Page_094.jpg
db42781bae7dec40ed750818b6d63524
e5610b6db5d7159f4291f80abff067e05e5d37fb
781242 F20110115_AABYWH elyacoubi_b_Page_074.jp2
91399422f242b374115cf8498439c232
5d684c73647670f49db014de0afdf5affd7d5ac2
86711 F20110115_AABZAY elyacoubi_b_Page_011.jp2
0a9615bc514ea7bcf7eff6b00e78de95
34ec06b9278cd207ccbffd4c0892a90d1fa78879
F20110115_AABYVT elyacoubi_b_Page_046.tif
8a25bcdfb7ec905a9961f8686d3169b5
009c9d182a77ae29ecb12d527cfc625cc3c7562e
6859 F20110115_AABZCC elyacoubi_b_Page_057thm.jpg
d05c4a27802c7df53f435eb089fb9a92
7b4169392ec553fb76d55f2d1e70256a6d271c61
95829 F20110115_AABZBO elyacoubi_b_Page_039.jp2
50684535072d18b65406b11215f031eb
bf436b6a746eaf93da9be93cbe7a957af89491a8
43576 F20110115_AABYWI elyacoubi_b_Page_064.jpg
1b60d6479c67780f730290a5b751eb94
c613ff27db54d6b0b4ef02330966c17de5c1a5f2
889 F20110115_AABZAZ elyacoubi_b_Page_097.txt
7337da25adde112359abe47d01e55748
d43f0cc2cab06b7ac11c268ed1fea0f61462476e
F20110115_AABZCD elyacoubi_b_Page_026.tif
9f04a6612d5c13cc0b4c68f7b9e4f2f3
6d7984a22a6bf1b0f84874f354b7b1688fafd168
19682 F20110115_AABZBP elyacoubi_b_Page_075.pro
ede432b5ca8389cbecd1cb9fea78ec2d
f05c0b85ee3e636d7bffb5c7fb3e2cec06d74c88
51504 F20110115_AABYVU elyacoubi_b_Page_079.pro
705bc2bc3e03bef00419ccafeb715658
1efc3de30ea7b8e2855937b7415f62f2d8eeb7ba
39536 F20110115_AABZCE elyacoubi_b_Page_042.pro
cb0df69d82b33eae36a88d011d1f3b69
c70cf44a40919326bc154fbce99acb23b9f9004d
1704 F20110115_AABZBQ elyacoubi_b_Page_011.txt
2dcdc7120c5dbc07740745c39df84632
a3dfadfa4d867a8ca968c364f38ea34ef743f29d
607 F20110115_AABYWJ elyacoubi_b_Page_092.txt
0751f80068539cb0ad797f23ee9eb21d
47ebb0e0c319c287f0fd0737fee7d8df50a41a52
F20110115_AABYVV elyacoubi_b_Page_031.tif
7fa4bd7a2e16e332eb4cb0b017301bad
bffba888dcb112cf123aca36420155ef915d35e1
43088 F20110115_AABZCF elyacoubi_b_Page_103.jp2
887dc36778e19b18e0fc856ac6cc3a7a
3cdad32baeb1cc94c0f713f914b12bffc4d63a16
25343 F20110115_AABZBR elyacoubi_b_Page_027.jpg
9495166bd2ea9173e38f2d13f7eb39ca
ef3f3bb2d0b3eae948f6de5c772af662de2a5c5f
1051985 F20110115_AABYWK elyacoubi_b_Page_009.jp2
b3be6f09d223bf4cceee91a5ef808e57
0fede094b9396888d8243d3a9e1699ce917833ef
F20110115_AABYVW elyacoubi_b_Page_017.tif
6870619496ed1673e9d714d12a5f6d57
e15282c790e3589870af1b845b16d3015bc39505
7094 F20110115_AABZCG elyacoubi_b_Page_027.pro
8ee97097008a4205894005ea2e5dd215
1df0290bebca000298de663a1aeb7ab73e533ef9
46642 F20110115_AABYXA elyacoubi_b_Page_019.pro
dad691c74428101edeafa09b550416f8
e9bf97ff0c205eac3b22e8fa98f6333f2c0a5d3f
25600 F20110115_AABZBS elyacoubi_b_Page_066.jpg
70f423c2ab0b17aa84227595f9dc93a2
1b3220a3b595d2f2c2f4a1190c93b26643e8eb52
4223 F20110115_AABYWL elyacoubi_b_Page_077thm.jpg
94f3186a1f07505bb478ab97fd4bb86e
b8ab5f28d605f20b223a1a870f771ec6fe2cfcc7
46015 F20110115_AABYVX elyacoubi_b_Page_017.pro
0b17974bd485eb88006bc9a1981bdfe4
f606067210f16c21169d4fae20de0546d206a4fc
85132 F20110115_AABZCH elyacoubi_b_Page_099.jpg
234a489bff771b6d22ef61622b9e0d49
8a04f39157c4ff75870d4619e1506a9473f2a4d9
25147 F20110115_AABYXB elyacoubi_b_Page_054.QC.jpg
fe316e9c3c176efe49abdfa7a5d8dc6a
dfd390cb33f3b67e834ce1de08a72663de092f8d
F20110115_AABYWM elyacoubi_b_Page_043.tif
3bfc2b858bc55cbb93a726b77951c04a
979c9866ef70bad1fd9aa08e9ef1fc79bd060f28
6728 F20110115_AABYVY elyacoubi_b_Page_018thm.jpg
36c982c1883c90eaba46138a449ffcb0
733abcdd6bb55bbff26385c69d6f5b91d57b2dfe
1051933 F20110115_AABZCI elyacoubi_b_Page_072.jp2
516159f985e9c123b7337d9bdcc15e61
28ab3f57a8f45d04eedc84dada29050effb821fe
1318 F20110115_AABYXC elyacoubi_b_Page_026.txt
e981b6c446175028853ef24da7b95a7a
eb811129d5fbf1419847444783bbed2c7a4032fb
6543 F20110115_AABZBT elyacoubi_b_Page_099thm.jpg
5649c7943fc439b07442c63601e804bf
54bd7d4d55d78339a89aa26908b730468f32e84f
697 F20110115_AABYWN elyacoubi_b_Page_077.txt
5ead06160de428e08952522023dc5e06
e2da6326eddc1d20d1f995d111a98d4ba9e4b0d3
109167 F20110115_AABYVZ elyacoubi_b_Page_046.jp2
1148caed79a8450cff624eb2a79becef
1dc00d3788699808744bc8e1778aeee479938136
103426 F20110115_AABZCJ elyacoubi_b_Page_081.jp2
79d9311545e1197e31b337250d8cc384
a404b226c84c1aaf54206765152c7ad3a0b2c7fe
12255 F20110115_AABYXD elyacoubi_b_Page_007.pro
b61938741683bb7d0ad42f818c5653f8
2e70ca1946ec6886585296ac52952537cb39cf42
1808 F20110115_AABZBU elyacoubi_b_Page_078.txt
689cc41e241f4b3da558f7455ebe5333
09d840048d14f7e8802190b8683af61b3afda411
20318 F20110115_AABYWO elyacoubi_b_Page_078.QC.jpg
96b670d866b89491533976a2a899e37c
3100e13a570b285a9233d9576c25dd467651ec10
20827 F20110115_AABZCK elyacoubi_b_Page_039.QC.jpg
2d76bdfd61f55297807f8e2d68d95a7d
827aa524cf586ddf185aa3f9b9bb6bf50939182d
1140 F20110115_AABYXE elyacoubi_b_Page_075.txt
91a4041ede9a9280eba53eb139d0be73
c0a3c6a856102830a066cff1c5b1158ce5e35637
52338 F20110115_AABZBV elyacoubi_b_Page_055.pro
6c0069c4cb384166ca93c604dd70e308
957630bc05f6183c863d93962f683edc32199a6b
11024 F20110115_AABYWP elyacoubi_b_Page_073.pro
9e3ec403523982eb32a5e7376df1677c
203c42ed597fabf331881b116a449636e31cdb53
47697 F20110115_AABZCL elyacoubi_b_Page_045.pro
74ca34c9b3f8fb735ddd7574ce56610c
3db75d5e610e743df3206f5fb8a55d5a75baf098
366 F20110115_AABYXF elyacoubi_b_Page_076.txt
18d699c07bcd4be17cb94c7cdfa99b54
c161533b7be46c766a7f53d146a0126b2ac7f5f5
F20110115_AABZBW elyacoubi_b_Page_011.tif
1d0dea5147c9eaa08136b0f6ce2539ce
85e3addd75f3b2487ec7da79ce1e11571958eb70
F20110115_AABYWQ elyacoubi_b_Page_062.tif
94677ae11ec7e66d6595390e5597adb4
f82b5c30d4213bd8aa6b7c7cd7ece68cbda9459d
69978 F20110115_AABZCM elyacoubi_b_Page_045.jpg
957c87b3ea85df74dc4a127d09f6bba2
8d90b2d1ea6a409ce4bc783d12be83d137c028ed
3290 F20110115_AABYXG elyacoubi_b_Page_103thm.jpg
f85959f5c1aac303f440ecb37ecf4618
a9cac19038b29c9f6555a55731d81bff4a38ed08
1910 F20110115_AABZBX elyacoubi_b_Page_053.txt
9d834279130c8fe308e71b3631f377b8
0982f145aad985540dd0d69b73f69ad1966d759f
2011 F20110115_AABYWR elyacoubi_b_Page_014.txt
63a9335c57c13b42f62856da651a10cc
fb457ba7748bf950e07da5f2e56e02ab5d952018
1869 F20110115_AABZDA elyacoubi_b_Page_016.txt
10259690c1aece15d5509307ef84699d
fe0dae650a67a634963c4fd25aecd42e7fef85e4
66680 F20110115_AABZCN elyacoubi_b_Page_035.jpg
514e12931a5fc025c5dfd2bea8e69c86
655a70471846838938d30161e6d36bf4fd06ad35
19081 F20110115_AABYXH elyacoubi_b_Page_091.pro
efbaedde2437759bd8b7497746a30fef
be1577b6fc2915ead5133b5db648cd125ad27789
F20110115_AABZBY elyacoubi_b_Page_052.txt
298461eeba5f981696009bde10278263
e5ac8187a1906b63e9dc611d9d394bec91f5597a
109893 F20110115_AABYWS elyacoubi_b_Page_058.jp2
519135a774ca4b013966ea1598bbaea8
8fac196311731f8646b2ca3bae7caef0deb51f4b
9195 F20110115_AABZDB elyacoubi_b_Page_071.QC.jpg
871ed34fce7f2a7742d8b1ce73f8fd6d
45146593d9133bb6807e85b5369371db7f8359a0
974759 F20110115_AABZCO elyacoubi_b_Page_032.jp2
bf0f9c9c0c5cac856cce0bbdc8cb25c7
e3be8f02521b2e3c1dc69e59aa04c29037337e29
71378 F20110115_AABYXI elyacoubi_b_Page_038.jpg
935cd8a335f4fa38249771d67cab391a
06265f698bed77378e80b232cecda97b44a3db42
F20110115_AABZBZ elyacoubi_b_Page_041.tif
868258e484c7af17bd29e7bb3e57e9b0
480d8665355c3663934c711232d6ca284327646d
1947 F20110115_AABYWT elyacoubi_b_Page_080.txt
bab2cc254deab6256cb9bd4c020dedc6
57556711e359533dbd6116f48092c7a8bba17c92
3579 F20110115_AABZDC elyacoubi_b_Page_028thm.jpg
8c74f3d8ac1462cce8cbf7eb410586aa
cbea69444d1a7d00e62985f56571f23121d8a6d9
45808 F20110115_AABZCP elyacoubi_b_Page_083.pro
4d03c1b1bbaa8974ced021481e82bf90
6337df4a27efda7771d84683a91386be388e42ee
12478 F20110115_AABYXJ elyacoubi_b_Page_064.QC.jpg
bcd988345b4329600ad7b506d66f3433
4b83b8bd1501ec60448379bd61a42a096760de07
F20110115_AABYWU elyacoubi_b_Page_096.tif
6c1c114c369b6f196323933c2f872867
70a915699727b7e754f4472595518ce0fc20f840
5976 F20110115_AABZDD elyacoubi_b_Page_098thm.jpg
835a293bb9950430293c70ae72f144be
232aae31bedbd2fcc86812b5c36ea0df6906c19a
68843 F20110115_AABZCQ elyacoubi_b_Page_098.jpg
3a53bad90438da6abffbdf0b09bb9c42
4b608ab8675ad592e93585470d2c5cdd333962cc
105830 F20110115_AABYWV elyacoubi_b_Page_045.jp2
96b5109e28c2cf6c0d8b26dd14cc999d
3f92aa1842ca2c8c16ac5566f94f5cfa45da523d
67164 F20110115_AABZDE elyacoubi_b_Page_041.jpg
f4894c9c284ee8ac822117f9a599161c
dbfe784f911f565e92b158d16c130de0869a5e78
2543 F20110115_AABZCR elyacoubi_b_Page_101.txt
4b49f1c9f596d3ccefc9d441a2536777
eaf08f87251da01a32f2f71678cce3fed0ee183f
984 F20110115_AABYXK elyacoubi_b_Page_070.txt
c729ce559acdca5b487836b7965515b2
9b33282e536e9c77c868b8599e10147859a8618d
76344 F20110115_AABYWW elyacoubi_b_Page_072.jpg
a0701db245c7ebceff8cf9184a8fe47b
3f6e1a0a566a511bf03c50d9fceed432a9193a28
F20110115_AABZDF elyacoubi_b_Page_006.jp2
5c3eb7aa64fb892c7e81995f25771853
a4a3a5e5436899e3f9aff49d6d11df56fd6da30f
1948 F20110115_AABYXL elyacoubi_b_Page_058.txt
7e0366a70b06d54caee1cd0c12c169dc
8d5c94ab6e47cf06c9f3976be4fb8b1b32949353
12317 F20110115_AABYWX elyacoubi_b_Page_004.QC.jpg
a87996a7a987020a9026a49c4cc1df20
026bccae7f8e2c98ec67147f9dda64f74c81d3f1
3741 F20110115_AABZDG elyacoubi_b_Page_064thm.jpg
78d0bf747a568c1f5d6b58b48d09ded8
99fd33e9cff266d29d72315941fc12b1208a5b90
42775 F20110115_AABYYA elyacoubi_b_Page_047.pro
ba13eb36ff442c0cf32ce6bdc7ad1572
3d2e306960efde15112397505db35c52327976b1
31632 F20110115_AABZCS elyacoubi_b_Page_093.jpg
9f9b9103b25ebae9b346d1797387d99e
1ef68f4525db03d5e61913715f91d096fb86ca9f
1752 F20110115_AABYXM elyacoubi_b_Page_096.txt
06900f0392d049bbeb46170efca475c9
f5cb00eab494403abce21965550a352c3829e9bf
44711 F20110115_AABYWY elyacoubi_b_Page_068.jpg
08e0628c313cf267683c71d8d5763014
521f5d97da139b97b181c1627b62849d7ca68e5b
1890 F20110115_AABZDH elyacoubi_b_Page_061.txt
cbc25506f395c826a7cbf846538ef895
ec276f90b66498c71b3e3a52fba9a97187447719
385404 F20110115_AABYYB elyacoubi_b_Page_070.jp2
20b4696385a67b56b8e5acd6c397c576
d28b9c55e86e281d23495b3bdd0b3bf742fd3811
2767 F20110115_AABZCT elyacoubi_b_Page_062.txt
1ae51119d0706b6ff564ac46ef20fc0d
9a6e86f59960ba5589d04483a0560287752631da
2760 F20110115_AABYXN elyacoubi_b_Page_065thm.jpg
97c0d1f4abb1c30b1b20c5ac3be12f8e
b9f2ecdf777d4827b4646f365778ca9c32d540ca
10766 F20110115_AABYWZ elyacoubi_b_Page_103.QC.jpg
bb31f9d5368348742da266c1af844987
f1a9e1a2146f2f40291d6b650173163ef32c5f24
23385 F20110115_AABZDI elyacoubi_b_Page_060.QC.jpg
b7f3ce31afb5d84bee4c3699b8cd14fe
b6a4d9a9c43dd8ec449fd4958bee4c2a6761ac1f
609697 F20110115_AABYYC elyacoubi_b_Page_028.jp2
cd8c0ec15e6b81fedc584f2272eed704
97f42e56e98c414ddc98cb434526c2d800eafe63
11443 F20110115_AABYXO elyacoubi_b_Page_028.QC.jpg
5c58e7071dea9a2765a0a8998606c373
4983f0475d0f91d841b072d5052af64f52e0bfda
2058 F20110115_AABZDJ elyacoubi_b_Page_059.txt
ae745427ec53c98e9d5b52aec9797c54
8750f3a359628818114ab43ed2a4705b949d0346
100947 F20110115_AABYYD elyacoubi_b_Page_040.jp2
db6a8aebc3ededf316ea8fd693b7e086
0bd5974690bb85aa5a258b0b5fda76c8b24c1a0a
63092 F20110115_AABZCU elyacoubi_b_Page_033.jpg
abc113939ca9fd87319787b9ffe965ca
018c019cc72839c4c02c41fc7a7017800889c9c6
F20110115_AABYXP elyacoubi_b_Page_013.tif
b486550f2a5e2a3883d6549334469c25
7604f720603755b29e71e9b8cf39affc34caf9f3
34488 F20110115_AABZDK elyacoubi_b_Page_033.pro
ded735483d6e87d1b99aca340381db47
829e376f80cb52ab67f49884064ed39be8282c76
25842 F20110115_AABYYE elyacoubi_b_Page_101.QC.jpg
a55fcb73f54a338bb50022649ff6e41f
5978becb62d34cd3916d53e61778fc9abedc09c3
33352 F20110115_AABZCV elyacoubi_b_Page_063.pro
c735817e718cfa49045c0f2487ac34b2
29372fadedfeadf3a0d3bba0024691ac8051bf9a
102340 F20110115_AABYXQ elyacoubi_b_Page_082.jp2
c5f077db35df006f9cadec9413dd026a
25bf5757926f9f8bc9b23c9707fff0ec27c08e15
24442 F20110115_AABZDL elyacoubi_b_Page_070.jpg
0ac99027266c9935b94a56615371e645
097679fea7e424538f008124ebf872a516ecf4a5
2570 F20110115_AABYYF elyacoubi_b_Page_069thm.jpg
6a7b1afbe703ef3e8b1465380dc2c2ed
18a46e7644e7cbd904decb6a9cb1ac133dc2a348
F20110115_AABZCW elyacoubi_b_Page_085.tif
d2ffb6643bf654abadf461dac2799be5
e308c25d5bb72a9f335db3afec1c1328c093f56b
6772 F20110115_AABYXR elyacoubi_b_Page_024.QC.jpg
65bf1ef92ad8648e5e951ebc579d48e9
2c0f576e72e24124aa9e3532aad2fef16717b46e
23550 F20110115_AABZEA elyacoubi_b_Page_048.QC.jpg
b2172e69ca9d230f2e72d86d4499a556
27c88871f0e8a06b879b9a95d0d96292ce31e8ef
F20110115_AABZDM elyacoubi_b_Page_079.tif
f91dce301dbc45797012652c7242577d
b3f9275c8b48207a3af966fd8a53fda7c9ceaf08
5630 F20110115_AABYYG elyacoubi_b_Page_007.QC.jpg
d4e269543615dd1f3fd73ad0b34f8420
307013870056a5143a5ee2cb65a6571120ea43f4
1838 F20110115_AABZCX elyacoubi_b_Page_035.txt
f2b96120a7b26dea5a767fbf57b99ec4
88adb9eba2f636e4611bb0a120b106efb7f631d8
F20110115_AABYXS elyacoubi_b_Page_064.tif
8facded89fd1450f7281b44494c924dd
315fa4d54fee3561f254c05105fbd77d714388a3
F20110115_AABZEB elyacoubi_b_Page_061.tif
a8c21163082f4457f53609290e5e7061
8cc2cdf3b0417a74bb270d84db996a1571819563
2291 F20110115_AABZDN elyacoubi_b_Page_089thm.jpg
55acd5d37631eb4f36c7426e288519df
f8e790868dd2458aeb34a402b1aa3eb3fe24a91f
50066 F20110115_AABYYH elyacoubi_b_Page_010.pro
3904d245651eff7859d091bb1b122e17
9828df0e50295c0c83c1f712209977e594ed4ede
F20110115_AABZCY elyacoubi_b_Page_042.tif
79fdb9778f5892ea019afa1d3b4d2598
310c571a50336c1bad030fcc9467102535177177
21824 F20110115_AABYXT elyacoubi_b_Page_021.QC.jpg
11b7d2ea01ff5673758bf6e62798722d
1f069c77d52f00bbeedc4a62109e4a2e743d73d3
F20110115_AABZEC elyacoubi_b_Page_021.txt
f3b004b5917b67bc07c17d6b1eca5c79
5e86fd049dc2535deecada11b6512229ed24cb9d
72341 F20110115_AABZDO elyacoubi_b_Page_060.jpg
45eb8cabe3e0e35cf79545d7a0d2fc77
59c21c1ea6b8fd96b70fc6d37af4a1dfc097f700
2051 F20110115_AABYYI elyacoubi_b_Page_098.txt
7d6290c4e2b634bcf19967fdf942af15
d932ed755ed05159d5ae3fb582006da5bccbf7da
109569 F20110115_AABZCZ elyacoubi_b_Page_048.jp2
568571042a4a8cca21c4942ef3795b5c
447c4f09bdda21c510058580f32f87aa0b209b31
10700 F20110115_AABYXU elyacoubi_b_Page_064.pro
13c29b2f97a0456325dfefab83566c58
b6b97c77bf72756b320989c83a2b19d07fe273ac
F20110115_AABZED elyacoubi_b_Page_035.tif
0354d95285607a4e7af50a5bb5554321
c71727283ffe156e579eb1dceb2af190849bb1fb
4180 F20110115_AABZDP elyacoubi_b_Page_067thm.jpg
02d3b236ef157b9f89daa291be319710
576d54d4dc31f69975e6df8843c014b99086b646
1764 F20110115_AABYYJ elyacoubi_b_Page_041.txt
04c1bfa945823ded062af94e84dc87e5
13d81f0ff185a2ade65f02fda7a7480ba563c048
2041 F20110115_AABYXV elyacoubi_b_Page_043.txt
7cc521b343ca4456fa9bfe56d2f9ea8f
9bf8c061897f61f08c84903c2bb3127f3a8b383c
24405 F20110115_AABZEE elyacoubi_b_Page_087.QC.jpg
98e8a03be4e1da739c595e07618bcab1
49aa2520397e87e9d01856b6542e5d6d21e94cf3
111224 F20110115_AABZDQ elyacoubi_b_Page_062.jp2
d4028666c8b69d9e38b87904ea1089f0
525b42d00d3c59918b41b6cd676ba179e3e99988
111877 F20110115_AABYYK elyacoubi_b_Page_015.jp2
3a0f17acee80960af728b8984dc2beef
3158c9d6687f1c2bfcdf03fc69ddeb8b198b603b
1832 F20110115_AABYXW elyacoubi_b_Page_084.txt
c4ec61c3803be162e142d736bb69310e
e09428d0bcfdac0c8e86916efe21809480d4f6c4
32623 F20110115_AABZEF elyacoubi_b_Page_103.jpg
9c7ecca006ceab4165df2686d5e992e2
c33cff8591d5afd77dfe45d58aae7daa8e699df6
4281 F20110115_AABZDR elyacoubi_b_Page_031thm.jpg
b4da758fe161716c505637275c3d29fb
310575dd1a7749b59e87245a5cdc57ee7d070d2a
77526 F20110115_AABYXX elyacoubi_b_Page_005.pro
2ca874c3b586ce0b6500f14154f52d53
5b2329e827492486addfd6b05803216fcaabf8a3
47329 F20110115_AABZEG elyacoubi_b_Page_081.pro
4ab2cd2544f6d78c59528bd9390eb1ee
2b5300f7774bbba3098f7112aaeb7fd967564330
46337 F20110115_AABYZA elyacoubi_b_Page_102.jp2
25dc91fbc56802adbfeb0c5713cbe56d
86d3734f200a1f155d4bba4ea739a0086c4b2697
F20110115_AABZDS elyacoubi_b_Page_004.tif
3fb12cf8e81f5f935b7dfb20fd089952
3262c39b3cbcafcdd3e76ce92fd55fce28d107b2
4838 F20110115_AABYYL elyacoubi_b_Page_076thm.jpg
af3da99bc64c70f910d1e05e2c2c6870
9b4e00f0a680d0c2f63afbb9fad856c575fee400
18831 F20110115_AABYXY elyacoubi_b_Page_011.QC.jpg
a4cf53265b5b21d8395c2e99ea8c5832
707fbf32458157712761aadc7ccc9f4646dc72d1
22188 F20110115_AABZEH elyacoubi_b_Page_084.QC.jpg
a7d4c54e7ffc22964370082ffb5418f2
7703340718e71edbbcaa57c097011670b5a51239
6633 F20110115_AABYZB elyacoubi_b_Page_055thm.jpg
775e1d1b79898eac469d3e798dfd1f58
b085074797bbf69b1198873aa8d475e2d1a410d8
1985 F20110115_AABZDT elyacoubi_b_Page_036.txt
9e2b8589a74aff807908bea2670ae3ef
d6f6852200899c7c9c58b0f8b71f67cf97349da6
2020 F20110115_AABYYM elyacoubi_b_Page_079.txt
d00bea8bb25f9bb6e9dcf7a853229634
eff01d6e2e17589547b47217840a9cb6047895b5
5195 F20110115_AABYXZ elyacoubi_b_Page_062thm.jpg
09bddda9f877234fac4ded708465d52c
b86aa422843dd9672610ddd66f490b4a5d496c51
58596 F20110115_AABZEI elyacoubi_b_Page_095.jpg
361f75dbc0adb5b784aede9120e19464
13f0a66d2e513cad680c00ed2eb9f925aefcb779
23830 F20110115_AABYZC elyacoubi_b_Page_014.QC.jpg
d099cde4d03754be1c18e02007687696
50ab8b2529b2f6685a4d6e655fcbc4a3134bb14f
20766 F20110115_AABZDU elyacoubi_b_Page_068.pro
77c9aa1b280827b418172d6f581cb193
79185512a63f287132b80e8cfefa2671f23b8855
557 F20110115_AABYYN elyacoubi_b_Page_093.txt
9c7bb79d213853d587940fea058ceaa0
dc8c42a4facdc1e4bd3c1dc90921901ce79b9539
133909 F20110115_AABZEJ elyacoubi_b_Page_100.jp2
433de08c2ad986c40f3719534c051d94
b5197cd05d35cae7d9651f9b9ccf3b71e7a5698c
F20110115_AABYZD elyacoubi_b_Page_027.tif
cd2d43521a70851c9197dcc5cbeb657f
188c32073a62d45086e58f6de2b1c8f6fd376f0f
106409 F20110115_AABYYO elyacoubi_b_Page_098.jp2
830a15c60ff77e3fe4bc8d804d5db6ce
147406d66f577845d4aa0e894eabe57e9fb8e327
21480 F20110115_AABZEK elyacoubi_b_Page_098.QC.jpg
99db8ba7ceaefd426b4053d192030d04
95bd0b7a9eb5f4c3b77933e6e57cf27f94d8c102
5557 F20110115_AABYZE elyacoubi_b_Page_042thm.jpg
85a670e84fce6c57ff09344847abde6f
621ded56186442a16cf9c1df33b6247246d57869
863980 F20110115_AABZDV elyacoubi_b_Page_033.jp2
4c6b9435bd38f4ebb7f55b55ee52d160
445c911e2a8fe23ad56e8d647f4028c02d84689e
F20110115_AABYYP elyacoubi_b_Page_074.tif
6bc599c6891954688f654b20dd5ef1e8
3f9b415f58ed0d8891c930de37e357872c4af44b
3225 F20110115_AABZEL elyacoubi_b_Page_005.txt
66a180a53f30f07332394147338077f2
2e44f316df97b2097336ae321fba37531037f3c4
105766 F20110115_AABYZF elyacoubi_b_Page_006.jpg
ec94e3063932731b593cb39664cdac44
1d6e76fa43fda02f0fdb9a59ceafd371fd95b82b
23635 F20110115_AABZDW elyacoubi_b_Page_058.QC.jpg
c56664a742901d51c46c48ec53bbca83
7b9a783b2c0a350f18f3bf1d7e4a1f481920ba8e
130665 F20110115_AABYYQ elyacoubi_b_Page_101.jp2
4d443b804cd484337b72c3459a9c1804
8beb80416762081ca5d9b0730b8d25bd430d4126
F20110115_AABZEM elyacoubi_b_Page_010.tif
4878b812513c9397934769edd93c1915
ab05c4254aa5f2242e7372a933595ba3be193e42
105226 F20110115_AABYZG elyacoubi_b_Page_022.jp2
af63111bcfce4f5351cdfcb7f2350be6
32b5708587ce0493c0a0cd61ba1180f9d0e07603
23573 F20110115_AABZDX elyacoubi_b_Page_015.QC.jpg
56c94b01dbf45870f68b032e872fa13e
adbb49ce8f24953fda3e1eb7ea04d26cf3bf9d3b
23272 F20110115_AABYYR elyacoubi_b_Page_080.QC.jpg
501f2c74abad838318cdd5e8438d6194
a38429738646e163421fd2c01a7331de9c988755
57784 F20110115_AABZFA elyacoubi_b_Page_096.jpg
e9841ce02d6dbf54ceae3e494e0157c7
1e6eb11c58b19d53033883ca9e3c99dca93d68a2
2054 F20110115_AABZEN elyacoubi_b_Page_055.txt
63c50459b2041b68341b88bec8bca888
17d0b264a46771ac8219c892eb0a4725b9c4e6d4
74919 F20110115_AABYZH elyacoubi_b_Page_055.jpg
b17e3519c8bcca33b53dad92f3031dcb
31fbb44ad3ec40308f522aafb68410ad39d4a54d
6258 F20110115_AABZDY elyacoubi_b_Page_081thm.jpg
4c8fb260092f0a7ffc1b6b915f877c42
eb569e711747969c0e1302059ad9feb29535cf81
6368 F20110115_AABYYS elyacoubi_b_Page_006thm.jpg
50539a3092861fb4d3cdb75b140fc072
dcb116954e14f9366074a52de5abb7bfe8958da1
F20110115_AABZFB elyacoubi_b_Page_101.tif
7938c7b7581348cbd6d2ac4fe24919b7
a82c593d8589ca72c45616309c47329e2301e1cb
3807 F20110115_AABZEO elyacoubi_b_Page_026thm.jpg
a8c4e7bf1d070f931891107c79ce7419
1bddf7db0cc0788fde48d4f2d6119e0c14e4dc4e
F20110115_AABYZI elyacoubi_b_Page_036.tif
9b7e554626bef3b98e716a7a7edf2950
6b7eec485592624aa83979adb9575f6de28b9661
73685 F20110115_AABZDZ elyacoubi_b_Page_015.jpg
684efc96f76e6a430ad12e7f7f589e6e
b48a865aae8d0eaaf618d65f138616973ca0e7d6
14069 F20110115_AABYYT elyacoubi_b_Page_067.QC.jpg
ef90596fe174d2e1e8144e3b5133a9d3
2e82fddfed1f21adae7dee0cf95df0794927af0c
6120 F20110115_AABZFC elyacoubi_b_Page_040thm.jpg
a41568f5d3f1998fe07618c9552b5ed4
975874d852428946de69388f66c91f62d399b385
68879 F20110115_AABZEP elyacoubi_b_Page_017.jpg
4df53d8ca13497976bf71f9e6ba244a6
5a684eb479b621a57409f04dfd36656999000249
74382 F20110115_AABYZJ elyacoubi_b_Page_051.jpg
32190c58e59efc101c9369ad8fb94377
48478e893f6fd1f3d6432ae8ef9acbfcd1d887f4
21313 F20110115_AABYYU elyacoubi_b_Page_072.QC.jpg
a72e698cf4f5ecd10558cde6cccf2e69
21dee57cae2556e4800037774b911fedeb3ef65e
73377 F20110115_AABZFD elyacoubi_b_Page_043.jpg
ac3f85f4f333f975d818d754d71e9621
279f193373e0f25ea261d8d4d18ad7f1504954a6
68349 F20110115_AABZEQ elyacoubi_b_Page_084.jpg
e24d507d6894f6c0d5ca022e748cbe8f
88d6b15c1abc4bb1c522c03b0f6b6d607d5cbf83
6740 F20110115_AABYZK elyacoubi_b_Page_056thm.jpg
b939674c127b91ed14c75c16056d4f4e
4fe0ca2e93f4651e3adf9fc6087964ebe48a1f98
3058 F20110115_AABYYV elyacoubi_b_Page_092thm.jpg
d0206385cd281010a7d27eed0b6b784a
ebda5ff98b49edddfeee34a41e3e99989a1cf042
66762 F20110115_AABZFE elyacoubi_b_Page_088.jpg
53992f0122374f5eed329308094f960d
e343f670870d0251b2dd160947b8cfdbc5863e95
F20110115_AABZER elyacoubi_b_Page_076.tif
2b93731761446c5c9a0398aa7b6a16ac
c294dbd256d7fa255cbd2bebd7e019a0af0f1c78
1245 F20110115_AABYZL elyacoubi_b_Page_068.txt
d797ed356de73b3b013cda990880c23d
6dcc4122ee3a7478e8e181517d71de695ab27a9e
F20110115_AABYYW elyacoubi_b_Page_022.tif
34d1c5b3d133a2dc2812d7679209e0de
4532801c7a7ed56bdbc84f90f2306fc0465a999a
499625 F20110115_AABZFF elyacoubi_b_Page_090.jp2
bda5b5ea6ba2b0de4b17ae8a8d4e8610
13c054167e511c04f5199ac23cb759385b8fa3c2
1548 F20110115_AABZES elyacoubi_b_Page_063.txt
9beb4b295db7d768942587a7c972c4b9
60361e0bf1b5a39533bab0f5aba5c7f731efa36b
113622 F20110115_AABYYX elyacoubi_b_Page_079.jp2
2508f47d7fa60230c839ea9a7adfdb1c
090ae5713e8319f35845a4cd0d4929b70bd2b768
F20110115_AABZFG elyacoubi_b_Page_023.txt
11ed273cfd88b0fd45a7566fbdac08de
26111076512a422f4d5ec141053842f686d2a2f5
98423 F20110115_AABZET elyacoubi_b_Page_047.jp2
b0eaddae5da1eaca8b98ea41cd54a2f5
bb9ac0550b107efec139e5678fc6d8b9565aafbd
488 F20110115_AABYZM elyacoubi_b_Page_007.txt
5f2e8bb55ab16a15cdb4096774892f7d
4d3ebc3d8350f52dd40917681d2a9598ce94b714
109980 F20110115_AABYYY elyacoubi_b_Page_038.jp2
9e5f23fbc6216abe465609d5674ff850
422c1c3cf66a14a49dca575e1a6198d29b88e153
49157 F20110115_AABZFH elyacoubi_b_Page_049.pro
c4a9ae4a16d23fd42eb4e68084cfa336
15696182b3a28045466654faa65a41810f9addca
23867 F20110115_AABZEU elyacoubi_b_Page_050.QC.jpg
5ec355d34f5b3c8e05129834bfb327d3
0da7e5916216e3548e47a85699d585badaea168a
793 F20110115_AABYZN elyacoubi_b_Page_090.txt
5c3dc16c1ca5d50199126c0f1d0feea8
e5265426c8969ed82a420b2b51d7c3fcd215bab5
3871 F20110115_AABYYZ elyacoubi_b_Page_091thm.jpg
603ec64595dcaa2374cb33bde0bc1c23
c10c453e2c9805825420e40a9e5cb19d0fbdfabf
18891 F20110115_AABZFI elyacoubi_b_Page_008.jpg
015436ff6b76125c263ab682e83ba424
6abf09f33cb03ca5679818115652fdb8c471a5a4
26147 F20110115_AABZEV elyacoubi_b_Page_006.QC.jpg
45db17bf2278b3a1a48b516eff6d09ac
96d1901340742023daebc37f5a8c4369583130dd
F20110115_AABYZO elyacoubi_b_Page_051.tif
bd85064337f79789fe26265f89f512bb
14215202044897152fa40b95f63132c105ae357c
1817 F20110115_AABZFJ elyacoubi_b_Page_088.txt
1790d5914211d58df698a488fac494b3
a54d10bc47bea81d0707424060b0e2244128aba1
540 F20110115_AABYZP elyacoubi_b_Page_073.txt
d1537b8ef47740900fa6b621f3cbb981
015c81173a80b8a7665e9883ac8a9c67a9ab91b4
104265 F20110115_AABZFK elyacoubi_b_Page_084.jp2
de3012980a846a463a979bf817912cf3
558ce847ccde326744972777083845deacd10760
11132 F20110115_AABZEW elyacoubi_b_Page_063.QC.jpg
bb08a8e843f960b1204d050ca332ebe8
1c9ad47105097c3de3e6c49d7d36f06ea4eac9f9
451213 F20110115_AABYZQ elyacoubi_b_Page_094.jp2
efe01d35a0420de954b01e15149be08a
0e8dcf7bf59b8e12b878ee89b8c0d551a0715680
5179 F20110115_AABZFL elyacoubi_b_Page_005thm.jpg
789b01727ad0b497df709ec8af922911
b8b42eacb3cef492b070eea9e606f07634a3cf18
23802 F20110115_AABZEX elyacoubi_b_Page_038.QC.jpg
876ea6704f5f90419eb4023b70aa3962
5c085d30c7e6306266351bf56267296925dcbfdb
102561 F20110115_AABYZR elyacoubi_b_Page_013.jp2
926f8dcbb2e95adf55c1981325c9f4b0
7de05d934aa8ec381fa0eeeffdb692e6f25230ce
21318 F20110115_AABZGA elyacoubi_b_Page_035.QC.jpg
1e0f99ced0658a0a336b942391e3038d
cc0a4652d1276f8ed2cbaaebfc26429e67e9f828
66657 F20110115_AABZFM elyacoubi_b_Page_062.jpg
5d5d35f2b3db1aff10d4bc677cb37818
df308f98b1d41e97c99a69b45ddb4f3aed736deb
400336 F20110115_AABZEY elyacoubi_b_Page_071.jp2
74bbce249de5515fa519284d10869c10
0dd46aedfeaa8bc4c3100b5e51cb3217425737df
51378 F20110115_AABYZS elyacoubi_b_Page_050.pro
ded4fb1f0d268e2d6f6dea2ecf2cb6bd
ca977d66963ad6dee89f7154d429cd9c334a32af
22706 F20110115_AABZGB elyacoubi_b_Page_016.QC.jpg
9a61f99655f89aa09a32e09454dc5d1c
2703ce584b3dfc9eae2bb059d272946c38610618
582 F20110115_AABZFN elyacoubi_b_Page_089.txt
962b504f32d3e4490a5a9a5da880f7e4
a03931f44a06f2f21b90e77c65a685e309143ba0
F20110115_AABZEZ elyacoubi_b_Page_103.tif
7b815323f3f0d5e7bffeaefd3b243910
85d6c6bde1a9af1e546120f9f94b0ebbf7fba1e6
F20110115_AABYZT elyacoubi_b_Page_090.tif
f45b14ab88a70cf75be1fc5b0a759a4d
4d32f6d000689a2fa2962c50e0b7033873bcb960
673511 F20110115_AABZGC elyacoubi_b_Page_026.jp2
c742ec75fd255424c7616a4e0490d3b0
1d36e41c81dabc7dad2dfa1f400906b431351b00
6641 F20110115_AABZFO elyacoubi_b_Page_058thm.jpg
047b2b11f4c162f17a47373bd1a0a5e6
fc78f2200cf74681a9c79de7c2f6d3ac26bac999
F20110115_AABYZU elyacoubi_b_Page_021.tif
5f363895d74dbbaf5719676a3ded4311
b7645a96d741e49aae1af3381654ffc3784c4efc
22547 F20110115_AABZGD elyacoubi_b_Page_001.jpg
47ac3fdba01d4c2e9403b17ee4b3db43
e8bc59292eae12bf78dc7f4a7700bce97a0ffdfb
24324 F20110115_AABZFP elyacoubi_b_Page_099.QC.jpg
e484415df9a49cb793a9360a3deca7f4
fbbb7918e91cc3517d8a51c865ad3ba91a6a3341
33264 F20110115_AABYZV elyacoubi_b_Page_012.pro
e8888a206633f27e085e549eccda6072
10f6044a9ed35ec059b3144e65b27f9b74ef0f23
6669 F20110115_AABZGE elyacoubi_b_Page_087thm.jpg
911fc01a4aad0d0f7388a2ec8953b9a4
63359994c2def7a4f651ba378d617b957a130f5c
51079 F20110115_AABZFQ elyacoubi_b_Page_014.pro
54d487a46eb5adfb7396f8329bc983a4
a06ef9259673d0f00e6fffcba9584e23d623d2d7
45061 F20110115_AABYZW elyacoubi_b_Page_040.pro
b4406336d5a25e57a7cd02bac9ee4fcf
481f9e1aced094aa5ca2280da0f796acacdb3f1a
53042 F20110115_AABZGF elyacoubi_b_Page_051.pro
320deec7e4fc73cfaf21a5998ac0b972
8583451dcd9fbeaaceb776ab565343ef710f1a34
49642 F20110115_AABZFR elyacoubi_b_Page_046.pro
82a027b5c192fc506f08b2097feb0ca9
485d8590dad9570841144f69b34ff0ade222a2be
F20110115_AABYZX elyacoubi_b_Page_049.tif
c041502bdce17b3cd519b0ced43de75a
f57f89d0c53003d2045c2e12260150280f69bfb1
45772 F20110115_AABZGG elyacoubi_b_Page_013.pro
88c0158884eb1730237b83e22dfde310
1e3b1884c4cc19da181204158618e2964df5ba02
37757 F20110115_AABZFS elyacoubi_b_Page_102.jpg
733eab5a1d2a06dce813375008b86611
7d19b4f6476a9e9bbaf6f674baedde3d9cf840e4
F20110115_AABYZY elyacoubi_b_Page_003.tif
1bbeb69a9a22cddb29f634db619f3223
dda8c5318e043b4d676f96c3c6808ca9201e8f2e
10333 F20110115_AABZGH elyacoubi_b_Page_090.QC.jpg
038a75849133f32d616522ad19e363f2
4614ddf6b7703fc7e80ff9c3250c3dab17a0efc1
50491 F20110115_AABZFT elyacoubi_b_Page_098.pro
9a8d52d8f689ee66b5f992bbff34b151
8615b9f96f62b109a2e1f32e13d7a15481b3204b
68099 F20110115_AABYZZ elyacoubi_b_Page_081.jpg
9cc4845f0cffbedb0e40ed59e94b5157
49477afa7281b6c95a5980adc7bff867c8eb1798
334128 F20110115_AABZGI elyacoubi_b_Page_007.jp2
3eba581b354ffe0f305aefb46912f1ec
4faff9c33e464747f690d1aee91aa83f86899211
77039 F20110115_AABZFU elyacoubi_b_Page_012.jp2
2b192de357a1cdf2baa1863e9bb5edb8
9b964c8906f73250628d36dd9c05a48a161003ac
90085 F20110115_AABZGJ elyacoubi_b_Page_042.jp2
93334c4ccaa3e79432e36a5ee66e91c9
6ea32a837a0a4418a1640aabcbafc2e91aa78a95
4704 F20110115_AABZFV elyacoubi_b_Page_032thm.jpg
db9ceefb68bc7f3a9dd146e1099f2b94
41b67a44dac9623679c34d04d53e4eb6bd6fff28
69623 F20110115_AABZGK elyacoubi_b_Page_019.jpg
fd92bcd6af1f9447224552f97a750d89
880a63f5a0557aeae2e4916c63783438b2d5e23c
22979 F20110115_AABZFW elyacoubi_b_Page_009.QC.jpg
a275f19b19ef703d2cd62227e5cfebe2
b53ce3b7b2ad2d75e210007c41ed43c876e7b399
3135 F20110115_AABZGL elyacoubi_b_Page_093thm.jpg
7c9964650124e7424df7c6d78a84e8cc
fd118a887a268903bf8257c9b72795bba74d86e5
F20110115_AABZHA elyacoubi_b_Page_065.tif
988e461e66484938409d8e0cf96c5573
d2c1cb0dae33da7436e69d74df69755e0b82ad9b
656 F20110115_AABZGM elyacoubi_b_Page_034.txt
084430f0b5308b69b1b501fa6f0a9a96
cfbcd7b1240a946a0e6eeca653042bc36367d3f0
6483 F20110115_AABZFX elyacoubi_b_Page_036thm.jpg
18e939cbd000928018b9f078a5061579
e106958b7725500d66588b455d5134a03f5426c4
6189 F20110115_AABZHB elyacoubi_b_Page_047thm.jpg
eaaeae8b3f001e09d380908620bd3577
14b8f7cf127fbcba5b7df8207c67f1d5b565df74
70119 F20110115_AABZGN elyacoubi_b_Page_016.jpg
a6db27afabe2a44183f505cf1326eb48
e1c2964216a39afc324d8ca0ecd9a3db36e735c3
6015 F20110115_AABZFY elyacoubi_b_Page_008.QC.jpg
5c25aa858d6656dc7095b190ce0d4f3d
3b29f90fd661345cfac3f90fa90bf9458c10cec6
13930 F20110115_AABZHC elyacoubi_b_Page_034.pro
0b63bb8f5b03f9682a3d885386d55b13
f804b918ecd11d2070d86db6473db065661a794b
48264 F20110115_AABZGO elyacoubi_b_Page_026.jpg
eca82eeef560cf738ceba349ed92dad3
e8200eeaaa532376fac6bb3e9a35aa5714042f06
432 F20110115_AABZFZ elyacoubi_b_Page_008.txt
c8325dd2fbb2cdf278859ec36d5f781b
ef354fd5f991415306f76662140c9da72f9820c1
5991 F20110115_AABZHD elyacoubi_b_Page_022thm.jpg
d217a0b2667c3224295c81d1202ca60a
8e823b63985f399038c2c949f72b029c7d303085
F20110115_AABZGP elyacoubi_b_Page_067.tif
9fb6a6c92ec74ff482919b152b54e29e
5213f405b4a3a28efea72b011ebbc2ce4ebb0206
466491 F20110115_AABZHE elyacoubi_b_Page_093.jp2
731ff4ff048010c9bc46328bd4b0c079
0a7ab7f8b07289791e6b39956b9513b0bfdb2e9d
F20110115_AABZGQ elyacoubi_b_Page_089.tif
690d2c2590d107c8a930ebb4f8c44ddf
1ebae85e68d76afebff240592b960be79b141bd5
19517 F20110115_AABZHF elyacoubi_b_Page_069.jpg
edfacac5a87768e8ab0b56ee23da9365
7ea7aef4bf29fac9656cab4bdd366e0d967211af
33795 F20110115_AABZGR elyacoubi_b_Page_097.jpg
7c7e3756c902cc38dd27e528a951e235
c56eb54782b7f72116260c3d321a5a25ec89fb3d
25902 F20110115_AABZHG elyacoubi_b_Page_100.QC.jpg
dd7bf07c6b143ec8f91a91a8844af964
d2261d05310b8d6789f23d61206c0b85d7ede70d
5322 F20110115_AABZGS elyacoubi_b_Page_011thm.jpg
a0503719ca14692b7520d4d3aff66bb0
6df05e37d1db059a20e4dfccc41ed86baa1bf891
9316 F20110115_AABZHH elyacoubi_b_Page_092.QC.jpg
ba048ce49fad746ad514fc8dfd99be56
dbef5946d00e342455a7690acf216835f10621e2
523280 F20110115_AABZGT elyacoubi_b_Page_097.jp2
03df9c6caad22dbfa393a351b488871a
a01b9f05bdd684532fa1e27fb11653acd29f6bb8
719755 F20110115_AABZHI elyacoubi_b_Page_030.jp2
74e005e229a0ff50824fe119cbd17c02
c42e0f26e05cc1c69309299c3b936e1cb7b32433
61044 F20110115_AABZGU elyacoubi_b_Page_099.pro
f3b45d5621a08b8a6de1e7c5a6352618
a567aca2a5473a87f89fbbcfad51c8b31a252e17
24223 F20110115_AABZHJ elyacoubi_b_Page_051.QC.jpg
05dd09441f07cde9be6c088e5a5e3961
157c97fbd29c71dc708c41518ffa2ca0030857f9
6526 F20110115_AABZGV elyacoubi_b_Page_017thm.jpg
1d2ff1b949fdfb9e47bbe263d9b1bb58
2a29afe104f02f9ca2f370aa12f7ff22f2f4edbe



PAGE 1

BACTERIAL CITRUS CANKER: MOLECUL AR ASPECTS OF A COMPATIBLE PLANT-MICROBE INTERACTION By BASMA EL YACOUBI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

PAGE 2

Copyright 2005 by Basma El Yacoubi

PAGE 3

To Souad, Kamal, Aziz, Mouma, Ma mi, Nemat, and ma petite Shemsi

PAGE 4

iv ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. Dean W. Gabriel, my supervisor and committee chair and for his constant s upport and guidance during my years as a graduate student in his laboratory. I also extend my gratitude to Dr. John M. Davis, (member of my supervisory committee) for hi s valuable advice and for welcoming me in his laboratory each time I needed it. I also thank all other members of my committee, (Dr. Alice Harmon, Dr. Kenneth Cline, Dr. Bill Gurl ey, Dr. Jim Preston) for their valuable advice and guidance. I thank my mother, Souad Benchemsi, w ho always supported me. Without her none of this would have been possible. My husband, Nemat Keyhani, and my little daughter Shemsi Aida Keyhani, gave to my graduate student life a new dimension, and I thank them for that.

PAGE 5

v TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... xi CHAPTER 1 A 37 KB PLASMID FROM A SOUTH AMERICAN CITRUS CANKER STRAIN CARRIES A TYPE IV SECRETION SYSTEM ESSENTIAL FOR SELFMOBILIZATION.........................................................................................................1 Introduction................................................................................................................... 1 Materials and Methods.................................................................................................5 Bacterial Strains, Plasmids and Culture Media.....................................................5 Marker Integration Mutagenesis............................................................................5 Plasmid Conjugal Transfer Techniques.................................................................6 Recombinant DNA Techniques.............................................................................7 Plant Inoculations..................................................................................................7 Results........................................................................................................................ ...7 The Type IV Secretion System Fo und on pXcB is Required for SelfMobilization.......................................................................................................7 Involvement of the TFSS of pXcB in Pathogenicity of Xca B69.........................9 Discussion...................................................................................................................10 2 IDENTIFICATION OF CITRUS GENES SPECIFICALLY RESPONSIVE TO PATHOGENICITY GENE pthB OF Xanthomonas citri pv. aurantifolii..................23 Introduction.................................................................................................................23 Material and Methods.................................................................................................26 Plant and Microbial Material...............................................................................26 Bacterial Counts..................................................................................................27 Microscopy..........................................................................................................27 Differential Display-Reve rse Transcriptase PCR................................................28 Suppressive Subtractive Hybridiza tion (SSH) Library Construction..................28 Northern Blots.....................................................................................................29 Reverse Northern Blots.......................................................................................29

PAGE 6

vi Statistical Analysis..............................................................................................30 Results........................................................................................................................ .31 Macroscopic Disease Phenotype of Citrus Leaves Inoculated with X. c. aurantifolii B69 and Its Mutant Deriva tive BIM2 Lacking the Pathogenicity Gene pthB .........................................................................................................31 PthB-Dependent Transcriptional Reprogr amming Induced upon Infection with Xca ...................................................................................................................32 Construction of Two Libraries Enriched in pthB Responsive cDNAs................33 Transcript Analyses of CCRs..............................................................................33 Identity of cDNAs Identified as Up -Regulated by the Presence of pthB in X. citri Genome..................................................................................34 Identity of cDNAs Identified as Up-Regulated by X citri Lacking pthB ............35 Northern Blot Analysis of Representative CCRs................................................36 Microscopic Phenotype of B69 and BIM2 Inoculated Leaves............................36 Discussion...................................................................................................................38 PthB Induces Cell Division and Ce ll Expansion in Citrus Leaves......................39 PthB Induces the Expression of Cell Wall Remodeling Enzymes......................40 Enod8 and SAH7/LAT52 are a Link Between Canker Symptoms Development and Nodule Organogenesis and Pollen Tube Growth Respectively................42 PthB Induces Up-Regulation of a Tonoplast Aquaporin.....................................44 PthB Induces Up-Regulation of Tw o Components Involved in Vesicle Trafficking.......................................................................................................44 Hormone Pathways are Possibly I nvolved in Canker Symptoms Development...................................................................................................45 Conclusions and Future Prospects..............................................................................47 3 CHANGES IN SUMO CONJUGATION ARE ASSOCIATED WITH CITRUS CANKER DISEASE..................................................................................................66 Introduction.................................................................................................................66 Materials and Methods...............................................................................................69 Plant Inoculations................................................................................................69 Bacterial Strains and Culture Media....................................................................70 Marker Integration Mutagenesis..........................................................................70 Bioinformatics.....................................................................................................71 Protein Extraction a nd Western Blotting.............................................................71 Results........................................................................................................................ .72 SUMO Conjugation Profiles are Altered in X. citri -Infected Leaves.................72 SUMO Conjugation Profiles in Infected Leaves are Partially PthB Dependent.73 SUMO De-Conjugation Observed at 7 days Following Infection with B69 and BIM2 is Dependent on a Functi onal Type III SecretionSystem......................74 Discussion...................................................................................................................74

PAGE 7

vii APPENDIX A LIST OF PLASMID AND STRAINS........................................................................83 B NORTHERN BLOT ANALYSIS OF CCRS.............................................................85 LIST OF REFERENCES...................................................................................................86 BIOGRAPHICAL SKETCH.............................................................................................91

PAGE 8

viii LIST OF TABLES Table page 2-1 List of putative CCR identified by DD-PCR...........................................................49 2-2 List of CCRs confirmed by reve rse northern blot analysis......................................50 A-1 List of strains and plasmids used in this study.........................................................83

PAGE 9

ix LIST OF FIGURES Figure page 1-1 Organization of the type four secretion system ( virB operon) found on pXcB compared to other described TFS systems...............................................................13 1-2 Hybridization profiles of DNA from B69 integrativ e mutants interrupted in virB4 of B69 virB clusters. ................................................................................................14 1-3 Eco RI and Bam HI restriction digest profiles of plasmid pB13.1 and plasmid pB13.2, derivatives of pXcB0 and pXcB, re spectively, and integrated in gene virB4 .........................................................................................................................15 1-4 PCR profiles using primers AB65 and AB 66 specific of plasmid pXcB. 16 1-5 Self-mobilization of pXcB derivatives is dependent on a type IV secretion system.17 1-6 Construction of suicide vector pBY17.1..................................................................18 1-7 Scheme of FLP recombinase-mediated marker eviction..........................................19 1-8 PCR confirmation of suicide pl asmid pBY17.1 integration in gene virB4 ..............20 1-9 CR confirmation of Flp-mediated eviction of pBY17.1..........................................21 1-10 Pathogenicity phenotype of primary a nd secondary exconjugants disrupted in virB4 .........................................................................................................................22 2-2 Late B69 and BIM2 phenotypes. (A) BIM2 inoculated leaves 30 dpi and (B) B69 inoculated leaves 30 dpi...........................................................................................53 2-3 Quantification of bacterial population two days post inoculation with B69 and BIM2. (cfu: colony forming unit), Exp1: experiment 1, Exp2: experiment 2)........54 2-4 Diagram of PCR-Sel ect cDNA subtraction..............................................................55 2-5 Distribution of potential ci trus canker responsive genes.........................................56 2-6 Distribution and origin of the clones stamped on the nitrocellulose membranes used in reverse northern blot analysis.....................................................................57 2-7 Cluster analysis of genes di fferentially regulated by PthB. ....................................58

PAGE 10

x 2-8 Northern blot analysis of CCR gene s found differentially regulated by reverse northern blot analysis...............................................................................................59 2-9 Microscopic phenotype of leaves in oculated with B69 (wt) and BIM2 (nonpathogenic mutant lacking PthB). ....................................................................60 2-10 Microscopic phenotype of leaves in oculated with B69 (wt) and BIM2 (nonpathogenic mutant lacking PthB). ....................................................................61 2-11 Microscopic phenotype of leaves in oculated with B69 (wt) and BIM2 (nonpathogenic mutant lacking PthB) at 14 dpi. .....................................................62 2-13 Quantification of leaf thickening and cell division dur ing B69 and BIM2 infection on Duncan grapefruit leaves.....................................................................................63 2-13 Microscopic symptoms of rapidly developing canker. ...........................................64 3-1 Alignment of grapefruit SUMO (par tial sequence) with (PopSUMO1, gi:23997054, and AtSUMO1, At4g26840)....................................................................................77 3-2 SUMO profiles of B69and mo ck-challenged grap efruit leaves. ...........................78 3-3 SUMO de-conjugation occurs 7 days after infection...............................................79 3-4 Split leaf inoculation of Xanthomonas citri pv. aurantifolii (B69) and derivative BIM2 mutant. ..........................................................................................................80 3-5 B69 mutant derivative B23.5 lacks a functional Type III secretion system............81 3-6 SUMO de-conjugation at 7 dpi requires a functional TTSS....................................82 B-1 Northern blot analysis of CCR genes not found differentially regulated by reverse northern blot.............................................................................................................85

PAGE 11

xi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BACTERIAL CITRUS CANKER: MOLECUL AR ASPECTS OF A COMPATIBLE PLANT-MICROBE INTERACTION By Basma El Yacoubi May 2005 Chair: Dean W. Gabriel Major Department: Plant Pathology Canker is an important disease affecting citrus worldwide. It is caused by two phylogenetically distinct groups of strains of Xanthomonas citri (Xc), with all citrus cultivars being susceptible to at least one Xc strain. It is known that canker-causing xanthomonads carry at least one pathogenicity gene of the pthA (of Asiatic X. citri pv citri) gene family, which is required for causing canker on citrus. However little is known of the host molecular even ts leading to canker. Our go al was to understand host molecular mechanisms underlying disease development, and identify bacterial components related to phylogeny or pathoge nicity of canker-causing xanthomonads. First we identified on plasmid pXcB of the South American strain X. citri pv aurantifolii B69, a pathogenicit y island composed of previous ly identified pathogenicity gene pthB and a type IV secretion system (TFSS) This TFSS was shown to be required for self-mobilization of pXcB, which led us to propose that natural hor izontal transfer of a pth host-specific pathogenicity gene may acc ount for the two phylogenetically distinct

PAGE 12

xii groups of strains, (the Asiatic and the Sout h American group of strains), causing canker symptoms on citrus. Second, we investigated plant responses to PthB using differen tial display PCR and suppressive subtractive hybridi zation techniques. We identified forty-nine genes that were differentially regulated when RNA expr ession profiles of leaves inoculated with Xca B69 were compared to those of leaves inoculated with a B69 mutant carrying a disrupted pthB Among these were genes predicted to be involved in cell expansion, protein modification, biotic /abiotic stress responses and cell-wall metabolism. Finally, we focused on one canker-responsiv e gene with strong similarity to the small ubiquitin like modifier (SUMO) from Arabidopsis Analysis of B69 mutant strains lacking PthB or the type III secretion system (TTSS) component, HrpG, revealed PthBdependent and TTSS dependent/PthB–inde pendent changes in SUMO conjugation profiles after inf ection with B69. The genes and cellular processes that we identified reflect the molecular events leading to disease development. They contri bute to the general aim of understanding the mechanisms underlying the variety of diseas es caused by compatible interactions between xanthomonads and their host plants.

PAGE 13

1 CHAPTER 1 A 37 KB PLASMID FROM A SOUTH AMERICAN CITRUS CANKER STRAIN CARRIES A TYPE IV SECRETION SYSTEM ESSENTIAL FOR SELFMOBILIZATION Introduction The genus Xanthomonas is comprised of strains that exhibit a high level of hostspecificity; over 125 different path ogenic variants (pathovars) of X. campestris have been described that differ primarily in host range (Bergey, 1994). Host specificity in Xanthomonas can be due to gene-for-gene interac tions involving avirulence genes that act in a negative fashion to limit host ra nge (Keen, 1990; Gabriel, 1999; Leach and White, 1996), but also can be due to positive ac ting factors that condition host range in a host-specific manner. For example, pthN avrb6 of X. campestris pv. malvacearum (Yang and Gabriel, 1996), opsX of X. campestris pv. citrumelo (Kingsley et al, 1993) and pthA of X. citri pv. citri (Swarup et al., 1991 and 1992) act as positive effectors of host range. Interestingly, although a clonal population st ructure is observed among strains within many pathovars (Gabriel et al, 1988), some pa thovars are comprised of phylogenetically distinct groups that have an identical host range and caus e identical disease symptoms. Examples include 1) common bean bli ght, caused by two groups of strains ( X. phaseoli and X. campestris pv. phaseoli var. fuscans) that are only 20% related by DNA-DNA hybridization (Hildebrand et al ., 1990); 2) bacterial spot of tomato and pepper, caused by two major groups of strains within X. campestris pv. vesicatoria (Jone s et al., 2000) that are less than 50% related by DNA-DNA hybridiza tion (Stall et al., 1994 ), and 3) citrus canker disease, caused by two groups of st rains that are only 62 -63% related by DNA-

PAGE 14

2 DNA hybridization (Egel et al., 1991). Strains with 70% or greater DNA-DNA relatedness are usually defined as single spec ies (Wayne et al., 1987) The question arises however, as to how phylogenetical ly diverse strains can cause identical diseases on an identical range of hosts. To date, all pathogenic xanthomonads examined require hrp genes (reviewed by Alfano & Collmer, 1996, 1997; He, 1998; Cornelis & VanGijsegem, 2000) to cause disease. These genes encode a type III secret ion machine that is close contact-dependent (Marenda et al., 1998) and used to inject high ly adapted effector proteins into both host and nonhost cells (Silhavy, 1997; Kubori et al., 1998; and Jin and He, 2001). These effector proteins elicit the diverse programmed phenotypes of the plant hypersensitive response (HR) and various pa thogenicity responses. The hrp (h ypersensitive r esponse and p athogenicity) injection system is thus a ppropriately named, and it is also highly indiscriminate, injecting whatever effector proteins are available, even some from animal pathogens (Anderson et al., 1999 and Ro ssier et al., 1999). If identical hrp effectors are available within two phylogenetically distin ct xanthomonads, they can cause the same disease symptoms, provided both strains are comp atible (able to multiply in the host) and both carry functional hrp systems. For example, pthA was transferred from X. citri to X. campestris pv. citromelo and converted the latter strain from a leaf-s potting strain to a strain with ability to cause citrus canker di sease (Swarup et al., 1991). PthA appears to be an effector protein that is cr itical for citrus disease sympto ms and is likely injected by X. citri into citrus cells, cau sing hyperplastic cankers (Duan et al, 1999). Citrus canker disease is caused by two phylogenetically distinct and clonal groups of Xanthomonas strains; each group contains subgr oups that are distinguished on the

PAGE 15

3 basis of host range (Brunings and Gabriel, 2003). The first phylogenetically distinct group is the Asiatic group, named Xanthomonas citri pv citri ex Hasse (syn = X. campestris pv. citri Dye pathotype A and X. axonopodis pv. citri Vauterin, Xca -A). The second phylogenetically distinct gro up is the S. American group, named X. citri pv. aurantifolii Gabriel (syn = X. campestris pv. citri Dye and X. axonopodis pv. aurantifolii Vauterin, Xca -B). Both groups cause identical citrus canker disease symptoms circular, water soaked raised lesions, that become da rk and thick as canker progresses (Graham et al., 2004; Stall and Civerolo, 1991; Gottwald et al, 2002; Brunings and Gabriel, 2003). Significantly, pthA or homologues are present in every Xanthomonas strain tested that causes citrus canker disease, and have not been found present in xanthomonads isolated from citrus that do not cause canker (Gabri el, 1999; Cubero and Graham, 2002). Prior to this work, two pthA homologues, named pthB and pthB0 were found on two separate plasmids (pXcB and pXcB0, respectively) of a S. American canker strain (B69). Plasmid pXcB carrying the functional homologue pthB was then found to be readily cured from B69 (Yuan and Gabriel unpublished, and Bruning s, A.M., 2004 M.S. thesis University of Florida). Readily cured plasmids are ofte n mobilizable by conjugation. Since Asia is considered to be the center of origin of c itrus canker disease, and since Asiatic canker strains are more widespread in S. America than S. American canker strains, it was of interest to determine if pXcB could transfer horizontally. pX cB was found to horizontally transfer in-vitro and in planta (Yuan and Gabriel unpublished) from the S. American strain B69 to the Asiatic st rain B21.1 lacking a functional pthA restoring its capacity to cause canker (Yuan and Gabriel unpublished) Presence of the type III effector pthB on a

PAGE 16

4 self-mobilizing plasmid might explain the creat ion of the entire S. American group of canker strains, and why they are phylogene tically distinct from the Asiatic group. pXcB was fully sequenced (NC_005240, gi32347275), and besides gene pthB a complete Type IV secretion system (TFSS) was also found on the plasmid (Brunings, A.M., 2004 M.S. Thesis, University of Fl orida). TFSS are defined on the basis of homologies between the A. tumefaciens T-DNA transfer system, the conjugal transfer system Tra, and the Bordetella pertussis toxin exporter, Ptl (Winams et al., 1996 and Christie, 1997). Most members of the TFSS fa mily function primarily to mobilize DNA, either from bacteria to bacteria (bacterial conjugation system) or from bacteria to eukaryotic cells ( Agrobacterium oncogenic T-DNA transfer system) (Burns, 1999). In addition, several bacterial pat hogens utilize conjugation mach ines to export effector molecules during infection. Such systems are said to be Type IV “adapted” conjugation or secretion systems, for their involvement in pathogenicity. Many non-plant pathogens such as Bordetella pertussis Legionella pneumophila, Brucella spp. and Helicobacter pylori use a type IV “adapted” conjugation syst em to secrete effector proteins to the extracellular milieu or the cel l cytosol (Burns, 1999; Christie, 1996; Christie and Vogel, 2000). Type IV systems are composed of products with homology to the Agrobacterium virB operon (Vogel, 2000). Sequence similarity analysis revealed that the Type IV secretion system of pXcB encodes twelve open reading frames, te n of which contained high sequence similarities to ge nes of previously described virB operons as well as similar relative positions within the cluster (Bruni ngs, A.M., 2004 M.S. Thesis, University of Florida).

PAGE 17

5 In order to investigate whether the T FSS found on pXcB is involved in selfmobilization of pXcB, a plas mid derivative lacking a functional TFSS was generated in this study and tested for its ability to self-mobilize in vitro In addition, a B69 derivative lacking the TFSS was generated in a non-polar fashion to address whether this system was required for pathogenicity of B69. It wa s found that the TFSS of pXcB was required for self-mobilization of the plasmid. Howe ver pathogenicity tests involving TFSS insertional mutants were inconclusive, a nd it remains unknown whet her this secretion system is involved in pathogenicity of X. citri pv. aurantifolii. Materials and Methods Bacterial Strains, Plasmids and Culture Media Bacterial strains and plasmids used in this study are listed in Table 1. Xanthomonas spp. were cultured in PYGM medium at 30oC (De Feyter et al., 1990). Escherichia coli were grown in Luria-Bertani (L B) medium (Sambrook et al., 1989). Antibiotics were used at the following concentrations (in g/mL): Chloramphenicol (Cm), 35; Kanamycin (Kn) 12.5 or 25 (when used to grow Xanthomonas or E. coli respectively); Spectinomycin (Sp) 35 and Streptomycin (St) 35. Marker Integration Mutagenesis Gene-specific knockout mutations of Xanthomonas were created by triparental matings. An E. coli DH5 strain carrying an internal frag ment of the target open reading frame (ORF) cloned in suicide vect or pUFR004 was used as donor. A DH5 strain carrying pRK2013 was used as the helper. A si ngle crossover in the exconjugates results in duplication of the internal fragment at the integration site, and also results in interrupting the target gene with the vector. To disrupt virB4 a PCR-generated, 270 bp

PAGE 18

6 internal fragment of virB4 (virB4 270 ) was cloned in pGEM–T Easy and recloned in pUFR004 creating pBY13. Plasmid Conjugal Transfer Techniques Plasmid transfer by triparental mating from E. coli strains HB101 or DH5 to various Xanthomonas strains, using helper strain pR K2013 were performed essentially as described in De Feyter a nd Gabriel (1991). For plasmid transmission experiments on artificial media, ove rnight cultures of E. coli strains grown without antibiotics were mixed with 50X concentrated overn ight, mid-log phase cultures of Xanthomonas strains, grown without antibiotics. Drops (10 l each) of recipient donor and helper cells were placed on PYGM agar medium one after the ot her and without antibiotics. In each case excess liquid was allowed to absorb into the plate before addition of the next cell type. The mating plates were incubated at 30oC overnight, and the spots were then streaked on PYGM selection medium supplemented with the appropriate antibiotics. In Xanthomonas to E. coli matings, B69 carrying pB13.2 (pBY13 integrated in virB4 of pXcB) or B69 carrying pB13.1 (pBY13 integrated in virB4 of pXcB0) were used as donor strains (in inde pendent matings) with DH5 as the recipient strain. After selection against Xanthomonas on MacConkey agar (DIFCO laboratories, Detroit MI, USA) with 35 g/mL chloramphenicol, DH5 exconjugants were screened for the presence of pBY13.2 or pBY13.1 by DNA mini-prep analysis. In E. coli to E. coli matings, DH5 /pBY13.2, DH5 /pBY13.1 and DH5 /pBIM2 (pYY40.10 integrated in pthB ) were used as donor stains in indepe ndent matings with HB101 as recipient. For frequency of transfer assays from one E coli strain to another, donor and recipient strains were grown overnight at 37 oC to an O.D. 600nm of 0.5. Twenty

PAGE 19

7 microliters of each culture were combined in a 1.5 ml Eppendorf tube containing 160 l of LB and grown overnight at 37 oC. Cells were then resuspended in 1 ml of LB, pelleted and then serially diluted on medium containing chloramphe nicol and streptomycin to select for HB101 transconjugants. All conjugati on experiments were performed at least twice with duplicate samples in each expe riment, and the numbers were averaged. Recombinant DNA Techniques Plasmid and total DNA were prepared from Xanthomonas as described by Gabriel and De Feyter (1992). E. coli plasmid preparation, restrictio n enzyme digestion, alkaline phosphatase treatment, DNA ligation, and ra ndom priming reactions were performed using standard techniques (Sambrook et al., 1989). Southern hybrid ization was performed using nylon membranes as descri bed by Lazo and Gabriel (1987). Plant Inoculations All citrus plants ( Citrus paradisi ‘Duncan’, grapefru it) were grown under greenhouse conditions. Plant inoculations involvi ng all citrus canker strains were carried out under quarantine at the Divi sion of Plant Industry, Florid a Department of Agriculture, Gainesville. Bacterial cells were harvested from log phase cultures by centrifugation (5,000 x g, 10 min.), washed once and resuspended in sterile tap water or distilled water saturated with calcium carbonate to 108cfu/mL. Inoculations were performed by pressureinfiltration into the abaxial leaf surface of the plants. Experimental inoculations were repeated at least three times. Results The Type IV Secretion System Found on pXcB is Required for Self-Mobilization Gene virB4 of the TFSS cluster of pXcB wa s chosen as target for marker insertional mutagenesis (Figure 1-1). Fo r that, a 270 bp integral fragment of virB4

PAGE 20

8 (virB4270) was cloned in pUFR004 (pBY13) and used in triparental matings to generate virB4 insertion mutants. Southern blots were us ed to verify integration events in the resulting transconjugants. These results dem onstrate the existence of two copies of virB4 in the B69 strain (using virB4270 as probe Figure 1-2). One copy was carried by pXcB (as determined by sequencing) and was absent in the cured strain B69.4 [Rifamycin resistant strain cured of plasmid pXcB but carrying plasmid pXcB0, Yuan and Gabriel, unpublished (Lane 3)]. A second putative copy carried by pXcB0, was maintained in B69.4 (Lane 3). Marker insertion resulted in two categories of exconjugants. Exconjugant strain B13.1 appeared to carry an interruption of the putative virB4 of pXcB0 ( virB40) (Lane 7), while exconjugants B13.2, B13.4 and B13.5 appeared to carry interruptions of the virB4 gene of pXcB (Lanes 4, 5 and 6). Plasmids pB13.1 and pB13.2 of strains B 13.1 and B13.2 (marker interruptions in the virB4 homologues found on pXcB0 and pXcB, re spectively) were further analyzed for their ability to transfer to E. coli Matings with and without the helper strain resulted in DH5 exconjugants carrying plasmids that were chloramphenicol resistant, indicating that both plasmids were still mobilizing. Re striction enzyme digests of plasmid DNA extracted from the Xanthomonas (B13.2) to the E. coli exconjugant (DH5 /pB13.2), corresponded to the expected profile of pXcB integrated with pBY13 (Figure 1-3). Restriction enzyme digests of plasmid DNA extracted from DH5 /pB13.1 did not corresponded to the profile expected for a pXcB insertional deri vative. Therefore, p13.1 is a derivative of a second native plasmid of B6 9, smaller in size than pXcB and inserted in a putative virB4 copy. Th ese results were confirmed by PCR using primers specific to pXcB. As shown in Figure 1-4, when pB13.2 wa s used as template with pXcB specific

PAGE 21

9 primers AB65/AB66 a 2014 bp band was obtained, while non specific bands were obtained when pB13.1 was used as template The ability of pB13.1 and pB13.2 to self-mobilize was then analyzed by performing matings from DH5 to E. coli HB101. Using DH5 /pBIM2, and DH5 /pBIM6 [pBIM6 is a derivative of pX cB where pUFR004 was inserted in a nonORF region, (Yuan and Gabriel, unpublished)] as a control, transfer of pBIM2 and pBIM6 from DH5 to HB101 was found not to require the presence of a helper strain and the transfer frequency was 7x10-03 and 6.6x10-05 per donor, respectively. By contrast, E. coli to E. coli transconjugants harboring pB13.1 or pB13.2 were only recovered when matings were performed in the presence of a helper strain (Figur e 1-5). These results indicated that the self-mobilization capacity of pXcB depended on the presence of an intact virB cluster. Involvement of the TFSS of pXcB in Pathogenicity of Xca B69 Non-polar knock out mutants of virB4 were generated using marker insertion followed by FLP recombinase mediated marker eviction. Plasmid pBY17.1 was generated so that a virB4 homology region was flanked by two FRT recognition sites (See Figure 1-6 for illustration) After marker integration of suicide vector pBY17.2 into primary transconjugants, the FLP recombinas e plasmid pJR4, was used to evict the marker, and generated non-polar seconda ry transconjugants (See Figure 1-7 for illustration). Several primary transconjugants (before FL P-mediated eviction of marker) (Figure 1-8) as well as secondary tr ansconjugants (after FLP mediat ed eviction of marker) were tested for integration events in a virB4 homologue using PCR. Bacterial cells directly

PAGE 22

10 from the selection plates were used as template for PCR (Figure 1-9). PCR positive colonies were then grown in liquid culture a nd tested for pathogenicity on citrus. In all cases, primary exconjugants showed a decrea se in pathogenicity while, unexpectedly, secondary exconjugants lost th eir potential to trig ger canker disease on citrus (Figure 110). When the secondary exconjugants used in pathogenicity assays were tested by PCR for presence of pXcB it was found th at the plasmid and therefore gene pthB were lost upon culturing. Discussion The putative TFSS of pXcB (Brunings A.M ., M.S. Thesis, University of Florida and Brunings and Gabriel, 2003) was functi onally investigated to determine its involvement in plasmid transfer as well as in pathogenicity of B 69. To investigate the role of this TFSS in plasmid transfer, gene virB4 was marker-interrupted and by consequence the whole system rendered dysfunctional. Self-mobilization experiments revealed that pXcB relied on a functional T FSS to self–mobilize. In the process a second putative virB4 homologue was identified on a se cond plasmid of B69, pXcB0. pB13.1, carrying a single insertion in virB40 of pXcB0 and pB13.2, carrying a single insertion in virB4 of pXcB were each able to mobilize from B13.1 and B13.2, respectively, to DH5 in biparental matings (without helper st rain), indicating that the two putative virB systems co-existing in B69 might be compensatory. The characterization of pXcB as a se lf-mobilizing plasmid carrying a TFSS and gene pthB suggests that the canker causing and p hylogenetically distinct South American strains may have arisen from horizontal gene transfer of an “ancestral” pthA member. This horizontal transfer likely w ould have occurred from an Asiatic Xanthomonas citri

PAGE 23

11 strain to a compatible TTS system-carryi ng xanthomonad residing on the same host. B69 was indeed shown to carry a functional TTS system required for pathogenicity (see Chapter 3). The type IV secr etion system together with pthB on pXcB of S. American Xanthomonas citri strains can therefore be considered an “aut o-mobile” pathogenicity island (Hacker et al., 1997) capable of spreading am ong compatible bacteria by horizontal gene transfer. Since pXcB from the South American strain is smaller, yet very similar to pXAC64 from the Asiatic strain, pXcB could be a deletion derivative of pXAC64 (Brunings and Gabriel 2003). However, while many genes on pXcB were found to be similar to genes on pXAC64, there were differen ces significant enough to concl ude that a simple deletion cannot account for pXcB. More likely, seve ral independent events were probably responsible for its divergence away from pXAC64. Horizontal gene transfer is proposed to be a major mechanism explaining rapid genetic diversificati on in bacteria (Falcow, 1996; Syvanen and Kado, 1998; Lawrence and Roth, 1999). It has been proposed to expl ain the apparent enigma of why pathogens carry dispensable avirulence genes (Yang and Gabriel, 1996 and Gabriel, 1999). For example, avrBs3 of Xanthomonas campestris pv. vesicatoria was found on a mobilizing plasmid carrying copper resistance, and th erefore wide horizontal transfer of avrBs3 to X. campestris pathovars may be due to coincidental linkage with copper resistance (Stall et al, 1986, Yang and Gabriel, 1996). The TFSS of pXcB was also analyzed for its involvement in pathogenicity. Primary exconjugants carrying a marker integration in virB4 showed a decrease in pathogenicity while non-polar secondary exconjugants, resul ting from marker eviction of the suicide

PAGE 24

12 plasmid, lost all pathogenicity. This was then found to be possibly due to a loss of pXcB upon curing of secondary transconju gant strains. Another explan ation is the presence of a large insertion vector in the native plasmi d decreasing the copy number in the population. Further examination of the TFSS is necessary to access its role in pathogenicity if any.

PAGE 25

13 Figure 1-1: Organization of the type four secretion system ( virB operon) found on pXcB compared to other described TFS system s. ORF106 shows no similarity to any virB cluster gene of Agrobacterium tumefaciens and is shown as an insertion. Orf106 B2B3B4B5 B7B6B8B9 B10 B11 B1 X.a.a ( pXcB) virB virB A tumefaciens (pAtC58) avhB Orf106 B2B3B4B5 B7B6B8B9 B10 B11 B1 Self-mobilization Transfer of T-DNA Ti-plasmid Conjugal transferOrf106 B2B3B4B5 B7B6B8B9 B10 B11 B1 X.a.a ( pXcB) virB virB A tumefaciens (pAtC58) avhB Orf106 B2B3B4B5 B7B6B8B9 B10 B11 B1 Self-mobilization Transfer of T-DNA Ti-plasmid Conjugal transfer

PAGE 26

14 6969.413.213.413.513.1 9.4 6.6 4.4 virB40 ( pXcB0) virB4( pXcB) 6969.413.213.413.513.1 9.4 6.6 4.4 virB40 ( pXcB0) virB4( pXcB) Figure 1-2: Hybridization prof iles of DNA from B69 integrat ive mutants interrupted in virB4 of B69 virB clusters. Total DNA was digested with Hin dIII and probed with a 32P-labelled 270 bp internal fragment of virB4 The same fragment was used as a homology region for integra tion of suicide vector pBY13. B13.2, B13.4 and B13.5 were marker integrated in virB4 of pXcB, and B13.1 was marker integrated in virB4 of pXcB0. Hin d III digestion results in splitting one restriction fragment harboring th e targeted region in to two hybridizing fragments. Therefore there ar e two bands hybridizing to the virB4270 probe in the wild type strains, while there are th ree bands in the insertional strains. The only band hybridizing to the virB4270 in the B69.4 lane corresponds to a putative virB4 copy present on a second native plasmid of B69. Indeed pXcB was lost upon curing in B69.4 and ther efore one hybridizing band is lost.

PAGE 27

15 pBIM2 p13.2p13.1Eco RI Bam HI pBIM2p13.2p13.1 23 9.4 6.6 4.4 2.1 2.3 pBIM2 p13.2p13.1Eco RI Bam HI pBIM2p13.2p13.1 23 9.4 6.6 4.4 2.1 2.3 Figure 1-3: Eco RI and Bam HI restriction digest profiles of plasmid pB13.1 and plasmid pB13.2, derivatives of pXcB0 and pXcB, re spectively, and integrated in gene virB4

PAGE 28

16 B69 B69.4 pB13.1 pB13.2 B69 B69.4 pB13.1 pB13.2 Figure 1-4: PCR profiles using primers AB65 and AB66 specific of plasmid pXcB. Plasmid DNA isolated from DH5 /pB13.1, DH5 /pB13.2, and total DNA isolated from B69 and B69.4 were us ed as templates. [AB65: CAG CCG CAA GTG TCT CAG GTC; AB66: GGC AAG AAA CCG TCC GAG TA (Tm 56 C)]. When B69.4 and pB13.1 DNA are used as template in the PCR reaction non-specific bands of low inte nsity are the resulting products. When B69 and pB13.2 (both derivatives of plas mid pXcB) are used as template, a specific band of 2014 bp is the resu lting product of the PCR reaction. ( ): Amplification fragment specific to pX cB when AB65/AB66 primers are used.

PAGE 29

17 0% 0.5% 1% pB13.1pB13.2pBIM2 Plasmid ( in donor strain DH5 ) Frequency of transfer to HB101 (per input donor) p13.1 0 p13.2 0 pBIM2 7.08E-03 pBIM6 6.57E-05 a b cA BFrequency of transfer (per input donor) 0% 0.5% 1% pB13.1pB13.2pBIM2 Plasmid ( in donor strain DH5 ) Frequency of transfer to HB101 (per input donor) p13.1 0 p13.2 0 pBIM2 7.08E-03 pBIM6 6.57E-05 a b c a a b cA BFrequency of transfer (per input donor) Figure 1-5: Self-mobilization of pXcB deriva tives is dependent on a type IV secretion system. ( A ) Mobilization of pXcB deriva tive, pBIM2 and pB13.2 and pXcB0 derivative pB13.1 from E. coli DH5 to E. coli HB101. Matings were carried with and without helper strain carrying plasmid pRK2013, and HB101 transconjugants were selected on LB supplemented with streptomycin and chloramphenicol. Each selection plate was separated in two sections. Results of matings with helper strain are sh own on the left section, and results of matings without helper are show n on the right. Matings: (a) DH5 /pB13.1 with HB101; (b) DH5 /pB13.2 with HB101; (c) DH5 /pBIM2 with HB101. ( B) Frequency of transfer of pXcB derivatives, pB13.2, pBIM2, pBIM6 and pXcB0 derivative, pB13.1 from E. coil DH5 into E. coli HB101.

PAGE 30

18 Figure 1-6: Construction of su icide vector pBY17.1. PCR was used to amplify an internal virB4 fragment using primers BY13 (g atcaggatcctatgcgcctcgttgaggt) and BY14 (cggtccgtcagtcagtcagagctctgaccaggtagtgcagga). Rsr III and Bcl I restriction sites were incorporated in the primer sequences respectively in the forward and reverse primer. The Rsr IIIBcl I fragment was used as the driver for homologous recombination and was cloned between FLP sites in pUFR012 (pUFR004 derivative carrying kanamycin resistance). FLP sites were obtain by PCR using plasmid p KD4 (gi:15554332) (1.5 Kb fragment). Primers FRTKn F (gaattcgctgct tcgaagttcctatac) and FRTKn R (aagcttatcctccttagttccaattcc) carried an Eco RI and a Hin dIII site for subcloning from pGEMT-ez (Promega) into pUFR012. FRTKn PCR fragment FRTKn F FRTKn R FRTKn PCR fragment FRTKn F FRTKn R PCR fragment from template pKD4 was cloned in pGEMT-ez BY13BY14PCR fragment from template pXcB was cloned in pGEMT-ez RsrIIIBclI pBY3.1 (pUFR012::FRTKn) Eco RI Hin dIIIRsrIII BclI Eco RI pBY1.1 Hin dIII pBY2.1RsrIIIBclI pBY17.1 (pBY3.1:: virB4 ) RsrIIIBclIPCR fragment internal to virB4 FRTKn PCR fragment FRTKn F FRTKn R FRTKn PCR fragment FRTKn F FRTKn R PCR fragment from template pKD4 was cloned in pGEMT-ez BY13BY14PCR fragment from template pXcB was cloned in pGEMT-ez RsrIIIBclI pBY3.1 (pUFR012::FRTKn) Eco RI Hin dIIIRsrIII BclI pBY3.1 (pUFR012::FRTKn) Eco RI Hin dIIIRsrIII BclI Eco RI pBY1.1 Hin dIII Eco RI pBY1.1 Hin dIII pBY2.1RsrIIIBclI RsrIIIBclI pBY17.1 (pBY3.1:: virB4 ) RsrIIIBclI RsrIIIBclIPCR fragment internal to virB4 FRTKn PCR fragment FRTKn F FRTKn R FRTKn PCR fragment FRTKn F FRTKn R PCR fragment from template pKD4 was cloned in pGEMT-ez BY13BY14PCR fragment from template pXcB was cloned in pGEMT-ez RsrIIIBclI pBY3.1 (pUFR012::FRTKn) Eco RI Hin dIIIRsrIII BclI Eco RI pBY1.1 Hin dIII pBY2.1RsrIIIBclI pBY17.1 (pBY3.1:: virB4 ) RsrIIIBclIPCR fragment internal to virB4 FRTKn PCR fragment FRTKn F FRTKn R FRTKn PCR fragment FRTKn F FRTKn R PCR fragment from template pKD4 was cloned in pGEMT-ez BY13BY14PCR fragment from template pXcB was cloned in pGEMT-ez RsrIIIBclI pBY3.1 (pUFR012::FRTKn) Eco RI Hin dIIIRsrIII BclI pBY3.1 (pUFR012::FRTKn) Eco RI Hin dIIIRsrIII BclI Eco RI pBY1.1 Hin dIII Eco RI pBY1.1 Hin dIII pBY2.1RsrIIIBclI RsrIIIBclI pBY17.1 (pBY3.1:: virB4 ) RsrIIIBclI RsrIIIBclIPCR fragment internal to virB4

PAGE 31

19 FRT FRThomology to virB4 pBY17.1 (Kn, Cm) 800bp800bp 700bp bes03 virB4 F bes04 inserted vector virB4 M13R bes03 bes04 virB4 F 22mer FLP recombinase plasmid Figure 1-7: Scheme of FLP recombinase-me diated marker eviction. Suicide plasmid pBY17.1 is marker integrated in gene virB4 via homologous recombination, generating a virB4 disruption. The light blue box represents the homology region targeted for recombination, and is found duplicated af ter insertion of the suicide plasmid pBY17.1 in pXcB After transformation of the B69 derivative carrying pXcB::pBY17.1 (prima ry transconjugant) with plasmid pJR4 carrying a FLP recombinase gene, pBY17.1 is evicted (secondary transconjugants). pJR4 [derived from pFLP (gi:1245114) (Ready and Gabriel, unpublished)] is cured by culturing secondary transconjugants on PYGM supplemented with 5% sucrose. VirB4F and bes03 and bes04 are virB4 specific primers and their locations ar e shown by arrows. 22mer and M13R are primers specific to the polylinker region of suicide vector pBY17.1 and are used to verify integration and ev iction events. FRT sites recognized the FLP recombinase are symbolized by yellow circles and flank the internal fragment with homology to virB4 cloned in pBY17.1. The green boxes symbolize DNA stretches carried over during sub-cloning steps.

PAGE 32

20 123456789 22mer/bes03M13R/BES04M13R/bes03DS Lane 1,4,7: B69, Lane 2,5,8: B18.12, Lane 3,6,9: B18.15 123456789 22mer/bes03M13R/BES04M13R/bes03DS Lane 1,4,7: B69, Lane 2,5,8: B18.12, Lane 3,6,9: B18.15 vector pBY17.1 virB4 M13R bes03 bes04 virB4 F 22mer vector pBY17.1 virB4 M13R bes03 bes04 virB4 F 22mer bes03DS Figure 1-8: PCR confirmation of suicid e plasmid pBY17.1 integration in gene virB4 22 mer (gttttcccagtgacgacg) and M13R ( agcggataacaatttcacac) are primer specific to the polylinker of pBY17.1. bes03(catcttggatcgtgcgtt) bes03DS, bes04 (catgttgctgagcatctt) and virB4 F(ggtaccacccatttgaaaacgtgtcc) are gene specific primers. Lanes 1,4,and 7, B69 bact erial cells were used as template source for the PCR. Lanes 2, 5 and 8, B18.12 (primary transformant with pBY17.1 inserted in virB4 ), bacterial cells were used as template source for the PCR. Lanes 3, 6 and 9, B18.5 (pri mary transformant with pBY17.1 inserted in virB4 ) bacterial cells were used as template source for the PCR. Primer combinations used in each lane are indicated in the figure. PCR bands resulting from using v irB4based primers in combination with suicide vector based primers are specific to an insertion in the targ eted region and should not appear when the wild type strain is used. The light blue box represents the homology region targeted for recombin ation, and is found duplicated after insertion of the suicide plasmid pBY1 7.1 in pXcB. FRT sites recognized the FLP recombinase are symbolized by yellow circles and flank the internal fragment with homology to virB4 cloned in pBY17.1. The green boxes symbolize DNA stretches carried over during sub-cloning steps.

PAGE 33

21 Lane 1: B69 Lane 2:B18.12 Lane 3:B18.12-1 123123123 BES03/BES04M13R/bes04virB4F/bes04 123 123123 123123 123 BES03/BES04M13R/bes04virB4F/bes04bes03/bes04: 470bp M13R/bes04: 1300 bp virB4 F/bes04: 950bp (wt), or 2500bp after FLP 800bp800bp 700bp bes03 virB4 F bes04 800bp800bp 700bp bes03 virB4 F bes04suicide vector inserted virB4 M13R bes03 bes04 virB4 F 22mer M13R bes03 bes04 virB4 F 22merB18.12-1 B18.12 Figure 1-9: PCR confirmation of Flp-medi ated eviction of pBY17.1. Genes specific primers (bes03, bes04, virB4F and vect or based primers were used in appropriate combinations. B69 and B18.12 (primary transconjugant with pBY17.1 inserted in virB4) were used as negative and positive control for the suicide vector integration respec tively. B18.1 2-1 is the secondary transformant resulting from suicid e vector eviction from primary transconjugant B18.12. Bacter ial cells from selection plates were used as template source for PCR. The expected size of each PCR band is indicated in the figure. The light blue box represen ts the homology region targeted for recombination, and is found duplicated af ter insertion of the suicide plasmid pBY17.1 in pXcB. FRT sites recognized the FLP recombinase are symbolized by yellow circles and flank the internal fragment with homology to virB4 cloned in pBY17.1. The green boxes symbolize DNA stretches carried over during sub-cloning steps.

PAGE 34

22 Figure 1-10: Pathogenicity phenotype of prim ary and secondary exconjugants disrupted in virB4 B18.12; primary exconjugant ( virB4 ::pBY17.1). B18.12-1 (secondary exconjugants, after eviction of pBY17.1) Picture on the left was taken 7 days post inoculation. The two pi ctures on the right were taken 15 days after inoculation. Note the dela y phenotype of primary transconjugant B18.12, and the total loss of pathoge nicity of transconjugant B18.12-1. B69 B18.12 B18.12-1 B69 B69 B18.12 B18.12-1 B18.12 B69 B18.12 B18.12-1 B69 B69 B18.12 B18.12-1 B18.12

PAGE 35

23 CHAPTER 2 IDENTIFICATION OF CITRUS GENES SPECIFICALLY RESPONSIVE TO PATHOGENICITY GENE pthB OF Xanthomonas citri pv. aurantifolii Introduction Many studies on plant-pathogen interacti ons have dealt with incompatible interactions using model plant systems (for example see Malek et al., 2000). Emphasis has been on dissecting signali ng pathways of resistance mechanisms, with few studies considering signaling pathways resulting in di seases of crop plants (Kazan et al., 2001). Therefore, the molecular events at the origin of disease induction by microbial effectors of pathogens remain obscure. Many Gram-negative, phytopathogenic bacter ia rely on a Type III secretion system (TTSS) to deliver effector pr oteins into the plant cells (H e et al., 2004). Inactivation of the TTSS of bacterial species th at utilize such a system results in loss of pathogenesis indicating that the proteins (named type III effectors) delivered by the TTSS are required for bacterial virulence (Rohmer et al., 2004). Most type III effectors identified to date were originally discovered and characterized by their avirulence f unction (Avr), while only few are recognized pathoge nicity factors [PthA from X. citri (Swarup at al., 1991), AvrB6 from X. campestris pv. malvacearum (Yang et al,. 1996), AvrXa7 from X. oryzae (Bai et al., 2000) and DspA from Erwinia amylovora (Gaudriault et al., 1997)]. A limited number of type III effectors have been assi gned proven or putative biochemical function (Collmer et al., 2000; Rohmer et al, 2000; Cha ng et al., 2004) and for a subset of these (principally avirulence effectors), a plant protein or cellu lar process has been identified as

PAGE 36

24 a possible target for pathoge nesis (Rohmer et al, 2004 and Chang et al. 2004). In two cases, a bacterial effector-triggered plant phe notype has been shown to be required for pathogenesis. In the case of pathogeni city factor DspA, a member of the P. syringae AvrE family, its induction of reactive oxygen species rele ase by the host cell has been shown to be required for successful colonizati on (Venisse et al., 2003) While in the case of pathogenicity factor PthA, a member of the Xanthomonas AvrBs3/PthA family, its induction of cell division and /or cell expans ion is required for pa thogenesis (Swarup et al., 1991) In this study the compatible interaction between citrus and Xanthomonas citri ( X. citri pv. aurantifolii syn X axonopodis pv. aurantifolii) was examined. Probably originating in Southeast Asia, citrus canker ha s now spread to most citrus producing areas of the world and causes severe economical losses (Civerolo, 1994). All canker strains induce similar disease phenotypes, including water soaked lesions, formation of large hyperplastic erumpent pustules (cankers) on a ll aerial plant parts, and rupture of the epidermis with accompanying cell death (Swa rup et al., 1991 and Duan et al., 1999). Specific members of the avrBs3/pthA gene family are required by strains of Xanthomonas citri to cause cankers on citrus (Swa rup and Gabriel, 1989; Swarup et al., 1990, Swarup et al., 1991). Members of the avrBs3 / pthA gene family are found in many xanthomonads (Gabriel, 1999; Vivian and Ar nold 2000), and all citrus canker strains examined carry multiple members of the gene family (Gabriel, 1999). All Xanthomonas avrBs3/pthA members described to date are 9097% identical in DNA sequence and are characterized by 1) a series of 12.5-25.5 almo st identical 34 amino acid repeats in the center of the protein that determines host specificity, pathogenici ty and/or avirulence

PAGE 37

25 phenotype (Herbers et al., 1992, Yang et al ., 1994, Zhu et al., 1998, and Yang et al., 2000), 2) three C-terminal nucl ear localization signals (Ya ng and Gabriel, 1995; Van den Ackerveken et al., 1996 and Sz urek et al., 2001) and 3) a Cterminal acidic region considered to function as an eukaryotic tr anscriptional activator (Zhu et al., 1998, Zhu et al., 1999, Yang et al., 2000, Szurek et al, 2001). Sequence and functional anal ysis of members of the avrBs3/pthA gene family showed that these proteins are type III effect ors, acting in the plant nucleus potentially as transcriptional regula tors (Yang and Gabriel, 1995, Zhu et al., 1998, Zhu et al., 1999, Yang et al., 2000, Szurek et al., 2001) When the pathogenicity gene pthA from X.citri was transiently expressed in susceptible plant cells (by Agrobacterium infection or particle bombardment delivery), it elic ited canker-like pustules, indicating that pthA alone was sufficient to trigger canker sy mptoms (Duan et al., 1999). Unlike pthA and its active homologues in other X. citri pv. citri and X. citri pv. aurantifolii strains, avrBs3 is not required for pathogenicity of X. c. pv. vesicatoria (Bonas, 1989). However, it was found to induce a subtle hypertrophy in the mesophyll of leaves inoc ulated with slow-growing strains of X. c vesicatoria, concomitant with the up-regulation of 13 plant genes (Marois et al., 2002). Taken together these results indi cate that members of this gene family are able to induce transcriptional reprogramming in both suscepti ble and resistant plant cells. In this study, the citrus canker system was used to probe the functions of pthB another member of the avrBs3/pthA gene family, that is isofunctional with pthA in eliciting host-specific symptoms. A comparativ e analysis of gene expression in citrus leaves inoculated with the wild type X. c aurantifolli strain B69 (carrying pthB ) and an Xca mutant derivative carrying a defective (marker interrupted) pthB was performed.

PAGE 38

26 Methodological approaches in this analys is included differential display-reverse transcriptase PCR (Liang and Pardee, 1992), suppressive subtrac tion hybridization, and microscopy. Forty-six clones of citrus canke r responsive genes belonging to several broad categories of cellular functions were identified as being sp ecifically regulated by pthB These categories included genes identified to be involved in cell wall loosening and growth, water homeostasis and vesicle traffi cking. In addition evidence is presented for the involvement of hormone signaling in canker disease development. Material and Methods Plant and Microbial Material Bacterial strains and plasmids used in th is study are listed in Appendix A. B69 and BIM2 were grown on PYGM (De Feyter et al. 1990) supplemented with 35 mg/L spectinomycin and 35 mg/L spectinomycin pl us 35 mg/L Chloramphenicol. All citrus plants ( Citrus paradisi ‘Duncan’, grapefruit) were gr own under greenhouse conditions. Plant inoculations involving all citrus canker strains were carried out under quarantine at the Division of Plant Industry, Florida Depart ment of Agriculture, Gainesville. Bacterial cells were harvested from log phase cultures by centrifugation (5,000xg, 10 min.), washed (1X) and resuspended in sterile ta p water or distilled water saturated with calcium carbonate to an OD600nm of 0.6-0.7, unless stated othe rwise. Inoculations were performed by pressure-infiltration into the abaxial leaf surface of the plants. Experimental inoculations were repeated at least three times. For differential display-reverse transc riptase PCR (DD-PCR) experiments and construction of the suppressive subtractive lib raries (SSH), inoculations were performed following a split leaf model. Strain B69 was inoculated on one side of the mid-vein; while BIM2 was inoculated on the opposite side of the mid-vein, in order to control for

PAGE 39

27 leaf to leaf variations. Tissue was harves ted 0, 2 or 7 days post inoculation (dpi) depending on the experiment. Bacterial Counts B69 and BIM2 bacterial cells were normalized to an OD600 of 0.7 and infiltrated as described previously. At 0 and 2 dpi, a tota l of 9 discs (0.28 mm in diameter) from 3 leaves (3 discs per leaves) were harveste d for each treatment and ground in 1ml of tap water. After serial dilution, the bacterial populations of wild type strain B69 and mutant strain BIM2 were counted. B acterial cell count determinati ons represent th e average of three replicate experiments. Microscopy Fresh, tender and half-expanded leaves were inoculated with a high inoculum of B69 or BIM2. At 0, 2, 7 and 14 dpi, leaf samples of an area of approximately 6 mm2 were harvested and fixed in 2% glutaraldehyde in phosphate buffer saline (PBS) for 48 hr at 4 C. They were then washed three times for 15 min each and fixed in 1% buffered osmium tetroxide overnight at 4 C. This was followed by one wash in PBS for 10 mi and by two washes in distilled water. A stepwise dehydr ation was conducted after these washes using ethanol (25%, 50%, 75%, 95% and 100%) for 10 min each step, followed by three washes in acetone for 15 min each. Samples were then infiltrated at room temperature in 30% acetone/EMbed (Electron microscopy scie nces, Pennsylvania) for 1 hr, followed by 50% acetone/EMbed for 1 hr and 70% acetone/EMbed for 2 hr. Samples were subsequently incubated in 100% EMbed overnig ht at room temperature to complete the infiltration and polymerized in fresh 100% EMbed in a 75 C oven overnight.

PAGE 40

28 Differential Display-Reverse Transcriptase PCR Two and seven days after inoculation, l eaf tissue was harvested, pooled and frozen in liquid nitrogen for total RNA extraction as described (Chang et al., 1993). Potential canker responsive (CCR) cDNAs were clone d as fragments by differential displayreverse transcriptase PCR (DD-PCR) of mRNA using 48 primer combin ations (Liang and Pardee, 1992) with the R NAimage kit from Genhunter (Nashville, TN, USA). Suppressive Subtractive Hybridizatio n (SSH) Library Construction For polyA mRNA isolation, leaves were fro zen in liquid nitrogen and stored at -80 C until extraction. PolyA mRNA was isolated from leaves using the FastTrack mRNA isolation kit (Invitrogen) according to the ma nufacturer’s protocol. SSH was constructed using a cDNA subtraction kit (Clontech PCR-Se lect, Palo Alto, CA). For construction of the forward subtraction library (FS), the te ster was chosen to be the pool of mRNA isolated from B69 inoculated l eaves at 2 dpi while the driver was chosen to be the pool of mRNA isolated from BIM2 inoculated leav es, and therefore, the FS was enriched in transcripts up-regulated by pthB For the reverse subtracti on library (RS), transcripts isolated from BIM2 inoculated leaves (2 dpi) were used as tester, and therefore, while the driver was chosen to be the pool of mRNA is olated from B69 inoculated leaves. the RS library was enriched in transcripts up-regulated in the absence of pthB Potential differentially regulated clon es were sent for sequencing to the Interdisciplinary Center for Biotechnology Research (ICBR) co re at the University of Florida. Putative functions were assigne d based on annotation derived by BLAST analysis.

PAGE 41

29 Northern Blots For RNA sample prepartion, NorthernMax Formaldehyde Load Dye was used as recommended by the manufacturer (Abion Austin, TX) with 5-10 g of RNA. Samples were loaded on a denaturing formaldehyde agarose gel (1%) and electrophoresis was conducted at 5 V/cm. RNA was blotted on Ge neScreen Plus hybridization transfer membrane (NemTM Life Science Products, MA) using 20X SSC as transfer buffer. Hybridization and washes were done as recommended by the manufacturer (Ultrahyb, Ambion Austin, TX). Probes were made w ith DECA primeTMII (random priming), (Ambion Austin, TX) as recommended. Reverse Northern Blots For reverse northern blots, cDNAs iden tified by DD-PCR or SSH were amplified using vector primers and purified using Qi aquick columns in plate format (Qiagen, Valencia CA). Membrane arrays were made essentially as described by Desprez et al., (1998). cDNAs were arrayed onto Hybond N+ membranes (Amersham Biosciences, Piscataway, NJ) using a 96-pin colony repli cator (V&P Scientific, San Diego CA). Six replicate arrays were generated and used to analyze transcript abunda nce of a subset of potential canker responsive genes or CCRs. Each cDNA was spotted in two locations, and several cDNAs were represented by more than one clone. Three replicate membranes for each treatment (B69 or BIM2 infection) we re used in hybridization experiments (total of six membranes or 3 pairs). Each memb rane was probed with radiolabelled cDNA synthesized from RNA isolated from one of three split leaf-experiments conducted, 2 dpi. Each membrane pair was one of three biol ogical replications. Signal intensities were statistically compared after normalization.

PAGE 42

30 For probe preparation, first strand cDNA probes were prepared from 10 g of total RNA by reverse transcription using MMLV-RT (Gibco-BRL, Gaithersburg MD) in the presence of 32P-dCTP. Unincorporated nucleotides were separated from first strand cDNA using Sephadex G-50 columns (Amers ham Pharmacia Biotech, Ithaca NY) and quantified using a liquid scinti llation counter (Beckman C oulter, Fullerton CA). Prehybridization, hybridization and low and high stringency washes were carried out at 65 C. Membranes were exposed to phosphorim ager screen for visualization. Spot intensities (called volumes) on the membrane arrays were quantified using a BioRad Molecular Imager FX run with the associ ated Quantity One software (Bio-Rad Laboratories, Inc. Hercules, CA). Data were imported into Microsoft Excel (Microsoft Corp., Redmond, WA, USA) for further analysis. Statistical Analysis A mixed model analysis (SAS Proc Mixed) was run on th e log base 2 transformed (normalization) local background adjusted volumes. cDNAs th at did not exhibit a mean value greater than 120 from either treatment we re not included in the analysis. The linear model used included replication (three biologi cal replications), trea tment (B69 treated or BIM2 treated) and gene (CCRs or Citrus Canker responsive clones). Least square means for the treatment by gene interaction were saved and used to form by-gene contrasts between treatments. Significance of these c ontrasts was controlled for an experimentwide alpha level.

PAGE 43

31 Results Macroscopic Disease Phenotype of Citrus Leaves Inoculated with X. c. aurantifolii B69 and Its Mutant Derivative BIM2 Lacking the Pathogenicity Gene pthB Xanthomonas strains B69 (wt) and its nonpathog enic mutant derivative BIM2, carrying a marker integration in gene pthB ( pthB ::pUFR004), were i noculated at high levels (OD = 0.7) on tender half-expanded leav es of new flushes of Duncan grapefruit and the corresponding induced disease phenotyp e analyzed. At day two post-inoculation, no symptoms were visible and no macroscopi c differences were observed among leaves inoculated with tap water, B69 or BIM2. By se ven days post-inoculati on, leaves that were mock inoculated showed no symp toms, while leaves inoculated with the wild type strain B69 showed a whitish canker phenotype, typical of South American canker disease. On the abaxial side of the leaf, the entire inocul ated area became raised, with a soft, velvetlike appearance, while a few individualized pustules appeared at the margins of inoculated areas. Pustules po ssibly corresponded to areas wher e bacteria were infiltrated at low density (Figure 2-1, A and B). On th e adaxial side of th e leaf, no raising was apparent; instead some yellowing develope d. This rapid symptom development is typically observed when a high inoculum is used on fresh, young expanding leaves. By contrast, at 7 dpi, no ma jor symptoms were visible on leaves inoculated with BIM2. Limited raising of the epidermis occu rred at the margins of some inoculation zones, with development of mi nimal pustule-like structures re miniscent of those seen in canker (Figure 2-1, C and D). These symptoms were not observed in mock-inoculated leaves. BIM2 inoculated leaves ultimately displayed attenuated canker phenotypes after thirty days (Figure 2-2). This is possibl y due to the week can ker-inducing activity of pthB0 the second pthA homologue found in the B strain, B69.

PAGE 44

32 PthB-Dependent Transcriptional Reprogr amming Induced upon Infection with Xca A small scale DD-PCR was conducted to co mpare transcript levels of leaves inoculated with B69 to those of leaves inoculated with BI M2 at two and seven dpi. To maximize the homogeneity and the intensity of the response, B69 and BIM2 were inoculated at high levels (OD600 of 0.7). In order to minimi ze leaf-to-leaf variation, a split-leaf inoculation strategy wa s used. An average of fifteen leaves (from three trees) were inoculated with B69 on one side of th e mid-vein and with BIM2 on the other side. Two and seven days after inoculation, ha lf-leaves were harvested, pooled into “B69 treated” or “BIM2 treated” samples and R NA extracted from both samples. Since B69 (carrying pthB and pthB0 ) differs from BIM2 (carrying pthB ::pUFR004 and pthB0 ) only by the presence of a single e ffector, PthB, differentially re gulated transcripts (named c itrus c anker r esponsive or CCRs) were PthB respons ive. Transcripts identified by DDPCR appeared differentially re gulated as early as two days post-inoculation despite a complete lack of symptoms. Twenty cDNAs were identified by DD PCR (Table 2-1), including six with homology to biotic or abio tic stress response genes (CCR20.2 to PR-1 proteins and CCR9.5, CCR15.1 to PR-5 prot eins, CCR2.2, CCR17.2 to peroxidases and CCR12.1 to catalases). One cDNA, CCR6.4 di splayed homology to cell wall remodeling enzymes of the cellulase family. CCR25.1 wa s homologous to the small ubiquitin like modifier SUMO. To remove the possibility that potential changes in transcript level were due to differences in the number of bacteria present in B69 inoculated leaves compared to BIM2 inoculated leaves, both bacter ial populations were monito red at 0 and 2 dpi. B69 and BIM2 bacterial populations were found to be comparable with almost no growth observed during the first two days post-inocula tion (Figure 2-3). Bacter ial growth at 2 dpi

PAGE 45

33 will occur if bacteria are inoculated at lower initial levels (OD600 of 0.3-0.4) (data not shown). However, when inoculated at lower levels, growth of BIM2 is very poor (data not shown and discussed later). Construction of Two Libraries Enriched in pthB Responsive cDNAs Following the same split leaf scheme as for the DD-PCR experiment, forward and reverse libraries were constructed by suppres sive subtraction hybridiz ation (see Figure 24 for illustration of the methodology), extend ing the collection of putative CCRs. The forward subtraction library ( FS) was constructed to be enriched in transcripts upregulated by PthB while the re verse subtraction library was co nstructed to be enriched in transcripts up-regulated in the absence of Pt hB (see Materials and Methods for design of the SSH). Approximately 500 clones were sequenced and annotated using homology based searches. Figure 2-5 illustra tes the distribution of CCRs for each of the FS and RS libraries according to their putative function. Categories representing genes of unknown function (8 %) and genes involved in cell gr owth and division (10%) were found more frequently in the forward library (up-regulated in the presence of pthB ) compared to the reverse library (1 % and 2 % respectively) while genes in the category representing abiotic and biotic stress responses were found mo re frequently in the reverse library (15% vs 6% in the FS) Transcript Analyses of CCRs cDNAs from each of the forward (131) a nd reverse (161) libra ries, as well as 20 clones identified by DD PCR (total of 312 cDNAs ) (Figure 2-6) were chosen for reverse northern-blot analysis. CCRs homologous to genes of known function were preferentially selected. Six replicate arrays were generated as described in materials and methods, and

PAGE 46

34 used to analyze transcript abundance of a subset of potential CCRs. Three membranes per treatment were probed with radiolabelled cDNA synthesized from RNA isolated from three split leaf-experiments, 2dpi with B69 and BIM2 (three biological replicates). For each experiment, inoculated leaves were samp led from new and older flushes, were half to fully expanded and were all tender (min imal cuticle). Signal intensities were statistically compared after normalization as described in material and methods. Forty-six clones were identified as diffe rentially regulated at (p < 0.05 ) (Figure 2-7). Only fifteen out of forty-six clones were found up-regul ated in the absence of PthB, while the remaining thirty-two were found up-regulated by PthB. Ratios of transcript abundance were calculated for each cDNA. Ratios range d from -3.5 to +34.5 (sign indicating overexpression of the gene in the absence of PthB and + sign indicating an up-regulation in the presence of PthB) (Table 2-2). Identity of cDNAs Identified as Up-Regulated by the Presence of pthB in X. citri Genome Of the forty-six clones identified as di fferentially regulated, all but four clones showed significant (e-value >2e-03) matches with sequences in available databases (Table 2-2). Thirty CCRs out of forty-six were found up-regulated by the presence of pthB in the bacterial genome i.e up-regulated in B69 infected leav es compared to BIM2 infected leaves. These are listed in Table 2-2. Cell growth. Twelve clones were highly similar to genes involved in cell growth (cell wall loosening and expansion): CCR 339 was similar to cellulases; CCR1511, CCR113 were similar to expansins; CCR 889 was similar to mannanendo-1,4-beta mannosidases; CCR571 and CCR1453 were sim ilar to pectate lyases and CCR313 was similar to tonoplast aquaporin s (TIP3). Another clone of interest, CCR575, had homology

PAGE 47

35 to the early nodulin gene Enod8 (predicted cel l wall localized estera se). An additional gene represented by CCR 109, CCR959 and CC R501 had homology to a secreted cellwall-associated pollen-specific allerg en of the ole e 1 family (SAH7). Giberellic acid pathway Two CCRs had homology to the GAST1 (GA responsive genes of unknown function) family of genes. Vesicle trafficking Several clones had homology to proteins involved in vesicle trafficking. For example, CCR673 had homology to a small GTPase of the Rab family (RAB8B, Vernoud et al. 2003), and CCR1258 had homology to the beta COP protein of the COPI complex. Unknown function Another eight clones found up-regulated had either no significant homology to any sequences in av ailable databases or had sequence homology to genes of unknown function. Identity of cDNAs Identified as Up-Regulated by X citri Lacking pthB Sixteen CCRs out of forty-six were found up-regulated by X. citri lacking pthB i.e up-regulated in BIM2 infected leaves as compared to B69 inoculated leaves. These are listed in Table 2-2. Cell growth CCR243, was the only BIM2 up-regul ated gene involved in cell wall metabolism. CCR243 is homologous to ca ffeic acid methyl transferases and is involved in phenylpropanoid metabolism. GA pathway CCR 237 was homologous to cytP450 ent-keuren oxidase and CCR105 was homologous to another cytP450 (p ossibly ent-kautenoi c acid oxidase). Protein modification and stability For example, CCR409 had homology to RD21a, a drought responsive cysteine pr oteinase, and CCR915 had homology to the small ubiquitin modifiers (SUMO).

PAGE 48

36 Transport. CCR1339 and CCR1435 were homologous to a mitochondrial import inner membrane translocase and a monos accharide-H+ symporter, respectively. Unknown function Another four clones found-up regulated in BIM2 infected leaves had either no significant homology to a ny sequences in availa ble databases or had sequence homology to genes of unknown function. Northern Blot Analysis of Representative CCRs Expression of several candidate CCR ge nes identified by reverse northern blot analysis was evaluated by northern blot analys is. Leaf tissue from split-leaf inoculations using B69 and BIM2 were harvested and pr ocessed for RNA extraction. Several labeled cDNA fragments were used to probe RNA blots (Figure 2-8). As in reverse northern blot analysis, clones corresponding to expans in, cellulase, SAH7/LAT52, GAST1, Enod8 and pectate lyase showed high levels of induction. Microscopic Phenotype of B69 and BIM2 Inoculated Leaves In order to characterize the microscopi c phenotype of B69 and BIM2 infected leaves, leaf discs mock inoculated and inf ected with BIM2 or B69 were harvested and processed for light microscopy analysis (Fig ure 2-9, 2-10, 2-11 and 2-12). Leaves were pooled as fast-responding to canker when di sease symptoms were fully developed by seven dpi (see figure 2-1, A and B). Leaves were pooled as slow-responding to canker when disease symptoms were fu lly developed by 12 to 14 dpi. Slow-responding leaves At 2 dpi B69, BIM2 and mock inoculated leaves looked identical at both the macroscopic and the mi croscopic level. At 7 dpi, while mock and BIM2 inoculated leaves showed no phenotypic signs at both the microscopic or macroscopic level (data not shown and Figure 2-9, A), the first signs of canker became visible on B69 inoculated leaves, i.e regions of darker green color around the veins and

PAGE 49

37 slight swelling. At the microsco pic level, B69 leaves showed high levels of cell division occurring across all the inoc ulated area (Figure 2-9, compare B, Cto A). Intense cell expansion and cell division phase resulted in complete filling of the air spaces of the spongy mesophyll in B69 infected leaves (Figure 2-9, compare B, C to A). The number of mesophyll cells from the abax ial to the adaxial epidermis more than doubled compared to the day 0 control or day 7 BIM2 inoculated leaves, while some cells almost tripled in size (Figure 2-10, compare A, B and D to C, a nd Figure 2-12). At later stages (14 dpi), increased raising of the epidermis and whitish coloration with soft or velvety appearance were observed at the macroscopic level. Th ese phenotypes coincided with a phase of increased cell expansion (dat a not shown and Figure 2-11, co mpare A, B to C and D). While areas of cell division were still visible, a significant su bset of cells became much larger and the leaf dramatically thickened (t wice that of the contro l leaf, see Figure 2-12). A critical preliminary conclusion from these an alyses indicated that the earliest visible canker phenotype was mainly due to cell divi sion, with a moderate cell expansion, while late onset phenotypes were due to scattered but dramatic increases in cell expansion. Fast-responding leaves Macroscopic analysis indicated that cell expansion was the primary phenotype with very little cell di vision occurring (Figure 2-13, compare B, C and D to A). Furthermore, several areas of ce ll lysis, were visible immediately under the abaxial epidermis. Bacterial growth in B69 and BIM2 infected leaves Canker visible symptoms (cell division, cell expansion and resulting cell death) appeared necessary for B69 growth as very few bacteria were vi sible in BIM2 infected tissue 14 dpi while numerous pockets

PAGE 50

38 of bacteria were seen in B 69 infected tissue (Figure 2-11, compare B to C and D and data not shown). Discussion In this study, we have used macroscopi c and microscopic phenotypic analysis in combination with targeted gene discovery techniques to understand how the pathogenicity factor pthB of X. c. aurantifolii belonging to the avrBs3/pthA gene family elicits host-specific citrus canker symptoms in a compatible plant microbe interaction. The nonpathogenic mutant BIM2, lacking pthB was used in combination with the wild type strain, Xca B69, to study th e specific effects of PthB on the plant cell transcriptome. A split-leaf inoculation experimental design wa s used to minimize leaf-to leaf variations in gene expression. In addition, bacterial cells were inoculated with high inoculum to: (1) ensure near saturation of infection sites, (2) maximize the synchronicity of the host response, and (3) artificially no rmalize the levels of bacter ial populations (wild type and mutant) present during early infec tion stages of the plant leaves (up to 2dpi). In order to obtain a collection of gene s potentially responsive to PthB, two complementary techniques, DD-PCR, and forward and revers e SSH, were used to enrich for: (1) transcript up-regulated when PthB is secreted in plant cells by X citri and (2) those upregulated in the absence of a functional PthB. Transcript analysis of a subset of 312 clones was conducted using reverse northern blot technique. Statistical analysis was used to identify a list of forty-ni ne PthB responsive genes and differential regulation for a subset of these was verified by northern blot analysis. Northern blot analysis was also conducted on several CCR that did not show differential regulation by reverse northern blot analysis (Appendix B). Several of th ese showed differential regulation when northern analysis was used suggesting a bett er sensitivity than with reverse northern

PAGE 51

39 analysis. This implies that the subtracti on libraries contain additional CCR that need identification. PthB Induces Cell Division and Cell Expansion in Citrus Leaves When inoculated on citrus leaves, Xanthomonas citri pv. aurantifolii was able to cause cell division and cell expansion, c onsistent with previous reports on pthAinduced phenotypes (Duan et al. 1999). Quantification of the three visible phenotypes of canker i.e cell division, cell expansion and the resulti ng thickening of the leaves was difficult due to (1) the heterogene ity of the cells in the spongy mesophyll and (2) the heterogeneity in distribution of the abundant air filled spaces in citrus leaf tissue. Therefore, as first approximation of the phenotype, quantification measurements were performed on areas where cellular activity wa s the most dramatic (areas of intense cell expansion, cell cycle activity and thicker leaf areas). Analysis of PthB induced symptoms over time revealed that the earliest visibl e phenotype associated with canker was cell division in the infected spongy mesophyll, whereas heterogeneous but massive cell expansion was observed at later stages of the infection. Interestingly, when canker developed rapidly, i.e advanced canker symptoms at 7 dpi versus 12 to 14 dpi, symptoms of cell division were found to be reduced co mpared to slower developing canker. In addition, cell expansion was the major phenot ype, primarily affecting mesophyll cells directly under the abax ial epidermis layer. This supports the hypothesis that the primary cellular mechanism affected by PthB alterati on of the plant cell tr anscriptome is the integrity of the cell wall and the induction of cell expansion. In turn, cell division could either be: (1) a consequence of modifi cation associated with cell expansion ( e.g. changes in cell volume) and (2) due to a second and dist inct effect of PthB. However, because cell expansion constituted a major phenotype in both rapid and slow developing canker,

PAGE 52

40 induction of cell expansion may be the primary consequence of pthB functions in the plant cell. Furthermore, a specific set of genes with homology to genes involved in cell growth were identified as responsive to PthB. PthB Induces the Expression of Cell Wall Remodeling Enzymes In order to understand PthB-induced phenotypes on citrus leaves, we have identified a set of forty-six genes (CCRs) sp ecifically regulated by the presence of this effector in the plant cell. Consistent w ith the PthB-induced morphological phenotypes, several CCRs were homologous to genes invol ved in plant cell wall modifications. Expansins Among these PthB-up-regulated plan t genes, two were homologous to -expansins. The role of the expansin gene fa mily in wall loosening (polymer creep) and cell expansion has been widely docum ented (Cosgrove, 2000). Expansins are extracellular proteins that facilitate ce ll wall expansion probably by altering hydrogen bonds between hemicellulosic wall components and cellulose microfibrils (Coscrove, 1998). These can act alone to induce cell wall extension in vitro however, in vivo they act with a suite of enzymes capable of rest ructuring the plant ce ll wall (Cosgrove, 1998). Consistent with this, several CCRs homol ogous to genes associated with cell wall remodeling were also identified. Pectate lyases Among CCRs associated with cell wall remodeling, CCR571 and CCR1453 were similar to pectate lyases (PLs). These enzymes are involved in hydrolysis of wall polymers, via cleavage of de-esterfi ed pectin, thereby fac ilitating cell expansion (Carpita and Gibeaut, 1993 and Domingo et al., 1998). Although the role of bacterial secreted PLs in cell wall degradation is we ll known (Collmer and Keen, 1998), the role of endogenous plant PLs in development has not been extensively examined. In pollen,

PAGE 53

41 plant PLs are thought to initiate the looseni ng of the cell wall enabling the emergence and growth of the pollen tube (Cosgrove et al ., 1997). PLs also medi ate cell wall breakdown in the style’s transmitting tissue, allowing pe netration of the pollen (Taniguchi et al., 1995, Wu et al., 1996). Thus, induction of plant PLs by PthB can help account for aspects of the disease phenotype. Cellulases Another PthB up-regulated CCR wa s homologous to the cellulase family, another class of ce ll wall remodeling enzymes. Cellulases catalyze the cleavage of internal 1,4 linkages of cellulose and are involved in seve ral aspects of plant development involving cell wall modifications including abscission, fruit softening and cell expansion (Lewis and Koehler, 1979, a nd Fisher and Bennet, 1991). Relevant to PthB induced phenotypes, it has been shown that constitutive expression of a poplar cellulase in A. thaliana led to a significant increase in cell size (Park et al., 2003). Beta-endo-mannanase In addition to CCRs homologous to expansins, PL, and cellulases, a fourth type of cell wall remodeling enzyme, a mannan endo-1,4D mannosidase (endo-beta-mannanase) was also identified as up-regulated by PthB. This enzyme catalyzes the hydrolysis of 1-4D mannosidic linkages in mannans, galactomannans, glucomannans and gala ctoglucoomannans (Matheson and McCleary, 1985 and Matheson, 1986) and has been implicat ed in cell wall weak ening during anther and pollen development (Filichkin et al., 2004) and in seed ripening where it is involved in mobilization of the mannan-containing cell walls of the tomato seed endosperm (Mo and Bewley, 2003). Caffeic acid methyl transferase Only one gene involved in cell wall metabolism was down regulated by PthB, CCR243. This clone was homologous a caffeic acid methyl

PAGE 54

42 transferase (COMT), belonging to the phenyl propanoid pathway that leads to lignin biosynthesis. Its expression has been shown to be regulated by biotic and abiotic elicitors including infection by avirulent and virulent bacteria (Toquin et al., 2003). It is possible that down-regulation of this enzyme relates to down-regulation of defense responses by down-regulation of lignin deposition. This ev ent could occur due to alterations in the lignin content or composition. COMT down -regulation is in acco rd with the cell expansion induced by PthB since mature wa lls lack acid-induced extension (Cosgrove, 1989). It is also interesting that fully expanded mature leaves are more resistant to canker, whereas young leaves (one half to two-third expanded) are the most sensitive ones (Graham et al., 2004). This is consistent w ith the hypothesis that PthB targets the cell wall, inducing cell expansion ultimately resulting in di sease progression. A synthesis of our results i ndicates that type III effe ctor PthB triggers the upregulation of an array of proteins whose combined activities induce cell wall loosening and cell expansion. The roles of expansins, PL s and cellulases in ce ll wall loosening have been shown to be complementary in othe r systems (Cosgrove et al.,1998; Carpita and Gibeault; 1993, Domingo et al ., 1998, Inouhe and Nevins, 1991). Enod8 and SAH7/LAT52 are a Link Betw een Canker Symptoms Development and Nodule Organogenesis and Pollen Tube Growth Respectively Two additional classes of CCRs (C CR575 and CCR109, 959 and 501) identified as up-regulated by PthB also s upport the theory that this effector targ ets cellular growth. The first one, CCR575, was homologous to Enod8 an early nodulin gene associated with the development of rhizobial nodule structures prior to nitrogen-fixat ion (Dickstein et al., 1988, 1993). Enod8 has sequence similarity to ex opolygalacturonase and lanatoside 15’O-acetylesterase (Pringle and Dickstein, 2003) Intriguingly, the up-regulation of Enod8

PAGE 55

43 in response to X. citri and Rhizobium suggests some common steps between nodule formation and canker pustule formation. This is also supported by the fact that both infections trigger cellular reprogramming events that lead to cellular growth. The function of Enod8 is unknown, but in-vitro characterization and se quence analysis predict that it is a cell wall localized esterase with acetylated ol igoor polysaccharides as substrates (Pringle and Dickstein, 2004). T hus the enzymatic activity of Enod8, its cell wall localization and involvement in both nodul e and canker pustule formation point to its involvement in modification of cell wall components during cellular growth. The second class of CCRs reinforcing the hypot hesis that the cell wall is the target of PthB, displayed homology to SAH7 and LAT52 genes encoding for members of the ole e I family of proteins. Originally identified as pollen allergens, members of this family have also been found expre ssed in other tissues (e.g. SAH7 in leaves). A recent study of one homologue LAT52 (tomato), indicates that these genes may be involved in controlling hydration and pollen tu be growth (Tang et al., 2002). LAT52 interaction with the pollen receptor kinase LePRK2 (LRR kinase ) led to the hypothesi s that binding of LAT52 initiates a signal transduction pathwa y required for pollen germination and pollen tube growth (Tang et al., 2002 and Johnson and Preuss, 2003). The up-regulation of a LAT52like gene in canker might, therefore, be part of a signali ng pathway leading to cell growth (the phenotype of both canker and pollen tube). Interestingly, pollen tube growth, which occurs by tip extension, i nvolves expansion and deposition of cell wall precursors at the growing tip and requires th e concerted action of endo-beta-mannanase, expansins and pectate lyases (Marin -Rodriguez et al., 2002, Cosgrove, 1998 and Filichkin et al., 2004), also found up-regulat ed during canker symptoms development.

PAGE 56

44 PthB Induces Up-Regulation of a Tonoplast Aquaporin CCR313, identified as up-regulated by PthB, displayed sequence similarity to a tonoplast aquaporin of the TIPs family (Maurel, 1997 and 2002, and Hill et al., 2004). Besides cell wall loosening, expansion requires extensive solute and water uptake resulting in the formation of a prominent vacuolar compartment. This maintains the turgor pressure that drives cell expansion (Veytsman and Cosgrove, 1998). Expansion is thought to require high hydrolic permeability of the tonoplast in order to support water entry into the vacuole, and tonopl ast aquaporins (TIPs) play a cr itical role in this process (Ludevid et al., 1992; Chaumont et al., 1998) TIPs are enriched in zones of cell expansion (Tyerman et al. 2002) as well as in zones of active cell division where their upregulation is linked to vacuol e biogenesis (Marty, 1997). Whet her the identified tonoplast aquaporin is indeed a marker for cell divisi on and/or is actively i nvolved in driving the cellular expansion is unknown. PthB Induces Up-Regulation of Two Compon ents Involved in Vesicle Trafficking Cell expansion and cell division both re quire deposition of new wall components into the extending cell walls (Veytsman and Cosgrove, 1998) or into the cell plate of dividing cells (Staehlin and He pler, 1996 and Samuels et al 1995). This may be achieved via secretory processes involving vesicle traffi cking. However, most genes identified here suggest that in response to canker, cell walls are mainly extended w ithout the building of new cell wall components. This would imply th at walls become thinner as cells expand. This indeed has been observed at late stag es of canker (Figure 2-10 compare A, B to C and D). Although plant cell walls generally appear not to become thinner as they extend (Veytsman and Cosgrove, 1998), expansion with out new cell wall deposition could be at the origin of the cell lysis obser ved in advanced canker stages.

PAGE 57

45 Several CCRs identified as up-regulated by PthB are involved in vesicle trafficking. Among these, CCR673 and CCR12 58 have homology to RAB8B and beta COP respectively. RAB8B is a member of th e small GTPase gene family. The yeast homologue of Rab8 (also named RABE see Vernoud et al., 2003) regulates membrane trafficking to the daughter cell bud site (Salminen and Novick, 1987 and Goud et al., 1988). Interestingly, in tomato, members of this subfamily appear to be targeted by the Pseudomonas avirulence factor, AvrPto. This implie s that in susceptible plants, AvrPto may interfere with membrane trafficking pathways (Bogdanove and Martin, 2000). It has been suggested that RAB8B might be involve d in polarized secre tion of antimicrobial compounds (Bogdanove and Martin, 2000). In mammals, beta COP belongs to a larg e complex that coats COPI vesicles (Kreis et al. 1995). COPI vesicl es transport membrane proteins and soluble molecules in a retrograde, and possibly anterograde, direction through mammalian Golgi stacks (Nickel and Wieland, 1997 and Harter, 1999). In plants little is known about COPI vesicles. Recent evidence suggests that CO PI-like vesicles are functional in plant secretion and localize mainly to the Golgi appara tus as well as to the cell plate of dividing cells (Couchy et al, 2003). Hormone Pathways are Possibly Involved in Canker Symptoms Development Triggering of cell expansion as well as induction of expansins and pectate lyases constitutes a common point between the effect of pthB on citrus (this study) and that of the avirulence effector avrBs3 on susceptible pepper plants (Marois et al., 2002). Cell expansion induction by both effectors share similar features; however, several plant auxin-induced proteins of the SAUR family were found up-regulated by avrBs3 (Marois et al. 2002). Several clones identified as pthB responsive are regulated by auxin in other

PAGE 58

46 systems. These include the expansins (Cat ala et al., 2000, Civello et al., 1999, Hutchison et al., 1999) and the pectate ly ases (Domingo et al., 1998). In the pepper model, one of two identified -expansins was found up-regulated by exogenous application of auxin; whereas a second expansin as well as a p ectate lyase were not (Marois et al., 2002). These data suggest that an auxin-indepe ndent pathway might ope rate under certain conditions leading to cell expansion. In addi tion to a possible role of auxin in canker disease, there is evidence for the involvement of the gibberellic acid signaling pathway in the plant response to pthB CCRs with homology to entkaurenoic acid oxidase and possibly to ent-kaurene oxidase (KO) (of GA biosynthetic pathway) (Oszewski et al., 2002) and two clones with homology to the GAST1 family (GA induced genes) were identified. Interestingly the GAST1 homologues were up-regulated by pthB ; whereas the putative KO and KAO were down-regulated. This may be explained by feed-back regulation of KAO and KO expr ession by GA. However, feedback regulation of several enzymes of the GAs biosynthetic pathway has b een described in other systems, it has not been reported to occur in the case of KAO and KO (Olszewski et al., 2002). GA is known to regulate TIPs (Phill ips and Huttly, 1994, Ozga et al., 2002), expansins (Oka et al., 2001, Vogler et al., 2003, Lee and Kende, 2002, Chen and Bradford, 2000), GAST1-like genes (Kotilain en et al., 1999 and Aubert et al., 1998), endo-beta-mannanase (Dutta et al., 1997, Yamagu chi e al., 2001) and cellulases (Litts et al., 1990). Therefore, PthB may act on regulatory steps upstr eam of GA biosynthesis. The involvement of GA does not preclude that auxin is also involved since the latter is able to regulate the production of the bioactive GA1 in elongating shoots (Ross et al., 2000).

PAGE 59

47 Indeed, these two hormones are known to, in concert, promote cellular division and elongation (Cleland, 2001 and Davies, 1995). Conclusions and Future Prospects The tight relationship between cell divi sion and cell expansion makes it difficult to address the question of whether cell expansion or cel l division are the cellular pathways that are altered as a downstream c onsequence of PthB regulating the plant cell transcriptome. However, the following resu lts presented here s upport the hypothesis that cell wall loosening and expansion is the major plant cellular mechanism targeted by PthB: 1) cell expansion occurs whether canke r symptoms develop ra pidly or slowly, 2) genes involved in cell expansion have been identified as responsive to PthB, 3) cell expansion is triggered by one another member of the avrBs3/pthA gene family and 4) PthB responsive genes are involved in cell growth. Microscopic analysis of leaves show ing a slow canker symptom development indicated that cell division is the major visibl e phenotype in initial infection stages, while cell expansion remains at a moderate level. Du ring the late infection stage however, cells dramatically expanded leading to areas of cell lysis. It is possible that PthB induces cell expansion and cell division by targeting seve ral distinct cellular mechanisms. Another hypothesis is that PthB targets cell e xpansion by altering cell wall composition (loosening). This in-turn lead s to a cell autonomous respons e that mainly involves the triggering of cell division in the early st ages and massive cell wall loosening and expansion in later stages. The concentration of bacteria su rrounding infected cells and, therefore, the concentration of PthB protein secreted into the plant cells as well as the physiological state of the infected tissue (for example immature expanding leaves will readily expand) would modulate this response. When the conc entration of PthB is low,

PAGE 60

48 moderate expansion and the subsequent cha nge in cell volume would lead to cell division, while in later stages, elevated concentrations of Pt hB would lead to gross cell expansion and cell ly sis (Figure 2-14). The relationship between cell expansion a nd cell division in plant growth and development remains controversial. Whether gr owth starts by an increase in cell size, triggering division, or whether division occurs first followed by restorat ion of the original cell size (Foard, 1971 and Cleland, 2001) is mainly unknown. Studies on leaf primordial (LP) initiation may begin to resolve this issu e. Initially, since the first visible sign of a new LP is a periclinal division in the L1 or L2 layer of the shoot apical meristem, it was suggested that division occurs first (Steev es and Sussex, 1989). However, recent evidence indicates that cell enlargement is the first step in LP initiation since LPs can be induced by adding expansins either by microinjection of by up-regulation of e xpansin transcripts at the shoot apical meristem (Pien et al ., 2001, Fleming et al. 1997). Canker could follow a similar pattern where cells expand first a nd then divide in response to expansion. The canker phenotype is necessary for optimal growth and dispersal of X. citri (Swarup et al., 1991 and this study); therefore, induction of cell division and or expansion are key steps in canker disease development and, unlike AvrBs3 for Xcv PthA/B confer a benefit to X. citri strains carrying it The PTHA/B family of pa thogenicity effectors may prove to be a valuable tool in dissec ting the molecular events surrounding microbeinduced diseases since they are require d for pathogenesis and can induce canker symptoms alone. Finally, an understanding of the mechanisms by which PthB induces canker phenotypes could help unravel the intr icate relationship between cell division and cell expansion that occurs in plant development.

PAGE 61

49 Table 2-1: List of putative CCR identified by DD-PCR. CCR Homology e-value CCR23.2 Unknown protein [ A. thaliana] (NP_196103.1) 2e-37 CCR24.5 Putative protein [ A.thaliana ] (NP_195874.1) 3e-24 CCR1.1 Putative Transposase [ A. thaliana ] (NP_189803.1] 4e-33 CCR22.5 3-hydroxyisobutyryl-coA hydrolase [ A. thaliana ] (NP_193072.1) 1e-22 CCR27.1 Cytochrome P450 [soybean] (T05942) 5e-41 CCR11.4 Cytochrome f [ Nicotiana tabacum ] (NP_054512.1) 4e-53 CCR6.2 Phosphoribosyl pyrophosphate synthase [ Spinacia oleracea ] (CAB43599.1) 6e-25 CCR28.2 Putative mitochondri al carrier protein [ A. thaliana ] (NP_181124.1) 4e-34 CCR7.6 Copper Transport Protein [ A. thaliana ] (NP_200711.1) 4e-33 CCR8.2 Receptor-like protei n kinase-like (LRR) [ A.thaliana ] 6.8e-45 CCR6.4 Cellulase [ sweet orange ] (eC3.2.1.4) 1e-23 CCR28.4 Peroxidase [ A. thaliana ] (CAA66035.1) 2e-50 CCR12.1 Catalase [ Campylobacter jejuni ] (Q59296) 2e-10 CCR2.2 Bacterial-induced peroxidase [ Goss hirsutum ] (AF155124) 3e-26 CCR17.2 Peroxidase [ Nicotiana tabacum ] (BAA82306.1) 6e-63 CCR20.2 Pathogenicity-related protein 1a [barley] (AF245497) 2e-43 CCR15.1 Osmotin-like protein [ Fagus sylvatica ] (AJ298303) 2e-17 CCR9.5 Osmotin -like protein [ Fragaria x ananassa ] (AF1999508) 3e-61 CCR21.1 Auxin induced protein, putative [ A. thaliana ] (NP_176274.1) 1. 9e-3 CCR25.1 Ubiquitin-like protein [ A.thaliana ] (NP_194414.1) 4e-28

PAGE 62

50 Table 2-2: List of CCRs confirmed by reverse northern blot analysis. CCR Homology e value Ratio* ( 0.05) Ribosomal protein 1385 30S ribosomal protein S20 ( A. thaliana ) gi21592469 1e-28 -3.36 Unknown function 1065 EST ( O. sativa ) gi50919279 4e-11 4.79 1243 EST ( A. thaliana ) gi42569501 2e-40 -3.2 497 Putative protein ( O. sativa ) gi50919279 4e-27 4.89 767 Putative protein ( A. thaliana ) gi15241855 3e-24 -2.41 1111 No significant homology 2.95 171 No significant homology 3.60 137 No significant homology 3.09 809 No significant homology -3.14 1139 Ring Finger Protein ( A. thaliana ) gi26450511 5e-07 3.86 1061 Splicing factor RSZp22 ( A. thaliana ) gi21554419 2e-03 3.16 475 Zinc finger protein ( A. thaliana ) gi28416541 1e-07 -2.55 1312 LRR receptor kinase ( A. thaliana ) gi42562316 1e-43 3.25 Metabolism/energy 539 CytoF ( N. tabacum ) gi11465970 4e-53 -3.05 1415 RubisCO activase ( malus x domestica ) gi415852 9e-58 -2.60 1239 F1F0 ATPase inhibitor protein ( O. sativa ) gi 52077175 4e-10 -2.91 1057 Hydroxymet hyltransferase ( A. thaliana ) gi21593312 4e-73 3.53 901 UMP-kinase ( A. thaliana ) gi2497486 3e-38 2.64 1445 UMP-kinase ( A. thaliana ) gi2497486 1e-10 6.92 33 UDP-galactose epimerase ( A. thaliana ) gi9758701 4e-21 3.58 Transport 1339 Mitochondrial import inner membrane translocase S.U gi42568553 1e-19 -2.62 343 Copper T protein ( A. thaliana ) gi15237802 4e-33 4.92 1435 Monosaccharide-H+ symporter ( D. glomerata ) gi30349804 2e-13 -3.36 Protein modification/stability 915 Small ubiquitin-like modifier ( A. thaliana ) gi15236885 4e-28 -2.53 1262 Protease inhibitor/seed storage//LTP ( A. thaliana ) gi42567284 6e-04 5.35 1345 Aminopeptidase ( A. thaliana ) gi34098848 2e-21 3.09 409 Putative cysteine proteinase RD21A ( A. thaliana ) gi22136972 5e-32 -2.99 GA pathway 1535 GAST1-like protein ( A. thaliana ) gi25406361 3e-10 11.00 493 GAST1-like protein ( A. thaliana ) gi25406361 3e-34 6.10 237 Cyt. P450 ent-keuren oxydase ( Malus x domestica ) gi45551401 7e-44 -3.2 1051 Cyt P450 (possibly ent-kaurenoic acid oxidase) ( P. sativum ) gi27776451 1e-23 -2.46 Vesicle trafficking 673 RAB 8B ( Lotus corniculatus ) gi1370192 1e-28 6.68 1258 beta COP protein ( O. sativa ) gi50900798 7e-20 2.38 279 Phosphatase (put. membrane trafficking factor) ( A. thaliana ) gi21553471 3e-19 -2.51

PAGE 63

51 Table 2-2. Continued CCR Homology e value Ratio ( 0.05) Cell growth (cell wall metabolism and expansion) 889 Mannan endo 1,4 beta mannosidase ( O. sativa ) gi34912090 4e-15 3.53 113 Alpha expansin ( P. cerasus ) gi13898655 4e-49 4.06 1511 Alpha expansin ( P. cerasus ) gi13898655 4e-51 4.26 571 Pecate lyase ( malus x domestica ) gi 34980263 1e-54 7.89 1453 Pectate lyase ( A. thaliana ) gi21593312 12-15 4.69 243 Caffeic acid O-methyl transferase ( C. roseus ) gi 18025321 6e-59 -2.39 339 Cellulase (sweet orange) gi7488904 1e-23 34.53 575 Enod8 (early nodulin 8 like) ( A. thaliana ) gi26451820 3e-07 4.59 313 Tonoplast aquaporin gamma TIP (TIP3) ( A. thaliana ) gi3688799 5e-13 4.82 109 SAH7/LAT52 (ole e I allergen family) ( L. esculentum ) gi 295812 7e-17 3.78 959 SAH7/LAT52 (ole e I allergen family) ( L. esculentum) gi 295812 5e-11 3.73 501 SAH7/LAT52 (ole e I allergen family) ( L. esculentum ) gi 295812 9e-21 2.75 *: A positive ratio indicates up-regulation in B69 infected tissue compared to BIM2 infected tissue. A negative ration indicat es up-regulation in BI M2 infected tissue compared to B69 infected.tissue.

PAGE 64

52 Figure 2-1: Phenotype of B69 and BIM2 in fections on grapefruit leaves. BIM2 lacks PthB and induces formation of very sm all pustule like structures, reminiscent of canker pustules, at the edges of some inoculated areas. (A), (C) are BIM2 (pUFR004:: pthB ) inoculations on grapefruit leav es and (B), (D) are wt B69 inoculations. Pictures were taken 7 days post inoculation. AB C D AB C D AB C D

PAGE 65

53 Figure 2-2: Late B69 and BIM2 phenotypes. (A ) BIM2 inoculated l eaves 30 dpi and (B) B69 inoculated leaves 30 dpi. Note th e much attenuated phenotype of BIM2 infected leaves. B A B A

PAGE 66

54 Figure 2-3: Quantification of bacterial population two days post inoculation with B69 and BIM2. (cfu: colony forming unit), Exp1: experiment 1, Exp2: experiment 2). 1.E+00 1.E+02 1.E+04 1.E+060dpi2dpi0dpi2dpi0dpi2dpi0dpi2dpi B69B69BIM2BIM2 Exp1Exp2Exp1Exp2 cfu/cm2 1.E+00 1.E+02 1.E+04 1.E+060dpi2dpi0dpi2dpi0dpi2dpi0dpi2dpi B69B69BIM2BIM2 Exp1Exp2Exp1Exp2 cfu/cm2

PAGE 67

55 Figure 2-4: Diagram of PCR-Select cDNA subt raction. Type e molecules are formed only if the sequence is up-regulated in the tester cDNA. Solid lines represent the Rsa Idigested tester or driver cDNA. Solid boxes represent the outer part of the Adaptor 1 and 2R longer strands and corresponding PCR primer 1 sequence. Green boxes represent the inner part of Adaptor 1 and the corresponding Nested PCR primer 1 se quence; red boxes represent the inner part of Adaptor 2R and the corresponding Nested PCR primer 2R sequence. Poly A+RNA IsolationPoncirus trifoliata Cold Acclimated at 4 C for 2 days Poncirus trifoliata Non Acclimated ControlcDNA Synthesis by Reverse Transcriptase Double-stranded cDNA Synthesis Restriction Enzyme Digestion with Rsa I Adaptor Ligation to the Tester DNA Driver Tester Driver Adaptor 1Adaptor 2R AAAAAAA TTTTTTT TTTTTTTT AAAAAAA AAAAAAA TTTTTTTT TTTTTTTT AAAAAAA First Hybridization 68 C for 8 hrs a b c d a b c d a b c d e Second Hybridization 68 C for 16 hrs Driver a b c d e Fill in the ends a and d No amplification b b’ No amplification c Linear amplification PCR Amplification using an Adaptor Primer 5’ 3’ 5’ 3’ e Exponential amplification Poly A+RNA IsolationPoncirus trifoliata Cold Acclimated at 4 C for 2 days Poncirus trifoliata Non Acclimated ControlcDNA Synthesis by Reverse Transcriptase Double-stranded cDNA Synthesis Restriction Enzyme Digestion with Rsa I Adaptor Ligation to the Tester DNA Driver Driver Tester Driver Adaptor 1Adaptor 2R AAAAAAA TTTTTTT TTTTTTTT AAAAAAA AAAAAAA TTTTTTT TTTTTTTT AAAAAAA AAAAAAA TTTTTTTT TTTTTTTT AAAAAAA First Hybridization 68 C for 8 hrs a b c d a b c d a b c d a b c d a b c d e a b c d e Second Hybridization 68 C for 16 hrs Driver Driver a b c d e a b c d e Fill in the ends a and d No amplification b b’ No amplification b b’ No amplification c Linear amplification PCR Amplification using an Adaptor Primer 5’ 3’ 5’ 3’ e Exponential amplification 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ e Exponential amplification Citrus leaves infected with BIM2 Citrus leaves infected with B69 Poly A+RNA IsolationPoncirus trifoliata Cold Acclimated at 4 C for 2 days Poncirus trifoliata Non Acclimated ControlcDNA Synthesis by Reverse Transcriptase Double-stranded cDNA Synthesis Restriction Enzyme Digestion with Rsa I Adaptor Ligation to the Tester DNA Driver Tester Driver Adaptor 1Adaptor 2R AAAAAAA TTTTTTT TTTTTTTT AAAAAAA AAAAAAA TTTTTTTT TTTTTTTT AAAAAAA First Hybridization 68 C for 8 hrs a b c d a b c d a b c d e Second Hybridization 68 C for 16 hrs Driver a b c d e Fill in the ends a and d No amplification b b’ No amplification c Linear amplification PCR Amplification using an Adaptor Primer 5’ 3’ 5’ 3’ e Exponential amplification Poly A+RNA IsolationPoncirus trifoliata Cold Acclimated at 4 C for 2 days Poncirus trifoliata Non Acclimated ControlcDNA Synthesis by Reverse Transcriptase Double-stranded cDNA Synthesis Restriction Enzyme Digestion with Rsa I Adaptor Ligation to the Tester DNA Driver Driver Tester Driver Adaptor 1Adaptor 2R AAAAAAA TTTTTTT TTTTTTTT AAAAAAA AAAAAAA TTTTTTT TTTTTTTT AAAAAAA AAAAAAA TTTTTTTT TTTTTTTT AAAAAAA First Hybridization 68 C for 8 hrs a b c d a b c d a b c d a b c d a b c d e a b c d e Second Hybridization 68 C for 16 hrs Driver Driver a b c d e a b c d e Fill in the ends a and d No amplification b b’ No amplification b b’ No amplification c Linear amplification PCR Amplification using an Adaptor Primer 5’ 3’ 5’ 3’ e Exponential amplification 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ e Exponential amplification Citrus leaves infected with BIM2 Citrus leaves infected with B69

PAGE 68

56 Figure 2-5: Distribution of potential citrus ca nker responsive genes. ribosomal protein 20% no homology 24% abiotic and biotic stress response 6% unknown function 8% signaling 5% hormone metabolism and signaling 1% transport 4% protein stability and degradation 3% secondary metabolism 4% metabolism/energy 9% transcription and translation 6% cell growth and division 10% signaling 3% hormone metabolism and signaling 2% ribosomal protein 28% no homology 14% abiotic and biotic stress response 15% unknown function 1%metabolism/energy 14% transcription and translation 6% cell growth and division 2% secondary metabolism 6% protein stability/degradation 4% transport 5%A) FS B) RS ribosomal protein 20% no homology 24% abiotic and biotic stress response 6% unknown function 8% signaling 5% hormone metabolism and signaling 1% transport 4% protein stability and degradation 3% secondary metabolism 4% metabolism/energy 9% transcription and translation 6% cell growth and division 10% signaling 3% hormone metabolism and signaling 2% ribosomal protein 28% no homology 14% abiotic and biotic stress response 15% unknown function 1%metabolism/energy 14% transcription and translation 6% cell growth and division 2% secondary metabolism 6% protein stability/degradation 4% transport 5%A) FS B) RS signaling 3% hormone metabolism and signaling 2% ribosomal protein 28% no homology 14% abiotic and biotic stress response 15% unknown function 1%metabolism/energy 14% transcription and translation 6% cell growth and division 2% secondary metabolism 6% protein stability/degradation 4% transport 5%A) FS B) RS

PAGE 69

57 Figure 2-6: Distribution and origin of the clones stam ped on the nitrocellulose membranes used in reverse northern blot analysis. 131 16 161 4 0 50 100 150 200 DD SSH

PAGE 70

58 Figure 2-7: Cluster analysis of genes differentially regulate d by PthB. In green are genes down-regulated by PthB, and in red ar e genes up-regulated by PthB -level

PAGE 71

59 Figure 2-8: Northern blot an alysis of CCR genes found diffe rentially regulated by reverse northern blot analysis rRNA was used as control for loading. Enod8 B69 BIM2 rRNA 2 dpi 18 S TIP LAT52 Exp PL GAST1 CCR137 B69 BIM2 2 dpi cellulase B69 BIM2 rRNA 2 dpi B69 BIM2 rRNA 2 dpi Enod8 B69 BIM2 rRNA 2 dpi 18 S TIP LAT52 Exp PL GAST1 CCR137 B69 BIM2 2 dpi cellulase B69 BIM2 rRNA 2 dpi B69 BIM2 rRNA 2 dpi B69 BIM2 rRNA 2 dpi B69 BIM2 rRNA 2 dpi

PAGE 72

60 100 m 100 m 100 m 100 m 100 m 100 m 100 m 100 m 100 m A B C 100 m 100 m 100 m 100 m 100 m 100 m 100 m 100 m 100 m A B C Figure 2-9: Microscopi c phenotype of leaves inoculat ed with B69 (wt) and BIM2 (nonpathogenic mutant lacking PthB). 7dpi BIM2 infected leaves, A; 7dpi B69 infected leaves, B, C. By In canke r-infected tissue, by 7 dpi, air spaces of the spongy mesophyll are almost inexistent These spaces are replaced by new divided cells as well as by cell of larg er size, resulting in thickening of the leaves.

PAGE 73

61 Figure 2-10: Microscopic phenot ype of leaves inoculated with B69 (wt) and BIM2 (nonpathogenic mutant lacking PthB). 7dpi BIM2 infected leaves, C; 7dpi B69 infected leaves, A, B, D. At 40X magnification, pockets of bacterial cells are visible surrounding mesophyll cells of B69 infected tissue while almost no bacteria is present in BIM2 infected ti ssue. Also not the areas of cell lysis in B69 infected tissue. 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 mA B C D 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 m 50 mA B C D

PAGE 74

62 Figure 2-11: Microscopic phenot ype of leaves inoculated with B69 (wt) and BIM2 (nonpathogenic mutant lacking PthB) at 14 dpi. A, B: BIM2, and C, D:B69 infected leaves. Note High levels of bact eria in B69 infected leaves compared to BIM2 infected leaves, as well as possible wall thinning of cells in B69 infected tissue. A C B D 10 m 10 m 10 m 10 m A C B D 10 m 10 m 10 m 10 m 10 m 10 m 10 m 10 m 10 m 10 m 10 m 10 m

PAGE 75

63 Figure 2-13: Quantification of leaf thicke ning and cell division during B69 and BIM2 infection on Duncan grapefruit leaves These measurements where taken on “slow canker-developing” leaves, i.e leaves showing high rate of cell division when inoculated with B69. The number of cells from abaxial epidermis to adaxial epidermis was calculated by counti ng the number of ce lls that a virtual line perpendicular to the epidermal layers would cross. Ten lanes were used n the analysis and the number shown are averages. 0 5 10 15 20 25 30 02714 0 200 400 600 800 1000 02714 B69 BIM2 0 5 10 15 20 25 30 02714 0 200 400 600 800 1000 02714 B69 BIM2 0 5 10 15 20 25 30 02714 0 200 400 600 800 1000 02714 0 5 10 15 20 25 30 02714 0 200 400 600 800 1000 02714 B69 BIM2Days post inoculationLeaf thickness ( m) Number of cells from abaxial epidermis to adaxial epidermis (in cross section) 0 5 10 15 20 25 30 02714 0 200 400 600 800 1000 02714 B69 BIM2 0 5 10 15 20 25 30 02714 0 200 400 600 800 1000 02714 B69 BIM2 0 5 10 15 20 25 30 02714 0 200 400 600 800 1000 02714 0 5 10 15 20 25 30 02714 0 200 400 600 800 1000 02714 B69 BIM2Days post inoculationLeaf thickness ( m) Number of cells from abaxial epidermis to adaxial epidermis (in cross section)

PAGE 76

64 Figure 2-13: Microscopic symp toms of rapidly developing canker. 14dpi BIM2 infected leaves, A; 14dpi B69 infected leaves, B, C, D. Note the highly enlarged cells the large areas of cell lysis and the absen ce of high rate of cell division in B69 infected tissue. 50 m A B C D 50 m 50 m 50 m A B C D

PAGE 77

65 Figure 2-14: Possible model for PthB eff ects on susceptible citrus cell showing parallel pathways activati ng cell division and expansion. PthBSignaling molecules: LAT52, Auxin /GA Exps, PLs, cellulase, Mannanase, TIP3, Enod8, GAST1, RAB8B, BetaCOP?Cell wall loosening Cell Expansion Cell Division? Rapid multiplication of X. citri and Canker phenotypes PthBSignaling molecules: LAT52, Auxin /GA Exps, PLs, cellulase, Mannanase, TIP3, Enod8, GAST1, RAB8B, BetaCOPCell division Effector molecules:?Cell wall loosening Cell Expansion Cell Division? Rapid multiplication of X. citri and Canker phenotypes Cell expansion Effector molecules: PthBSignaling molecules: LAT52, Auxin /GA Exps, PLs, cellulase, Mannanase, TIP3, Enod8, GAST1, RAB8B, BetaCOP?Cell wall loosening Cell Expansion Cell Division? Rapid multiplication of X. citri and Canker phenotypes PthBSignaling molecules: LAT52, Auxin /GA Exps, PLs, cellulase, Mannanase, TIP3, Enod8, GAST1, RAB8B, BetaCOPCell division Effector molecules:?Cell wall loosening Cell Expansion Cell Division? Rapid multiplication of X. citri and Canker phenotypes Cell expansion Effector molecules:

PAGE 78

66 CHAPTER 3 CHANGES IN SUMO CONJUGATION ARE ASSOCIATED WITH CITRUS CANKER DISEASE Introduction Citrus canker is an important disease of citrus worldwid e (Civerolo, E., 1984). It is caused by several pathovars of Xanthomonas citri which differ mainly in their host range (Shubert et al, 2001, Verniere et al, 1998). Canker infecti ons cause defoliation, fruit blemishes, premature fruit drop and tree declin e, resulting in severe economical losses (Shubert et al, 2001). Considerable internati onal regulatory efforts are implemented to prevent the spreading of the already quar antined pathogen, with negative effects on national and international trad e of citrus (Timmer et al 1996; Shubert et al, 2002). Canker symptoms are characterized by erumpe nt corky lesions that can affect all aerial parts of citrus trees (Shubert et al, 2002). Microscopy studies showed that canker lesions result from hyperplasia (cell divisi on) and hypertrophy (cel l expansion) in the spongy mesophyll tissue, where the bacteria cont act plant cells (Swarup et al, 1991; Duan et al, 1999 and Chapter 2). Ultimately, this in tense increase in cellular growth ruptures the epidermis and causes necrosis. The rupture of the epidermis is thought to be crucial for bacterial dissemination a nd spread of the disease (Graham and Gottwald, 1991; Duan et al, 1999). A crucial step towards understanding citrus canker disease was the identification of a pathogenicity gene, pthA required by X. citri pv. citri to cause canker on citrus (Swarup et al., 1991). Since then, all canker-causing stra ins have been shown to carry at least two

PAGE 79

67 members of the pthA gene family, with one copy suffici ent for most or all pathogenicity (Yang and Gabriel, 1995; Al -Saadi and Gabriel unpublished). pthA found in X. citri pv. citri ( Xcc ) of the Asiatic group of strains, and pthB found in X. citri pv. aurantifolii B69 ( Xca ) of the South American group, have been shown to be interchangeable in their ability to elicit canker (Yuan a nd Gabriel, unpublished). As for pthA of Xcc pthB of Xca was also shown to be required for pat hogenicity on citrus (Yuan and Gabriel, unpublished, and Chapter 2), and therefore, the B69 derivative mutant strain BIM2 lacking pthB does not elicit the typical macroscopic symptoms associated with canker disease (Chapter 2). When transferred to other xanthomonads carrying a functional type III secretion system (TTSS), or tran siently expressed in leaf cells, pthA was found to induce cell division, cell expansion, and rupt ure of the epidermis the three most prevalent canker symptoms (Swarup et al 1991 and 1992; Duan et al, 1999). It was therefore concluded that pthA alone was able to cause canke r-like symptoms and that its delivery into the plant cell relies on a functional TTSS. Members of the pthA gene family are also found in non-canker causing strains of Xanthomonas Examples of genes belonging to this gene family include avrBs3 and avrBs3-2 of Xanthomonas campestris pv. vesicatoria (Bonas et al, 1989, and Bonas et al, 1993), avrXa10 and avrXa7 of Xanthomonas oryzae pv. oryzae (Hopkins et al, 1992); along with avrB4 avrb6 and avrb7 of Xanthomonas campestris pv. malvacearum (De Feyter and Gabriel, 1991 and 1993). Proteins encoded by memb ers of this gene family are 90 to 97% similar and are characterized by several structural features essential for their function in avirulence and/ or pathogenicity. Such features include 1) nearly identical 102-bp tandem repeats in their center, 2) C-te rminal nuclear localization signals (NLS),

PAGE 80

68 and 3) C-terminal eukaryotic acidic transcri ptional activator (Herbers et al, 1992; Yang et al, 1994; Zhu et al, 1998; Yang et al, 2000; Yang and Gabriel, 1995; Van den Ackerveken et al, 1996, Szurek et al, 2001). Little is known about how canker disease is initiated in planta In order to understand the molecular mechanism underlyi ng canker, a differential display PCR experiment was conducted to identify plan t genes potentially responsive to canker (Chapter 2). At two days pos t inoculation (dpi), transcri pts were compared between leaves inoculated with B69 and leaves inoc ulated with BIM2 (B69 derivative carrying a non-functional pthB ). One clone was related to AtSUMO1 from Arabidopsis SUMO belongs to the ubiquitin family of proteins that are conjugated to target proteins; however;its functions are distin ct from those of ubiquitin. SUMO conjugation has been shown to be an important regulatory step in processes such as protein stability, s ubcellular localizati on, and response to various stresses. SUMOylation is carried out in a ATP-dependa nt reaction cascade si milar to the E1-E2E3 reactions responsible for ubiquitin conj ugation (Melchior F., 2000; Kim et al, 2002; Kurepa et al, 2003). In addition, SUMO modifi cation has been shown to be important for cell cycle progression in yeast. Specifically, temperaturesensitive mutants lacking a functional SUMO conjugation pathway have been shown to arrest at the G2/M transition (Seufert et al, 1995; Johnson and Gupta, 2001). Such work is of interest, as Xanthomonas citri infection triggers division of mesophy ll cells contacted by the bacteria. Recent work has shown that strain s of the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria encode at leas t two type III effectors with demonstrated SUMO protease activity (Hot son et al, 2003; Roden et al, 2004). Though

PAGE 81

69 loss of these SUMO protease-like effectors di d not affect pathogenicity on susceptible plants, it raises the possibility that th e plant SUMO conjugation pathway could be targeted during infection by X. c vesicatoria (Hotson et al 2003; Roden et al, 2004). This study indicates that: 1) changes in plant protein SUMO ylation profiles occurred after host infection by Xanthomonas citri pv. aurantifolii, 2) these changes in SUMOylation profiles were of two types, gene pthB -dependent and independent, and 3) these changes in SUMOylation profiles di d not occur following challenge with a nonpathogenic mutant strain lacking a TTSS. Toge ther, these data indicate that the TTSS of Xca delivers one or more effectors that direct ly, or indirectly, de -conjugate SUMO from host proteins in vivo Materials and Methods Plant Inoculations All inoculations were done with needle-l ess syringes on the abaxial surface of the leaf. Plants ( Citrus paradisi ‘Duncan’ grapefruit) were grown under greenhouse conditions. Inoculations involvi ng strains B69 and its derivati ves were carried out in BL3P level containment (refer to Federal Re gister Vol.52 no 154, 1987) at the Division of Plant industry, Florida Department of Agri culture, Gainesville, FL. For inoculation, bacterial suspensions were standardized in sterile 10mM CaCO3 (mock) to an optical density of 0.5 and pressure -infiltrated. For phenotypic obs ervation, inoculations were repeated at least three times. For protein ex traction, a split leaf i noculation scheme was followed to normalize differences due to physiol ogical state of inoculat ed tissue. For each combination of treatments (i.e. mock/B69 and mock/BIM2), one treatment was inoculated on the right side of the mid-vein and the other strain on the left side of the

PAGE 82

70 mid-vein. For each split-leaf experiment thr ee trees were used, with an average of 10 leaves inoculated per tree (approximatel y 5 leaves per treatment combination). Bacterial Strains and Culture Media Bacterial strains and plasmids used in th is study are listed in Table 1 Appendix A. All Xanthomonas strains were cultured in PYGM medium at 30C (De Feyter et al. 1990). Escherichia coli were grown on Luria-Bertani (LB) medium (Sambrook et al., 1990). For culture on solid media, agar was adde d at 15 g/L. Antibiotics were used at the following concentrations: Spectinomycin (S p), 35 mg/L; Kanamycin (Kn), 12.5 mg/L; Chloramphenicol (Cm), 35 mg/L; Gentomycin (Gt), 3 mg/L. Marker Integration Mutagenesis hrpG gene knock-out mutation was generated by triparental matings (as described in Chapter 1). Briefly, a 550 bp internal fragment of hrpG was cloned in the suicide vector pUFR012 [derivative of pUFR004 ca rrying kanamycin resistance (Gabriel laboratory, unpublished)] creating pB Y23. Transconjugants resulting from E. coli DH5/pBY23, DH5/pRK2013 (helper plasmid) and B69 matings were selected on spectinomycin to select against E. coli and chloramphenicol and ka namycin to select for plasmid insertion events. Putative transconj ugants were purified to a single colony, and Southern hybridization was used to confirm the integration of suicide vector pBY23 in hrpG For complementation purposes, a Hin dIII to Kpn I fragment was cloned out of plasmid pXG8 (REF) and recloned in pU FR053 (Yuan and Gabriel, unpublished) creating pBY24. DH5/pBY24 was used in triparental matings to create B23.5/pBY24 (B23.5c and B23.5c1). Putative exconjugant s were purified to a single colony, and

PAGE 83

71 Southern hybridization was used to conf irm the presence of the complementation plasmid. Total DNA extractions were performed as described in Gabriel and De Feyter (1992). Southern hybridizations were perf ormed as described by Lazo and Gabriel (1987). Bioinformatics Alignments and box shading were carried out using Clustal W (http://clustalw.genome.jp). Protein Extraction and Western Blotting Citrus leaf tissue was harvested at 0, 2 or 7 days post inocula tion (dpi), depending on the experiment, and ground to a fine powder in liquid nitrogen. Soluble proteins were extracted in two volumes of extraction buffer (50mM Tris, pH = 8.0, 300mM sucrose, 2mM EDTA, 0.3% DIECA, 10mM N-ethylmaleimide, 1 g/ l pepstatin, 1 g/ l leupeptin, and 7.5% w/v PVPP). Extracts were vortexed and briefly sonicated, then clarified by two rounds of centrifugation at 16,000 x g for 10 min at 4C. Soluble proteins were quantified by the BCA assa y (Pierce Biotechnology, Rockford, IL). Proteins were separated by polyacryalmid e electrophoresis on a 15% Tris-Tricine gel, and transferred to PVDF membrane (Millipore, Bedford, MA). For immunoblot analysis, membranes were probed with 1:2,500 immunopurified polyclonal PopSUMO1 (gi:23997054) antiserum (Cocalico, Reamstown, PA) diluted in phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 1.4 mM K2HP O4, 10.1 mM Na2HPO4, pH 7.4) containing 0.1% Tween 20 (T-PBS) with 1% v/v goat seru m (Sigma, St. Louis, MO). The antibodies were raised against purified PopSUMO1, whic h also contained an additional N-terminal hexahistidine tag generated by PCR (Reed, J., Master’s Thesis Univ ersity of Florida, 2005). For secondary antibody, the membranes were probed with 1:25,000 horseradish

PAGE 84

72 peroxidase conjugated donkey anti-ra bbit secondary antibodies (Amersham, Buckinghamshire, England) diluted in 1X T-PBS. Chemilluminescence was carried out according to the manufacturer’s instructions using the ECL plus (+) kit (Amersham). Following chemilluminescence, each membrane was rinsed in 1X T-PBS and stained with Coomassie R250 as a loading control. Results SUMO Conjugation Profiles are Altered in X. citri-Infected Leaves The grapefruit partial cDNA, CCR915 was identified by differential display as being canker responsive. Following reverse no rthern blot analysis, CCR915 which shows homology to SUMO, was found up-regulated in leaves inoculated with BIM2 (lacking pthB ) compared to leaves inoculated with B69 (w t) (Chapter 2). To determine if shifts in SUMO transcript abundance reflected regula tion at the protein levels, a split-leaf experiment was conducted in which Duncan gr apefruit leaves were mock inoculated on one side of the mid-vein, and Xanthomonas citri pv. aurantifolii strain B69 was inoculated on the other side Soluble extracts taken from canker or mock –inoculated leaves were probed for CitSUMO and CitS UMO-conjugated protei ns using PopSUMO1 antibodies. The grapefruit sequence was highly similar to poplar SUMO isoform PopSUMO1 (gi:23997054) (Figure 3-1) and as expected, the grapefruit SUMO and its protein conjugates cross-reacted with anti bodies raised against PopSUMO1. Using anti PopSUMO1, it was found that at two days post inoculation, the profile of SUMO conjugation is noticeably altered (Figure 3-2). The amounts of free CitSUMO and high molecular weight CitSUMO conjugated proteins were higher in B69-in filtrated leaves as compared to mock-infiltrated leaves.

PAGE 85

73 SUMO Conjugation Profiles in Infected Leaves are Partially PthB Dependent To determine if SUMOylation patterns were associated with disease symptom development, a split leaf inoculation experi ment was conducted and the effects of three separate treatments examined over time. Split-le aves were mock infiltrated, or inoculated with wild type strain B6 9, or the non-pathogenic mutant strain BIM2, which lacks pthB At 0, 2, and 7 dpi, half-leaves were harveste d and soluble proteins examined by western blot analysis. SUMO profiles of leaves inoculated with B69 were compared to those of leaves inoculated with mutant BIM2 at two dpi. There were no cha nges in the abundance of free CitSUMO or SUMOylated proteins in BIM2 i noculated leaves (Figur e 3-3, lane 4 and 5). The expected changes were seen in leaves inoculated with B69, i.e an increase in the amount of free SUMO and SUMO-conjugated pr oteins (Figure 3-3, lane 7 and 8). SUMO profiles at 7 days post inoculation revealed that the majority of the high molecular weight conjugates seen at 2 dpi in canker infected leaves were lost (Figure 3-3, lane 8 and 9). Interestingly, this loss of high molecular weight conjugates was also observed in leaves inoculated with non-pathogenic mutant strain BIM2. Whether the identities of SUMOylated prot eins in canker infected leaves are similar to the ones in BIM2 infected leaves is unknown; howev er, in both cases, SUMO de-conjugation occurred 7 dpi. These findings suggest that the SUMO de-conjugation observed at 7 dpi, in both BIM2and B69-inoculated leaves is PthB-independent and is also independent of the development of the macromolecular di sease symptom of canker (Figure 3-4). Conversely, the increase in the amounts of free SUMO and SUMO-conjugated proteins seen at 2 dpi with B69 are PthB-dependent.

PAGE 86

74 SUMO De-Conjugation Observed at 7 days Following Infection with B69 and BIM2 is Dependent on a Functional Type III Secretion System To determine if the SUMO de-conjugation observed at day 7 post inoculation in both B69and BIM2-inoculated leaves is dependent on a functional type III secretion system, a hrpG integrative mutant, B23.5, was generated. B23.5 was no longer pathogenic on citrus, and the hrpGphenotype was complemented after transformation of B23.5 with pUFR057::Xcv hrpG (Figure 3-5). There was no SUMO de-conjugation at da y 7 following inoculation with B23.5 (Figure 3-6), indicating that SUMO de-conjugation relies on a functional TTSS. In addition, B23.5 inoculation stimulated accu mulation of a 45kDa SUMO conjugate. A SUMOylated product of similar size was obser ved in leaves inoculated with B69 and BIM2, but did not accumulate (Figure 3-3). Discussion A great deal of effort has been directed towards investigating the mechanisms by which plants mount defense responses toward s pathogenic bacteria. Most studied cases involve incompatible plant microbe interactio ns that lead to the classical hypersensitive response or HR (Malek et al., 2000; Kazan et al, 2001). However, far less effort has been invested in trying to elucidate the mechanis ms by which a specific pathogen, or a group of pathogens elicit a particular disease with specific sets of morphological and molecular symptoms. In an effort to understand the processes by which different pathovars of Xanthomonas citri trigger canker symptoms, a canker responsive gene with sequence similarity to the SUMO gene family was identified by differential display PCR. The SUMO conjugation pathway in canker di sease was investigated using a splitleaf inoculation experiment to normalize for leaf-to-leaf vari ations. It was found that at 2

PAGE 87

75 dpi, X. citri pv. aurantifolii infection induces an increase in free CitSUMO and an increase in the number of high molecular we ight SUMOylated proteins. These changes were not observed in mock-inoculated leav es. SUMO conjugation in plants and other systems has been shown to be up-regulated by various instances of biotic and abiotic stresses (Kurepa et. al., 2003, Lois et al., 2003 and O’Donnell et. al., 2003). In order to test if changes in SUMO conjugation observed were specific to X. citri pv. aurantifolii infection, two mu tant strains unable to cause ca nker on citrus were used in this study, BIM2 (interrupted in pathogenicity gene pthB ) and B23.5 (interrupted in the TTSS regulatory gene, hrpG ). Disruption of hrpG was previously shown to disable the type III secretion system in Xanthomonas (Wengelnik et al. 1996). Using split leaf inoculations, it was shown that in BIM2 i noculated leaves, at 2 dpi, there were no changes in the amount of free SUMO and SUMO ylated high molecula r weight proteins. Thus, the increase in free SUMO and in the number of SUMOylated proteins is likely to be a PthB-specific plant respons e rather than a general stre ss response. A large number of SUMO targets identified in ot her organisms are cell-cycle re lated (Melchior, 2000). It has been shown in yeast ( Saccharomyces cerevisiae ) that temperature-sensitive mutants corresponding to SUMO and the enzymes invol ved in its conjugation pathway arrest the cell cycle at the G2/M transition, therefore, showing a critical role for SUMO in cell cycle progression (Johnson and Gupta, 2001). It is possible that the observed upregulation of SUMOylated proteins and free SUMO reflects activation of the plant cell cycle by X. c. pv. aurantifolii in the early stages of infection. Remarkably, this increase in free SUMO and in the amount of high molecular weight SUMOyl ated proteins is lost 7 dpi, potentially indicating a transition to a second disease phase. The deconjugation

PAGE 88

76 phenotype observed at 7 dpi with B69 is al so observable at 7 dpi with BIM2, and therefore, the triggering f actor of de-conjugation is pr obably independent of PthB. The possibility that another effector c ould be the trigger of the de-conjugation observed at 7 dpi came from the finding that the TTSS mutant B23.5, did not induce deconjugation. Therefore it is po ssible that another type thre e effector, beside PthB is responsible for the de-conjugation observed at day 7. Alternatively, it is possible that the second PthA homologue, PthB0 (not required for canker, Ch apter 2), found in B69 and BIM2 is also able to trigger the de-conjugation observed 7dpi. It has been proposed that the abundance of SUMO proteases in X. campestris pv. vesicatoria could reflect an importa nt role of theses effectors in Xcv pathogenesis (Hotson et al. 2003 and Roden et al 2004) However, none of the iden tified proteases have been implicated in disease and are, in fact, disp ensable. Given the cri tical role of SUMO conjugation in cell cycle proce sses (Melchior, 2000), and th e lack of apparent SUMO proteases encoded by another canker causing strain X. citri pv. citri it is possible that the late de-conjugation phenotype is not directly triggere d by a type III effector of a protease nature, but rather that a type III effecto r(s) acts to induce endogenous citrus SUMO protease(s) leading to the de -conjugation observed in late stages of canker infection. Both hypotheses are not mutually exclus ive and characteriza tion of additional X. citri effectors as well as citrus pr oteins SUMOylated in respon se to canker are required to better characterize the involvement of SUMOyl ation in the infection process of canker causing xanthomonads.

PAGE 89

77 Figure 3-1: Alignment of grapefruit SUMO (partial sequence) with (PopSUMO1, gi:23997054, and AtSUMO1, At4g26840).

PAGE 90

78 B 6 9r S U M O Molecular Mass (kDa)20.4 29.6 37.4 54.6 7.0 98.0 206.7 115.8 A B M o c k B 6 9r S U M O Molecular Mass (kDa)20.4 29.6 37.4 54.6 7.0 7.0 98.0 206.7 115.8 A B M o c k Figure 3-2: SUMO profiles of B69a nd mock-challenged gr apefruit leaves. 10 g of crude protein from day 2 of the spl it leaf experiment was separated by electrophoresis, blotted to PVDF and (A) probed with purified Pop SUMO1 antisera. Lane 1, Mock treated leaf; la ne 2, B69 inoculated leaf; lane 3, 2 ng purified recombinant Pop SUMO1. ([]): high molecula r weight SUMOylated proteins. (): un-conjugated SUMO. (B) The membrane was stained with Coomassie R250 as a loading control (S hown is the small subunit of Rubisco).

PAGE 91

79 Molecular Mass (kDa)20.4 29.6 37.4 54.6 98.0 206.7 115.8 027027027 MockBIM2B69 DPI Treatment AMolecular Mass (kDa)20.4 29.6 37.4 54.6 98.0 206.7 115.8 027027027 MockBIM2B69 DPI Treatment A Figure 3-3: SUMO de-conjugation occurs 7 days after infecti on. Leaves were inoculated with Mock, BIM2, and B69 strains. 7.5 g of crude protein from 0, 2, and 7 dpi from each treatment of the spli t leaf experiment was separated by electrophoresis, blotted to PVDF and (Upper panel) probed with purified PopSUMO1 antisera. ([]): high molecu lar weight SUMOylated proteins. (): un-conjugated SUMO. (Lower panel) The membrane was stained with Coomassie R250 as a loading control (Show n is the small subunit of Rubisco).

PAGE 92

80 B B A A C C B B A A C C Figure 3-4: Split leaf inoculation of Xanthomonas citri pv. aurantifolii (B69) and derivative BIM2 mutant. Duncan grapef ruit leaf 7 dpi with B69 (shown on the left side of the mid-vein and BIM2 (shown on the right side of the mid-vein). (A) adaxial side and (B) abaxial side of the leaf. Note the whitish canker characteristic of the Xca strain and yellowing associated with the day 7 post inoculation canker phenotype. (C ) Advanced B69 canker phenotype.

PAGE 93

81 B69 B23.5c B23.5c1 B69 B23.5c B23.5c1AB Hi ndIII B69 B23.5 B23.5c Hi ndIII B69 B23.5 B23.5c B69 B23.5c B23.5c1 B69 B23.5c B23.5c1AB Hi ndIII B69 B23.5 B23.5c Hi ndIII B69 B23.5 B23.5c Figure 3-5: B69 mutant derivative B23.5 lacks a functional Type III s ecretion system. (A) Southern blot hybridization profile s contrast B69, B23.5 and B23.5c (B23.5/ hrpG ). DNA was digested with Hin dIII and probed with the same internal fragment of hrpG used as homology region for marker interruption. (B) B69 and B23.5c inoculation on Duncan grapefruit. hrpG complemented the hrpphenotype of B23.5

PAGE 94

82 20.4 29.6 37.4 54.6 7.0 98.0 206.7 115.8 Molecular Mass (kDa)027027 B23.5B69* 20.4 29.6 37.4 54.6 7.0 98.0 206.7 115.8 Molecular Mass (kDa)027027 B23.5B69 20.4 29.6 37.4 54.6 7.0 98.0 206.7 115.8 20.4 29.6 37.4 54.6 7.0 98.0 206.7 115.8 20.4 20.4 29.6 29.6 37.4 37.4 54.6 54.6 7.0 7.0 98.0 98.0 206.7 206.7 115.8 115.8 Molecular Mass (kDa)027027 B23.5B69027027 027027 B23.5B69* Figure 3-6: SUMO de-conjugati on at 7 dpi requires a functional TTSS. Leaves were inoculated with B23.5 and B69 strains. 7.5 g of crude protein from 0, 2, and 7 dpi from each split leaf treatment was separated by electrophoresis, blotted to PVDF and (A) probed with purif ied PopSUMO1 antisera. ([]): high molecular weight SUMOylated proteins. (): un-conjugated SUMO. (*) novel 70kDa protein unique to B23.5 7 dpi leaves. Equal amounts of protein was loaded in each lane.

PAGE 95

83 APPENDIX A LIST OF PLASMIDS AND STRAINS Table A-1: List of strains a nd plasmids used in this study. Strains or plasmids Relevant ch aracteristics Reference or source Escherichia coli DH5 F-, end A1, hsd R17(rk -mk -), sup E44, thi -1, rec A1, gyr A, rel A1, 80d lac ZM15, ( lac ZYAarg F)U169 Gibco BRL, Gaithesburg, MD HB101 sup E44, hsd S20(rk -mk -), rec A13, ara -14, pro A2, lac Y1, gal K2, rps L20, xyl -5, mtl -1, SmR Boyer and RoullandDussoix ED8767 sup E44, sup F58, hsd S3(rk -rk r), rec A56, gal K2, gal T22, met B1 Murray et al. 1977 Xanthomonas 3213T X. citri pv. citri A Gabriel et al, 1989 3213Sp X. citri pv. citri A, SpR derivative of 3213 Swarup et al., 1991 B21.1 pthA ::Tn5-gusA, marker exchanged mutant of 3213Sp, SpRKnR Swarup et al., 1991 B69 X. axonopodis pv. aurantifolii 69, ATCC, B form citrus canker type strain B69Sp Spntaneous SpR derivative of 69, SpR Unpublished BIM2 pthB ::CmR, marker integration mutant of B69Sp, SpRCmR Unpublished BIM6 Marker integration mutant of B69Sp, CmR integrated upstream of pthB SpRCmR Unpublished B13.2 VirB4 ::CmR, marker integration mutant of B69Sp, SpRCmR This study

PAGE 96

84 Table A-1. Continued Strains or plasmids Relevant ch aracteristics Reference or source B13.1 VirB40::CmR, marker integration mutant of B69Sp, SpRCmR This study B69.4 Unpublished pRK2013 ColE1, KmR,Tra+, helper plasmid Figurski and Helinski, 1979 pUFR004 ColE1, Mob+, Cmr, lacZ+ De Feyter et al, 1990 pUFR012 Derivative of pUFR004 with Kn resistence. ColE1, Mob+, KnRCmR, lacZ+ Unpublished pBY13 270 bp fragment of virB4 cloned in pUFR004, CmR This study pB13.1 virB4 ::pBY13 of pXcB0, CmR This study pB13.2, pB13.4, pB13.5 virB4 ::pBY13 of pXcB, CmR This study PXcB Natural plasmid of B69 carrying pthB Unpublished pXcB0 Natural plasmid of B69 carrying pthB0 Unpublished pBIM2 pthB ::Cm R (pYY40.10) of pXcB, CmR Unpublished pBIM6 pXcB::CmR(pQY92.1), pthB still functional, CmR Unpublished pBY23 550 bp fragment of hrpG cloned in pUFR012, KnR, CmR This study pBY23c HrpG from pXG8 (REF) cloned in pUFR53 This study B23.5 hrpG ::pBY23 of B69, KnR CmR This study B23.5c B23.5/pBY23c This study

PAGE 97

85 APPENDIX B NORTHERN BLOT ANALYSIS OF CCRS Figure B-1: Northern blot an alysis of CCR genes not found differentially regulated by reverse northern blot PR2 PR1 Mock BIM2 B69 Mock BIM2 B69 2 dpi7 dpi 18S PR2 PR1 Mock BIM2 B69 Mock BIM2 B69 2 dpi7 dpi Mock BIM2 B69 Mock BIM2 B69 2 dpi7 dpi 18S rRNA PDF1 Mock BIM2 B69 BIM2 B69 2 dpi7 dpi rRNA PDF1 Mock BIM2 B69 BIM2 B69 2 dpi7 dpi PDF1 Mock BIM2 B69 BIM2 B69 2 dpi7 dpi PR5 B69 BIM2 2 dpi PR5 B69 BIM2 2 dpiRD22 GST CHI rRNA rRNA rRNA B69 BIM2 2 dpi Exp rRNA B69 BIM2 2 dpi Exp rRNA B69 BIM2 2 dpi Exp rRNA B69 BIM2 2 dpi CaCO3 BIM2 B69 BIM2 B69 2dpi7dpi TIP Exp CaCO3 BIM2 B69 BIM2 B69 2dpi7dpi TIP Exp CaCO3 BIM2 B69 BIM2 B69 2dpi7dpi TIP Exp Frap/tor rRNA B69 BIM2 2 dpi Frap/tor rRNA B69 BIM2 2 dpi pip3 B69 BIM218 S2 dpiPR2 PR1 Mock BIM2 B69 Mock BIM2 B69 2 dpi7 dpi 18S PR2 PR1 Mock BIM2 B69 Mock BIM2 B69 2 dpi7 dpi Mock BIM2 B69 Mock BIM2 B69 2 dpi7 dpi 18S rRNA PDF1 Mock BIM2 B69 BIM2 B69 2 dpi7 dpi rRNA PDF1 Mock BIM2 B69 BIM2 B69 2 dpi7 dpi PDF1 Mock BIM2 B69 BIM2 B69 2 dpi7 dpi PR5 B69 BIM2 2 dpi PR5 B69 BIM2 2 dpiRD22 GST CHI rRNA rRNA rRNA B69 BIM2 2 dpi Exp rRNA B69 BIM2 2 dpi Exp rRNA B69 BIM2 2 dpi Exp rRNA B69 BIM2 2 dpi CaCO3 BIM2 B69 BIM2 B69 2dpi7dpi TIP Exp CaCO3 BIM2 B69 BIM2 B69 2dpi7dpi TIP Exp CaCO3 BIM2 B69 BIM2 B69 2dpi7dpi TIP Exp Frap/tor rRNA B69 BIM2 2 dpi Frap/tor rRNA B69 BIM2 2 dpi pip3 B69 BIM218 S2 dpi

PAGE 98

86 LIST OF REFERENCES Alfano, J.R. and Collmer, A. 1996. Bacterial path ogens in plants: life up against the wall. Plant Cell. 8: 1683-1698. Alfano, J.R. and Collmer, A. 1997. The type III (Hrp) secretion pathway of plant pathogenic bacteria: traffick ing harpins, Avr proteins and death. J. Bacteriol. 179:5655-5662. Anderson, D.M., Fouts, D.E., Collmer, A. and Schneewind, O. 1999. Reciprocal secretion of proteins by th e bacterial type III machines of plant and animal pathogens suggests recognition of mRNA targ eting signals. Proc. Nat. Acad. Sci. USA. 96:12839-12843. Bergey’s Mannual of Determinative Bacteriology, 9th Eddition, JG Holt (ed.), Williams and Wilkins, Baltimore, MD, USA. Bonas, U., Stall, R.E. and Staskawicz, B. 1989. Genetic and structural characterization of the avirulence gene, avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol. & Gen. Genet. 218:127-136. Boyer, H. W., and Roulland-Dussoix, D. 1969. A complementation analysis of the restriction and modi fication of DNA in Escherichia coli J. Mol. Biol. 41:459-465. Brunings A.M. and Gabriel D.W. 2003. Xant homonas citri: breaking the surface. Molec. Plant Pathol. 4(3):141-157. Burns, D.L., 1999. Biochemistry of type IV secretion. Curr. Opin. Microbiol. 1999. 2(1):25-29. Christie, P.J. 1997. Agrobacterium T-Complex tr ansport apparatus: a paradigm for a new family of multifunctional tran sporters in Eubacteria. J. Bacteriol. 179:3085-3094. Christie, P.J. 2001. Type IV secretion: in tracellular tran sfer of macromolecules by systems ancestrally related to conjuga tion machines. Mol. Microbiol. 40:294-305. Christie, P.J. and Vogel, J.P. 2000. Bacteria l type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol. 8:354-360. Cornelis, G.R. and VanGijsegem, F. 2000. Asse mbly and function of type III secretory systems. Annu. Rev. Microbiol. 54:735-774.

PAGE 99

87 Cubero, J. and Graham, J.H. 2002. Genetic relationship among worldwide strains of Xanthomonas causing canker in citrus species and design of new primers for their identification by PCR. Appl. E nviron. Microbiol. 68:1257-1264. da Silva, A. C., J. A. Ferro, et al. (2 002). “Comparison of the genomes of two Xanthomonas pathogens with differing host sp ecificities.” Na ture 417(6887): 45963. De Feyter, R., Kado, C. I., and Gabriel, D. W. 1990. Small st able shuttle vectors for use in Xanthomonas Gene 88:65-72. De Feyter, R., and Gabriel, D. W. 1991. At least six avirul ence genes are clustered on a 90-kilobase plasmid in Xanthomonas campestris pv. malvacearum. Mol. PlantMicrobe Interact. 4:423-432. De Feyter, R., Yang, Y., and Ga briel, D. W. 1993. Gene-for-g enes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes. Mol. Plant-Microbe Interact. 6:225-237. Duan, Y.P., Castaneda, A.L., Zaho, G., Erdos, G. and Gabriel, D.W. 1999. Expression of a single, host-specific, bacter ial pathogenicity gene in plant cells elicits division, enlargement and cell death. Mol. Plant-Microbe Interact. 12:556-560. Egel, D.S., Graham, J.H. and Stal l, R.E. 1991. Genomic relatedness of Xanthomonas campestris strains causing diseases of ci trus. Appl. Environ. Microbiol. 57:2724-2730. El Yacoubi, B., Brunings,A., Yuan, Q. and Gabriel, D.W. 2001. A self-transmissible plasmid isolated from Xanthomonas campestris carries a member of the avr/pth gene family and additional factor(s) requi red for pathogenicity. Abstract of the 10th International Congress of Mo lecular Plant-Microbe Inte ractions, Madison, WI, 1014 July 2000, #650. Falcow, S. 1996. The evolution of pathogenicity in Escherichia, Shigella and Salmonela ; in Cellular and Molecular biology (ed.) F.C. Neidhadz (Washington DC: American Society for Microbiology). 2723-2729. Figurski, D. H., and Helinski, D. R. 1979. Re plication of an origin-containing derivatives of plasmid RK2 dependent on a plasmid function provided in trans Proc. Natl. Acad. Sci. USA 76:1648-1652. Gabriel, D. W., Kingsley, M., Hunter, J. E ., and Gottwald, T. R. 1989. Reinstatement of Xanthomonas citri (ex Hasse) and X. phaseoli (ex Smith) and reclassification of all X. campestris pv. citri strains. Int. J. Syst. Bacteriol. 39:14-22.

PAGE 100

88 Gabriel, D. W., and De Feyter, R. 1992. RFLP analyses and gene tagging for bacterial identification and taxonomy. Pages 5166 in: Molecular Plant Pathology: A Practical Approach. Vol. 1. S. J. Gurr, M. J. McPherson, and D. J. Bowles, eds. IRL Press, Oxford. Gabriel, D.W. 1999. Why do plant pathogens carry avirulence genes? Physiol. Mol. Plant Pathol. 55: 205-214. Gottwald, T.R., Graham, J.H., Schubert, T.S. 2002. Citris canker: the pathogen and its inpact. Online. Plant health Progress. doi:10.1094/PHP-2002-0812-01RV.http://plant managementnetwork.org/pub/php/review/citruscanker/. Graham, J.H., Gottwald, T.R., Cubero, J., Achor, D.S. 2004. Xanthomonas axonopodis pv citri factors affecting successful eradic ation of citrus canker. Molec. Plant Pathol. 5:1-15. He, S.Y. 1998. Type III protein secretion system in plant and animal pathogenic bacteria. Annu. Rev. Phytopathol. 36: 363-392. Hildebrand, D.C., Palleroni, N.J. and Schroth, M.N. 1990. Deoxyribonucleic acid relatedness of 24 xanthomona d strains representing 23 Xanthomonas campestris pathovars and Xanthomonas fragariae J. Appl. Bacteriol. 68: 263-269. Jin, Q. and S.Y. He (2001). “Role of the Hr p pilus in type III protein secretion in Pseudomonas syringae .” Science 294(5551): 2556-2558. Jones, J.B., Bouzar, H., Stall, R.E., Almira E.C., Roberts, P.D., Bowen, B.W., Subderry, J., Strickler, P.M., and Chun, J. 2000. Sy stematic analysis of Xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesi ons. Int. J. Syst. Evol. Microbiol. 50:1211-1219. Keen N.T. 1990. Gene for gene complementr ity in plant-pathogen interactions. Annu. Rev. Genet. 24:447-63. Kingsley, M.T., Gabriel, D.W., Marlow G.C. and Roberts, P.D. 1993. The opsX locus of Xanthomonas campestris affects host range and bios ynthesis of lipopolysaccharide and extracellular polysaccharide. J. Bacteriol. 175:5839-5850. Kubori, T. Matsushima, Y., Nakamura, D., Uralil, J., Lara-Tajero, M., Sukhan, A., Galan, J.E., and Aizawa, S. 1998. Supramolecular structure of the Salmonella typhimurium type III pretein secretion system. Science. 280:602-605. Lawrance, J.G. and Roth, J.R. 1996. Selfish operons: horizontal transfer may drive the evolution of gene cluste rs. Genetics. 143: 1843-9417. Lazo, G. R., and Gabriel, D. W. 1987. Conservation of plasmid DNA sequences and pathovar identification of strains of Xanthomonas campestris Phytopathology 77: 448-453.

PAGE 101

89 Lazo, G. R., Roffey, R., and Ga briel, D. W. 1987. Pathovars of Xanthomonas campestris are distinguishable by restri ction fragment length polymorphisms. Int. J. Syst. Bacteriol. 37:214-221. Leach, J. E. and White F. F. 1996. Bacteria l avirulence genes. Annu. Rev. Phytopathol. 34:153-179. Leong, S. A., Ditta, G. S., and Helinski, D. R. 1982. Heme biosynthesis in Rhizobium: Identification of a cloned gene coding fo r aminolevulinic acid synthetase from Rhizobium meliloti J. Bio. Chem. 257:8724-8730. Lorian, V. (ed.) 1986. Antibiotics in Laborat ory Medicine, Second edition, Williams & Wilkins, Baltimore. 683-721. Marenda, M., Brito, B., Callard, D., Genin, S., Barberis, P., Boucher, C. and Arlat, M. 1998. Prha controls a novel regulatory pathway require d for the specific induction of Ralstonia solanacearum hrp genes in the presence of palnt cells. Mol. Microbiol. 27:437-453. Marois, E., Van den Ackerveken, G. and Bonas, U. (2002) The Xanthomonas type III effector protein AvrBs3 modulates pl ant gene expression and induces cell hypertrophy in the susceptible host. Mo l. Plant-Microbe In teract. 15(7), 637-46. Murray, N. E., Brammar, W. J., and Murra y, K. 1977. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150:53-61. Sambrook, J., Fritsch, E. F., and Maniatis, T. A. 1989. Molecular Cl oning: A Laboratory Manual. 2nd ed. Cold Spring Habor Laboratory, Cold Spring Habor, NY. Silhavy, T.J. 1997. Death by leatha l injection. Scie nce. 278:1085-1086. Stall, R.E., Loschke, D.C., and Jones, J.B. 1986. Linkage of copper resistance and avirulence loci on a self-transmissible plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology. 76:240-243. Stall, R.E. and Civerolo, E.L. 1991. Research relating to the recent outbreak of citrus canker. Annu. Rev. Phytopathol. 29:399-420. Swarup, S., De Feyter, R., Brlansky, R. H., and Gabriel, D. W. 1991. A pathogenicity locus from Xanthomonas citri enables strains from several pathovars of X. campestris to elicit canker-like lesion s on citrus. Phytopathology 81:802-809. Swarup,S., Yang, Y., Kingsley, M. K., and Gabriel, D. W. 1992. An Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhost. Mol. Plant-Microbe Interact. 5:204-213. Syvanen, M. and Kado, C.I. 1998. Horizontal gene transfer. London: Chapman and Hall.

PAGE 102

90 Vernoud, V., Horton, A.C., Yang, Z. and Nielse n, E. (2003) Analysis of the Small GTPase Gene Superfamily of Arabi dopsis. Plant Physio l. 131(3), 1191-1208. Wayne, L.G., Brenner, D.J., Colwell, R.R ., Grimont, P.A.D., Kandler, O., Krichevsky, M.I., Moore, L.H., Moore, W.E.C., Murra y, R.G.E., Stackebrandt, E., Starr, M.P. and Truper, H.G. 1987. Report of the Ad Hoc Committee on reconciliation of approaches to bacterial systematics. Int. J. Winans, S.C., Burns, D.L. and Christie, P. J. 1996. Adaptation of a conjugal transfer system for the export of pathogenic macr omolecules. Trends Microbiol. 4:64-68. Yang, Y., Yuan, Q. and Gabriel, D.W. 1996. Watersoaking function( s) of XcmH1005 are redundantly encoded by members of the Xanthomonas avr/pth gene family. Mol. Plant-Microbe Interact. 9:105-113.

PAGE 103

91 BIOGRAPHICAL SKETCH Basma El Yacoubi was born on Octobe r 12 1973, in Mekns, Morocco. She obtained her D.E.U.G and Licen se in cell biology and physio logy from University Joseph Fourier in Grenoble, France; and her Maitri se cell biology and physiology from Paris 7 University in Paris, France. In the summer of 1996, she joined the University of Florida, and attended the English Language Institute during fall 1996. In spring 1997, she began her graduate studies, and obtai ned a Master of Science degr ee from the Department of Environmental Horticulture. In fall 1999, sh e joined the Plant Molecular and Cellular Biology program, working on her Ph.D. in the department of Plant Pathology.


Permanent Link: http://ufdc.ufl.edu/UFE0008379/00001

Material Information

Title: Bacterial Citrus Canker: Molecular Aspects of a Compatible Plant Microbe Interaction
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008379:00001

Permanent Link: http://ufdc.ufl.edu/UFE0008379/00001

Material Information

Title: Bacterial Citrus Canker: Molecular Aspects of a Compatible Plant Microbe Interaction
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008379:00001


This item has the following downloads:


Full Text











BACTERIAL CITRUS CANKER: MOLECULAR ASPECTS OF A COMPATIBLE
PLANT-MICROBE INTERACTION

















By

BASMA EL YACOUBI


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Basma El Yacoubi

































To Souad, Kamal, Aziz, Mouma, Mami, Nemat, and ma petite Shemsi















ACKNOWLEDGMENTS

I would like to express my sincere gratitude to Dr. Dean W. Gabriel, my supervisor

and committee chair and for his constant support and guidance during my years as a

graduate student in his laboratory. I also extend my gratitude to Dr. John M. Davis,

(member of my supervisory committee) for his valuable advice and for welcoming me in

his laboratory each time I needed it. I also thank all other members of my committee, (Dr.

Alice Harmon, Dr. Kenneth Cline, Dr. Bill Gurley, Dr. Jim Preston) for their valuable

advice and guidance.

I thank my mother, Souad Benchemsi, who always supported me. Without her none

of this would have been possible. My husband, Nemat Keyhani, and my little daughter

Shemsi Aida Keyhani, gave to my graduate student life a new dimension, and I thank

them for that.
















TABLE OF CONTENTS
Page

A C K N O W L E D G M E N T S ................................................................................................. iv

L IS T O F T A B L E S ...................................................................... .......... .. ............. ...... v iii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

ABSTRACT .............. .......................................... xi

CHAPTER

1 A 37 KB PLASMID FROM A SOUTH AMERICAN CITRUS CANKER STRAIN
CARRIES A TYPE IV SECRETION SYSTEM ESSENTIAL FOR SELF-
M O B IL IZ A T IO N ............................................................................... ....................

In tro du ctio n .................................................................................................... ..... .
M materials and M methods ....................................................................................... 5
Bacterial Strains, Plasmids and Culture Media................. ............................5
M arker Integration M utagenesis....................................... ......................... 5
Plasm id Conjugal Transfer Techniques...................................... .....................6
Recom binant DN A Techniques....................................... .......................... 7
P lant Inoculation s ....................................................... 7
R esu lts .............................. .. ...................7..........
The Type IV Secretion System Found on pXcB is Required for Self-
M obilization ............. ............ ........ ....... ....... ... ......... 7
Involvement of the TFSS of pXcB in Pathogenicity of Xca B69 ......................
D iscu ssio n ....................................... ................... ............................ 10

2 IDENTIFICATION OF CITRUS GENES SPECIFICALLY RESPONSIVE TO
PATHOGENICITY GENE pthB OF Xanthomonas citri pv. aurantifolii ..................23

Introduction..................................... .................................. .......... 23
M material and M methods ................................................... ..... .............................. 26
Plant and M icrobial M aterial..................................... ........................ .. .......... 26
B bacterial C ounts ........................................ .... ....... .... ....... 27
M icroscopy ................................... ............................27
Differential Display-Reverse Transcriptase PCR.............................................28
Suppressive Subtractive Hybridization (SSH) Library Construction..................28
N northern Blots .................................... ..... .......... ...... ........ .. 29
R everse N northern B lots ............................................... ............................ 29









Statistical A analysis ...................................... .............................30
R e su lts ...................................... ........................................................ ... ............... 3 1
Macroscopic Disease Phenotype of Citrus Leaves Inoculated with X c.
aurantifolii B69 and Its Mutant Derivative BIM2 Lacking the Pathogenicity
Gene pthB .................................... ..................................... 31
PthB-Dependent Transcriptional Reprogramming Induced upon Infection with
X ca ................... ......... ...... ... ... .. .... ...... .. ........... ........... . 3 2
Construction of Two Libraries Enriched in pthB Responsive cDNAs ..............33
Transcript Analyses of CCRs ........................................ ...............33
Identity of cDNAs Identified as Up-Regulated by the Presence of
pthB in X citri G enom e ................................ ............ .... ................34
Identity of cDNAs Identified as Up-Regulated by X citri Lacking pthB ............35
Northern Blot Analysis of Representative CCRs.............. ..... .............36
Microscopic Phenotype of B69 and BIM2 Inoculated Leaves..........................36
Discussion .............. .... ... ......... .. ........... ........... ...............38
PthB Induces Cell Division and Cell Expansion in Citrus Leaves .................39
PthB Induces the Expression of Cell Wall Remodeling Enzymes.................40
Enod8 and SAH7/LAT52 are a Link Between Canker Symptoms Development
and Nodule Organogenesis and Pollen Tube Growth Respectively ................42
PthB Induces Up-Regulation of a Tonoplast Aquaporin..................................44
PthB Induces Up-Regulation of Two Components Involved in Vesicle
T trafficking ............. ............................................ ........ ........ .. ........ .... 44
Hormone Pathways are Possibly Involved in Canker Symptoms
Development .................................... .......................... .... ...... 45
Conclusions and Future Prospects .................................... ............................. ....... 47

3 CHANGES IN SUMO CONJUGATION ARE ASSOCIATED WITH CITRUS
C A N K E R D ISE A SE ......................................................................... ................... 66

Intro du action ...................................... ................................................ 6 6
M materials and M methods ............................................................... ..........................69
Plant Inoculations ................... ...... ............... ... ............ 69
Bacterial Strains and Culture M edia......................................... ............... 70
M arker Integration M utagenesis...................... ... .......................... 70
B ioinform atics ......................................................... ........................ 7 1
Protein Extraction and W western Blotting............................................... 71
R esu lts .................. ......... ....... ............................................. ..... 72
SUMO Conjugation Profiles are Altered in X citri-Infected Leaves .................72
SUMO Conjugation Profiles in Infected Leaves are Partially PthB Dependent.73
SUMO De-Conjugation Observed at 7 days Following Infection with B69 and
BIM2 is Dependent on a Functional Type III SecretionSystem.................74
D discussion ............... ........... .......................... ............................74









APPENDIX

A LIST OF PLASMID AND STRAINS..................................................................83

B NORTHERN BLOT ANALYSIS OF CCRS .................................. ...............85

L IST O F R E F E R E N C E S ........................................................................ .....................86

B IO G R A PH IC A L SK E T C H ..................................................................... ..................91
















LIST OF TABLES


Table page

2-1 List of putative CCR identified by DD-PCR. ........................ 49

2-2 List of CCRs confirmed by reverse northern blot analysis.....................................50

A-i List of strains and plasmids used in this study .....................................................83















LIST OF FIGURES


Figure p

1-1 Organization of the type four secretion system (virB operon) found on pXcB
compared to other described TFS systems. ....................................................... 13

1-2 Hybridization profiles of DNA from B69 integrative mutants interrupted in virB4
of B 69 virB clusters. .............................. ............. .. ........ .. ........ .... 14

1-3 EcoRI and BamHI restriction digest profiles of plasmid pB 13.1 and plasmid
pB13.2, derivatives of pXcB0 and pXcB, respectively, and integrated in gene
virB 4 ................................................................................ 15

1-4 PCR profiles using primers AB65 and AB66 specific of plasmid pXcB. 16

1-5 Self-mobilization of pXcB derivatives is dependent on a type IV secretion system. 17

1-6 Construction of suicide vector pBY17.1 ........... ............................ ............... 18

1-7 Scheme of FLP recombinase-mediated marker eviction............... ................... 19

1-8 PCR confirmation of suicide plasmid pBY17.1 integration in gene virB4 ............20

1-9 CR confirmation of Flp-mediated eviction of pBY17.1 ............ ................21

1-10 Pathogenicity phenotype of primary and secondary exconjugants disrupted in
virB 4. ............................ ...... ................ ......... ............. ........... ............... 22

2-2 Late B69 and BIM2 phenotypes. (A) BIM2 inoculated leaves 30 dpi and (B) B69
inoculated leaves 30 dpi. ........................... .................. ............... ...... ............53

2-3 Quantification of bacterial population two days post inoculation with B69 and
BIM2. (cfu: colony forming unit), Expl: experiment 1, Exp2: experiment 2)........54

2-4 Diagram of PCR-Select cDNA subtraction........................................................55

2-5 Distribution of potential citrus canker responsive genes. ..................................56

2-6 Distribution and origin of the clones stamped on the nitrocellulose membranes
used in reverse northern blot analysis. ....................................... ............... 57

2-7 Cluster analysis of genes differentially regulated by PthB. ............ .................58









2-8 Northern blot analysis of CCR genes found differentially regulated by reverse
northern blot analysis................................................................59

2-9 Microscopic phenotype of leaves inoculated with B69 (wt) and BIM2
(nonpathogenic mutant lacking PthB). ......................................... ............60

2-10 Microscopic phenotype of leaves inoculated with B69 (wt) and BIM2
(nonpathogenic mutant lacking PthB). ........................................ ............... 61

2-11 Microscopic phenotype of leaves inoculated with B69 (wt) and BIM2
(nonpathogenic mutant lacking PthB) at 14 dpi. ................... .................. .......... 62

2-13 Quantification of leaf thickening and cell division during B69 and BIM2 infection
on D uncan grapefruit leaves ......................................................... ..... .......... 63

2-13 Microscopic symptoms of rapidly developing canker. .........................................64

3-1 Alignment of grapefruit SUMO (partial sequence) with (PopSUMO1, gi:23997054,
and AtSUM O 1, At4g26840). .............................................................................. 77

3-2 SUMO profiles of B69- and mock-challenged grapefruit leaves. ...........................78

3-3 SUMO de-conjugation occurs 7 days after infection....................... ...............79

3-4 Split leaf inoculation of Xanthomonas citri pv. aurantifolii (B69) and derivative
B IM 2 m utant. ..................................................... ................. 80

3-5 B69 mutant derivative B23.5 lacks a functional Type III secretion system. ...........81

3-6 SUMO de-conjugation at 7 dpi requires a functional TTSS.................................82

B-l Northern blot analysis of CCR genes not found differentially regulated by reverse
northern blot. .........................................................................85















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

BACTERIAL CITRUS CANKER: MOLECULAR ASPECTS OF A COMPATIBLE
PLANT-MICROBE INTERACTION

By

Basma El Yacoubi

May 2005

Chair: Dean W. Gabriel
Major Department: Plant Pathology

Canker is an important disease affecting citrus worldwide. It is caused by two

phylogenetically distinct groups of strains ofXanthomonas citri (Xc), with all citrus

cultivars being susceptible to at least one Xc strain. It is known that canker-causing

xanthomonads carry at least one pathogenicity gene of thepthA (of Asiatic X citri pv

citri) gene family, which is required for causing canker on citrus. However little is

known of the host molecular events leading to canker. Our goal was to understand host

molecular mechanisms underlying disease development, and identify bacterial

components related to phylogeny or pathogenicity of canker-causing xanthomonads.

First we identified on plasmid pXcB of the South American strain X citri pv

aurantifolii B69, a pathogenicity island composed of previously identified pathogenicity

genepthB and a type IV secretion system (TFSS). This TFSS was shown to be required

for self-mobilization of pXcB, which led us to propose that natural horizontal transfer of

apth host-specific pathogenicity gene may account for the two phylogenetically distinct









groups of strains, (the Asiatic and the South American group of strains), causing canker

symptoms on citrus.

Second, we investigated plant responses to PthB using differential display PCR and

suppressive subtractive hybridization techniques. We identified forty-nine genes that

were differentially regulated when RNA expression profiles of leaves inoculated with

Xca B69 were compared to those of leaves inoculated with a B69 mutant carrying a

disrupted pthB. Among these were genes predicted to be involved in cell expansion,

protein modification, biotic/abiotic stress responses and cell-wall metabolism.

Finally, we focused on one canker-responsive gene with strong similarity to the

small ubiquitin like modifier (SUMO) from Arabidopsis. Analysis of B69 mutant strains

lacking PthB or the type III secretion system (TTSS) component, HrpG, revealed PthB-

dependent and TTSS dependent/PthB-independent changes in SUMO conjugation

profiles after infection with B69.

The genes and cellular processes that we identified reflect the molecular events

leading to disease development. They contribute to the general aim of understanding the

mechanisms underlying the variety of diseases caused by compatible interactions

between xanthomonads and their host plants.














CHAPTER 1
A 37 KB PLASMID FROM A SOUTH AMERICAN CITRUS CANKER STRAIN
CARRIES A TYPE IV SECRETION SYSTEM ESSENTIAL FOR SELF-
MOBILIZATION

Introduction

The genus Xanthomonas is comprised of strains that exhibit a high level of host-

specificity; over 125 different pathogenic variants (pathovars) ofX. campestris have been

described that differ primarily in host range (Bergey, 1994). Host specificity in

Xanthomonas can be due to gene-for-gene interactions involving avirulence genes that

act in a negative fashion to limit host range (Keen, 1990; Gabriel, 1999; Leach and

White, 1996), but also can be due to positive acting factors that condition host range in a

host-specific manner. For example, pthN, avrb6 ofX. campestris pv. malvacearum (Yang

and Gabriel, 1996), opsXofX. campestris pv. citrumelo (Kingsley et al, 1993) andpthA

of X. citri pv. citri (Swarup et al., 1991 and 1992) act as positive effectors of host range.

Interestingly, although a clonal population structure is observed among strains within

many pathovars (Gabriel et al, 1988), some pathovars are comprised of phylogenetically

distinct groups that have an identical host range and cause identical disease symptoms.

Examples include 1) common bean blight, caused by two groups of strains (X. phaseoli

and X campestris pv. phaseoli var. fuscans) that are only 20% related by DNA-DNA

hybridization (Hildebrand et al., 1990); 2) bacterial spot of tomato and pepper, caused by

two major groups of strains within X campestris pv. vesicatoria (Jones et al., 2000) that

are less than 50% related by DNA-DNA hybridization (Stall et al., 1994), and 3) citrus

canker disease, caused by two groups of strains that are only 62 -63% related by DNA-









DNA hybridization (Egel et al., 1991). Strains with 70% or greater DNA-DNA

relatedness are usually defined as single species (Wayne et al., 1987). The question arises

however, as to how phylogenetically diverse strains can cause identical diseases on an

identical range of hosts.

To date, all pathogenic xanthomonads examined require hrp genes (reviewed by

Alfano & Collmer, 1996, 1997; He, 1998; Cornelis & VanGijsegem, 2000) to cause

disease. These genes encode a type III secretion machine that is close contact-dependent

(Marenda et al., 1998) and used to inject highly adapted effector proteins into both host

and nonhost cells (Silhavy, 1997; Kubori et al., 1998; and Jin and He, 2001). These

effector proteins elicit the diverse programmed phenotypes of the plant hypersensitive

response (HR) and various pathogenicity responses. The hrp (hypersensitive response

and pathogenicity) injection system is thus appropriately named, and it is also highly

indiscriminate, injecting whatever effector proteins are available, even some from animal

pathogens (Anderson et al., 1999 and Rossier et al., 1999). If identical hrp effectors are

available within two phylogenetically distinct xanthomonads, they can cause the same

disease symptoms, provided both strains are compatible (able to multiply in the host) and

both carry functional hrp systems. For example, pthA was transferred from X citri to X

campestris pv. citromelo and converted the latter strain from a leaf-spotting strain to a

strain with ability to cause citrus canker disease (Swarup et al., 1991). PthA appears to be

an effector protein that is critical for citrus disease symptoms and is likely injected by X

citri into citrus cells, causing hyperplastic cankers (Duan et al, 1999).

Citrus canker disease is caused by two phylogenetically distinct and clonal groups

of Xanthomonas strains; each group contains subgroups that are distinguished on the









basis of host range (Brunings and Gabriel, 2003). The first phylogenetically distinct

group is the Asiatic group, named Xanthomonas citri pv citri ex Hasse (syn = X

campestris pv. citri Dye pathotype A and X axonopodis pv. citri Vauterin, Xca-A). The

second phylogenetically distinct group is the S. American group, named X citri pv.

aurantifolii Gabriel (syn = X campestris pv. citri Dye and X axonopodis pv. aurantifolii

Vauterin, Xca-B). Both groups cause identical citrus canker disease symptoms circular,

water soaked raised lesions, that become dark and thick as canker progresses (Graham et

al., 2004; Stall and Civerolo, 1991; Gottwald et al, 2002; Brunings and Gabriel, 2003).

Significantly, pthA or homologues are present in every Xanthomonas strain tested that

causes citrus canker disease, and have not been found present in xanthomonads isolated

from citrus that do not cause canker (Gabriel, 1999; Cubero and Graham, 2002). Prior to

this work, twopthA homologues, namedpthB, andpthBO were found on two separate

plasmids (pXcB and pXcB0, respectively) of a S. American canker strain (B69). Plasmid

pXcB carrying the functional homologuepthB, was then found to be readily cured from

B69 (Yuan and Gabriel unpublished, and Brunings, A.M., 2004 M.S. thesis University of

Florida). Readily cured plasmids are often mobilizable by conjugation. Since Asia is

considered to be the center of origin of citrus canker disease, and since Asiatic canker

strains are more widespread in S. America than S. American canker strains, it was of

interest to determine if pXcB could transfer horizontally. pXcB was found to horizontally

transfer in-vitro and in plant (Yuan and Gabriel unpublished) from the S. American

strain B69 to the Asiatic strain B21.1 lacking a functional pthA, restoring its capacity to

cause canker (Yuan and Gabriel unpublished). Presence of the type III effectorpthB on a









self-mobilizing plasmid might explain the creation of the entire S. American group of

canker strains, and why they are phylogenetically distinct from the Asiatic group.

pXcB was fully sequenced (NC_005240, gi32347275), and besides genepthB, a

complete Type IV secretion system (TFSS) was also found on the plasmid (Brunings,

A.M., 2004 M.S. Thesis, University of Florida). TFSS are defined on the basis of

homologies between the A. tumefaciens T-DNA transfer system, the conjugal transfer

system Tra, and the Bordetellapertussis toxin exporter, Ptl (Winams et al., 1996 and

Christie, 1997). Most members of the TFSS family function primarily to mobilize DNA,

either from bacteria to bacteria (bacterial conjugation system) or from bacteria to

eukaryotic cells (Agrobacterium oncogenic T-DNA transfer system) (Burs, 1999). In

addition, several bacterial pathogens utilize conjugation machines to export effector

molecules during infection. Such systems are said to be Type IV "adapted" conjugation

or secretion systems, for their involvement in pathogenicity. Many non-plant pathogens

such as Bordetella pertussis, Legionella pneumophila, Brucella spp. and Helicobacter

pylori use a type IV "adapted" conjugation system to secrete effector proteins to the

extracellular milieu or the cell cytosol (Burns, 1999; Christie, 1996; Christie and Vogel,

2000). Type IV systems are composed of products with homology to the Agrobacterium

virB operon (Vogel, 2000). Sequence similarity analysis revealed that the Type IV

secretion system of pXcB encodes twelve open reading frames, ten of which contained

high sequence similarities to genes of previously described virB operons as well as

similar relative positions within the cluster (Brunings, A.M., 2004 M.S. Thesis,

University of Florida).









In order to investigate whether the TFSS found on pXcB is involved in self-

mobilization of pXcB, a plasmid derivative lacking a functional TFSS was generated in

this study and tested for its ability to self-mobilize in vitro. In addition, a B69 derivative

lacking the TFSS was generated in a non-polar fashion to address whether this system

was required for pathogenicity of B69. It was found that the TFSS of pXcB was required

for self-mobilization of the plasmid. However pathogenicity tests involving TFSS

insertional mutants were inconclusive, and it remains unknown whether this secretion

system is involved in pathogenicity ofX. citri pv. aurantifolii.

Materials and Methods

Bacterial Strains, Plasmids and Culture Media

Bacterial strains and plasmids used in this study are listed in Table 1.

Xanthomonas spp. were cultured in PYGM medium at 30C (De Feyter et al., 1990).

Escherichia coli were grown in Luria-Bertani (LB) medium (Sambrook et al., 1989).

Antibiotics were used at the following concentrations (in [tg/mL): Chloramphenicol

(Cm), 35; Kanamycin (Kn) 12.5 or 25 (when used to grow Xanthomonas or E. coli

respectively); Spectinomycin (Sp) 35 and Streptomycin (St) 35.

Marker Integration Mutagenesis

Gene-specific knockout mutations of Xanthomonas were created by triparental

matings. An E. coli DH5a strain carrying an internal fragment of the target open reading

frame (ORF) cloned in suicide vector pUFR004 was used as donor. A DH5a strain

carrying pRK2013 was used as the helper. A single crossover in the exconjugates results

in duplication of the internal fragment at the integration site, and also results in

interrupting the target gene with the vector. To disrupt virB4, a PCR-generated, 270 bp









internal fragment of virB4 (virB4270) was cloned in pGEM-T Easy and recloned in

pUFR004 creating pBY13.

Plasmid Conjugal Transfer Techniques

Plasmid transfer by triparental mating from E. coli strains HB101 or DH5a to

various Xanthomonas strains, using helper strain pRK2013 were performed essentially as

described in De Feyter and Gabriel (1991). For plasmid transmission experiments on

artificial media, overnight cultures of E. coli strains grown without antibiotics were

mixed with 50X concentrated overnight, mid-log phase cultures of Xanthomonas strains,

grown without antibiotics. Drops (10 [l each) of recipient donor and helper cells were

placed on PYGM agar medium one after the other and without antibiotics. In each case

excess liquid was allowed to absorb into the plate before addition of the next cell type.

The mating plates were incubated at 30C overnight, and the spots were then streaked on

PYGM selection medium supplemented with the appropriate antibiotics.

In Xanthomonas to E. coli matings, B69 carrying pB13.2 (pBY13 integrated in

virB4 of pXcB) or B69 carrying pB13.1 (pBY13 integrated in virB4 of pXcBO) were used

as donor strains (in independent matings) with DH5a as the recipient strain. After

selection against Xanthomonas on MacConkey agar (DIFCO laboratories, Detroit MI,

USA) with 35 [tg/mL chloramphenicol, DH5a exconjugants were screened for the

presence of pBY13.2 or pBY13.1 by DNA mini-prep analysis. In E. coli to E. coli

matings, DH5a/pBY13.2, DH5a/pBY13.1 and DH5a/pBIM2 (pYY40.10 integrated in

pthB) were used as donor stains in independent matings with HB 101 as recipient.

For frequency of transfer assays from one E coli strain to another, donor and

recipient strains were grown overnight at 37 C to an O.D. 600nm of 0.5. Twenty









microliters of each culture were combined in a 1.5 ml Eppendorf tube containing 160 [tl

of LB and grown overnight at 37 C. Cells were then resuspended in 1 ml of LB, pelleted

and then serially diluted on medium containing chloramphenicol and streptomycin to

select for HB 101 transconjugants. All conjugation experiments were performed at least

twice with duplicate samples in each experiment, and the numbers were averaged.

Recombinant DNA Techniques

Plasmid and total DNA were prepared from Xanthomonas as described by Gabriel

and De Feyter (1992). E. coli plasmid preparation, restriction enzyme digestion, alkaline

phosphatase treatment, DNA ligation, and random priming reactions were performed

using standard techniques (Sambrook et al., 1989). Southern hybridization was performed

using nylon membranes as described by Lazo and Gabriel (1987).

Plant Inoculations

All citrus plants (Citrusparadisi 'Duncan', grapefruit) were grown under

greenhouse conditions. Plant inoculations involving all citrus canker strains were carried

out under quarantine at the Division of Plant Industry, Florida Department of Agriculture,

Gainesville. Bacterial cells were harvested from log phase cultures by centrifugation

(5,000 x g, 10 min.), washed once and resuspended in sterile tap water or distilled water

saturated with calcium carbonate to 108cfu/mL. Inoculations were performed by pressure-

infiltration into the abaxial leaf surface of the plants. Experimental inoculations were

repeated at least three times.

Results

The Type IV Secretion System Found on pXcB is Required for Self-Mobilization

Gene virB4 of the TFSS cluster of pXcB was chosen as target for marker

insertional mutagenesis (Figure 1-1). For that, a 270 bp integral fragment of virB4









(virB4270) was cloned in pUFR004 (pBY13) and used in triparental matings to generate

virB4 insertion mutants. Southern blots were used to verify integration events in the

resulting transconjugants. These results demonstrate the existence of two copies of virB4

in the B69 strain (using virB4270 as probe Figure 1-2). One copy was carried by pXcB (as

determined by sequencing) and was absent in the cured strain B69.4 [Rifamycin resistant

strain cured of plasmid pXcB but carrying plasmid pXcBO, Yuan and Gabriel,

unpublished (Lane 3)]. A second putative copy, carried by pXcBO, was maintained in

B69.4 (Lane 3). Marker insertion resulted in two categories of exconjugants. Exconjugant

strain B13.1 appeared to carry an interruption of the putative virB4 of pXcBO (virB4o)

(Lane 7), while exconjugants B13.2, B13.4 and B13.5 appeared to carry interruptions of

the virB4 gene of pXcB (Lanes 4, 5 and 6).

Plasmids pB13.1 and pB13.2 of strains B13.1 and B13.2 (marker interruptions in

the virB4 homologues found on pXcBO and pXcB, respectively) were further analyzed

for their ability to transfer to E. coli. Matings with and without the helper strain resulted

in DH5a exconjugants carrying plasmids that were chloramphenicol resistant, indicating

that both plasmids were still mobilizing. Restriction enzyme digests of plasmid DNA

extracted from the Xanthomonas (B13.2) to the E. coli exconjugant (DH5a/pB13.2),

corresponded to the expected profile of pXcB integrated with pBY13 (Figure 1-3).

Restriction enzyme digests of plasmid DNA extracted from DH5a/pB13.1 did not

corresponded to the profile expected for a pXcB insertional derivative. Therefore, p13.1

is a derivative of a second native plasmid of B69, smaller in size than pXcB and inserted

in a putative virB4 copy. These results were confirmed by PCR using primers specific to

pXcB. As shown in Figure 1-4, when pB13.2 was used as template with pXcB specific









primers AB65/AB66 a 2014 bp band was obtained, while non specific bands were

obtained when pB 13.1 was used as template

The ability of pB13.1 and pB13.2 to self-mobilize was then analyzed by

performing matings from DH5a to E. coli HB101. Using DH5a/pBIM2, and

DH5a/pBIM6 [pBIM6 is a derivative of pXcB where pUFR004 was inserted in a non-

ORF region, (Yuan and Gabriel, unpublished)] as a control, transfer of pBIM2 and

pBIM6 from DH5a to HB101 was found not to require the presence of a helper strain

and the transfer frequency was 7x10-03 and 6.6x10-05 per donor, respectively. By contrast,

E. coli to E. coli transconjugants harboring pB13.1 or pB13.2 were only recovered when

matings were performed in the presence of a helper strain (Figure 1-5). These results

indicated that the self-mobilization capacity of pXcB depended on the presence of an

intact virB cluster.

Involvement of the TFSS of pXcB in Pathogenicity of Xca B69

Non-polar knock out mutants of virB4 were generated using marker insertion

followed by FLP recombinase mediated marker eviction. Plasmid pBY17.1 was

generated so that a virB4 homology region was flanked by two FRT recognition sites

(See Figure 1-6 for illustration). After marker integration of suicide vector pBY17.2 into

primary transconjugants, the FLP recombinase plasmid pJR4, was used to evict the

marker, and generated non-polar secondary transconjugants (See Figure 1-7 for

illustration).

Several primary transconjugants (before FLP-mediated eviction of marker) (Figure

1-8) as well as secondary transconjugants (after FLP mediated eviction of marker) were

tested for integration events in a virB4 homologue using PCR. Bacterial cells directly









from the selection plates were used as template for PCR (Figure 1-9). PCR positive

colonies were then grown in liquid culture and tested for pathogenicity on citrus. In all

cases, primary exconjugants showed a decrease in pathogenicity while, unexpectedly,

secondary exconjugants lost their potential to trigger canker disease on citrus (Figure 1-

10). When the secondary exconjugants used in pathogenicity assays were tested by PCR

for presence of pXcB it was found that the plasmid and therefore gene pthB were lost

upon culturing.

Discussion

The putative TFSS of pXcB (Brunings A.M., M.S. Thesis, University of Florida

and Brunings and Gabriel, 2003) was functionally investigated to determine its

involvement in plasmid transfer as well as in pathogenicity of B69. To investigate the

role of this TFSS in plasmid transfer, gene virB4 was marker-interrupted and by

consequence the whole system rendered dysfunctional. Self-mobilization experiments

revealed that pXcB relied on a functional TFSS to self-mobilize. In the process a second

putative virB4 homologue was identified on a second plasmid of B69, pXcBO. pB13.1,

carrying a single insertion in virB40 of pXcBO and pB13.2, carrying a single insertion in

virB4 of pXcB were each able to mobilize from B13.1 and B13.2, respectively, to DH5ca

in biparental matings (without helper strain), indicating that the two putative virB systems

co-existing in B69 might be compensatory.

The characterization of pXcB as a self-mobilizing plasmid carrying a TFSS and

gene pthB suggests that the canker causing and phylogenetically distinct South American

strains may have arisen from horizontal gene transfer of an "ancestral" pthA member.

This horizontal transfer likely would have occurred from an Asiatic Xanthomonas citri









strain to a compatible TTS system-carrying xanthomonad residing on the same host. B69

was indeed shown to carry a functional TTS system required for pathogenicity (see

Chapter 3). The type IV secretion system together with pthB on pXcB of S. American

Xanthomonas citri strains can therefore be considered an "auto-mobile" pathogenicity

island (Hacker et al., 1997), capable of spreading among compatible bacteria by

horizontal gene transfer.

Since pXcB from the South American strain is smaller, yet very similar to pXAC64

from the Asiatic strain, pXcB could be a deletion derivative of pXAC64 (Brunings and

Gabriel 2003). However, while many genes on pXcB were found to be similar to genes

on pXAC64, there were differences significant enough to conclude that a simple deletion

cannot account for pXcB. More likely, several independent events were probably

responsible for its divergence away from pXAC64.

Horizontal gene transfer is proposed to be a major mechanism explaining rapid

genetic diversification in bacteria (Falcow, 1996; Syvanen and Kado, 1998; Lawrence

and Roth, 1999). It has been proposed to explain the apparent enigma of why pathogens

carry dispensable avirulence genes (Yang and Gabriel, 1996 and Gabriel, 1999). For

example, avrBs3 of Xanthomonas campestris pv. vesicatoria was found on a mobilizing

plasmid carrying copper resistance, and therefore wide horizontal transfer of avrBs3 to X

campestris pathovars may be due to coincidental linkage with copper resistance (Stall et

al, 1986, Yang and Gabriel, 1996).

The TFSS of pXcB was also analyzed for its involvement in pathogenicity. Primary

exconjugants carrying a marker integration in virB4 showed a decrease in pathogenicity

while non-polar secondary exconjugants, resulting from marker eviction of the suicide






12


plasmid, lost all pathogenicity. This was then found to be possibly due to a loss of pXcB

upon curing of secondary transconjugant strains. Another explanation is the presence of a

large insertion vector in the native plasmid decreasing the copy number in the population.

Further examination of the TFSS is necessary to access its role in pathogenicity if any.







13


Orf 06
B2B3 B4 B5 B7 B6 B8 B9 B10 Bll BI
X.aa (pXcB) virB .4 2 : 4 :4i. I: I4 0 ir> = c 0 Self-mobilization


A. tumefaciens (pAtC58) avhB [:::::::::
Ti-plasmid virB -:-:-:-:-:-:-:-:-:-


Conjugal transfer
Transfer of T-DNA


Figure 1-1: Organization of the type four secretion system (virB operon) found on pXcB
compared to other described TFS systems. ORF 106 shows no similarity to any
virB cluster gene ofAgrobacterium tumefaciens and is shown as an insertion.












69 69.4 13.2 13.4 13.5 13.1

9qr4 4virB40 (pXcBO)
9..-

6.6 --m -virB4(pXcB)

4.4






Figure 1-2: Hybridization profiles of DNA from B69 integrative mutants interrupted in
virB4 of B69 virB clusters. Total DNA was digested with HindIII and probed
with a 32P-labelled 270 bp internal fragment of virB4. The same fragment was
used as a homology region for integration of suicide vector pBY13. B13.2,
B13.4 and B13.5 were marker integrated in virB4 of pXcB, and B13.1 was
marker integrated in virB4 of pXcBO. Hind III digestion results in splitting
one restriction fragment harboring the targeted region into two hybridizing
fragments. Therefore there are two bands hybridizing to the virB420o probe in
the wild type strains, while there are three bands in the insertional strains. The
only band hybridizing to the virB4270 in the B69.4 lane corresponds to a
putative virB4 copy present on a second native plasmid of B69. Indeed pXcB
was lost upon curing in B69.4 and therefore one hybridizing band is lost.












EcoRI


BamHI


pBIM2 p13.2 p13.1 pBIM2


p13.2 p13.1


Figure 1-3: EcoRI and BamHI restriction digest profiles of plasmid pB 13.1 and plasmid
pB13.2, derivatives of pXcBO and pXcB, respectively, and integrated in gene
virB4.


23

9.4
6.6

4.4

2.3
2.1












B69 B69.4 pB13.1 pB13.2


Figure 1-4: PCR profiles using primers AB65 and AB66 specific of plasmid pXcB.
Plasmid DNA isolated from DH5a/pB13.1, DH5a/pB13.2, and total DNA
isolated from B69 and B69.4 were used as templates. [AB65: CAG CCG
CAA GTG TCT CAG GTC; AB66:GGC AAG AAA CCG TCC GAG TA
(Tm 560C)]. When B69.4 and pB13.1 DNA are used as template in the PCR
reaction non-specific bands of low intensity are the resulting products. When
B69 and pB13.2 (both derivatives of plasmid pXcB) are used as template, a
specific band of 2014 bp is the resulting product of the PCR reaction. ( ):
Amplification fragment specific to pXcB when AB65/AB66 primers are used.






















S1%



^-o

4 0%-


-1:
Y
I~ti
%!
~p~i
~t~i~l
i,


pB13.1


pB13.2


pBIM2


Plasmid Frequency of transfer to HB101
( in donor strain DH5a) (per input donor)
p13.1 0
p13.2 0
pBIM2 7.08E-03
pBIM6 6.57E-05


Figure 1-5: Self-mobilization of pXcB derivatives is dependent on a type IV secretion
system. (A) Mobilization of pXcB derivative, pBIM2 and pB 13.2 and pXcB0
derivative pB 13.1 from E. coli DH5a to E. coli HB101. Matings were carried
with and without helper strain carrying plasmid pRK2013, and HB 101
transconjugants were selected on LB supplemented with streptomycin and
chloramphenicol. Each selection plate was separated in two sections. Results
of matings with helper strain are shown on the left section, and results of
matings without helper are shown on the right. Matings: (a) DH5a/pB 13.1
with HB101; (b) DH5a/pB13.2 with HB 101; (c) DH5a/pBIM2 with HB 101.
(B) Frequency of transfer of pXcB derivatives, pB 13.2, pBIM2, pBIM6 and
pXcBO derivative, pB 13.1 from E. coilDH5a into E. coli HB101.










FRIKn PCR fragment

FRTKnF FRTKnR
PCR frag men t from temp late
pKD4 was cloned in pGEMT- ez
PCR fragment internal to virB4
RsrIII BclI
pBY1.1 BY13 BY14

\. PCR fragment from temp late
EcoRI HindlI pXcB was cloned in pGEMT- ez



pBY3.1 \
(pUFR012 FITKn) BY2.1

in dlI
Eco BclH RsrIII Bcll
RsrIII




pBY17.1
(pBY3.1 ::viMB4


RsrIII BclI

Figure 1-6: Construction of suicide vector pBY17.1. PCR was used to amplify an internal
virB4 fragment using primers BY13 (gatcaggatcctatgcgcctcgttgaggt) and
BY14 (cggtccgtcagtcagtcagagctctgaccaggtagtgcagga). RsrIII and BclI
restriction sites were incorporated in the primer sequences respectively in the
forward and reverse primer. The RsrIII-BclI fragment was used as the driver
for homologous recombination and was cloned between FLP sites in
pUFR012 (pUFR004 derivative carrying kanamycin resistance). FLP sites
were obtain by PCR using plasmid pKD4 (gi:15554332) (1.5 Kb fragment).
Primers FRTKn F (gaattcgctgcttcgaagttcctatac) and FRTKn R
(aagcttatcctccttagttccaattcc) carried an EcoRI and a HindIII site for subcloning
from pGEMT-ez (Promega) into pUFR012.






















virB4 F bes03 22mer M13R bes04
FLP recombinase I-
plasmid vr4 inserted vector
virB4

virB4 F bes03 bes04

800bp 700bp 800bp




Figure 1-7: Scheme of FLP recombinase-mediated marker eviction. Suicide plasmid
pBY17.1 is marker integrated in gene virB4 via homologous recombination,
generating a virB4 disruption. The light blue box represents the homology
region targeted for recombination, and is found duplicated after insertion of
the suicide plasmid pBY17.1 in pXcB. After transformation of the B69
derivative carrying pXcB::pBY17.1 (primary transconjugant) with plasmid
pJR4 carrying a FLP recombinase gene, pBY17.1 is evicted (secondary
transconjugants). pJR4 [derived from pFLP (gi: 1245114) (Ready and Gabriel,
unpublished)] is cured by culturing secondary transconjugants on PYGM
supplemented with 5% sucrose. VirB4F and bes03 and bes04 are virB4
specific primers and their locations are shown by arrows. 22mer and M13R
are primers specific to the polylinker region of suicide vector pBY17.1 and
are used to verify integration and eviction events. FRT sites recognized the
FLP recombinase are symbolized by yellow circles and flank the internal
fragment with homology to virB4 cloned in pBY17.1. The green boxes
symbolize DNA stretches carried over during sub-cloning steps.










22mer/bes03 M13R/BES04 M13R/bes03DS

1 2 3 4 5 6 7 8 9









Lane 1,4,7: B69, Lane 2,5,8: B18.12, Lane 3,6,9: B18.15
viB4 F bes03 mer M13R bes04 bes03DS

vector pBY17.1
virB4

Figure 1-8: PCR confirmation of suicide plasmid pBY17.1 integration in gene virB4. 22
mer (gttttcccagtgacgacg) and M13R ( agcggataacaatttcacac) are primer
specific to the polylinker of pBY17.1. bes03(catcttggatcgtgcgtt) bes03DS,
bes04 (catgttgctgagcatctt) and virB4 F(ggtaccacccatttgaaaacgtgtcc) are gene
specific primers. Lanes 1,4,and 7, B69 bacterial cells were used as template
source for the PCR. Lanes 2, 5 and 8, B18.12 (primary transformant with
pBY17.1 inserted in virB4), bacterial cells were used as template source for
the PCR. Lanes 3, 6 and 9, B 18.5 (primary transformant with pBY17.1
inserted in virB4) bacterial cells were used as template source for the PCR.
Primer combinations used in each lane are indicated in the figure. PCR bands
resulting from using virB4-based primers in combination with suicide vector
based primers are specific to an insertion in the targeted region and should not
appear when the wild type strain is used. The light blue box represents the
homology region targeted for recombination, and is found duplicated after
insertion of the suicide plasmid pBY17.1 in pXcB. FRT sites recognized the
FLP recombinase are symbolized by yellow circles and flank the internal
fragment with homology to virB4 cloned in pBY17.1. The green boxes
symbolize DNA stretches carried over during sub-cloning steps.












BES03/BES04 M13R/bes04 virB4F/bes04
1 2 3 1 2 3 1 2 3
Lane 1: B69
Lane 2:B18.12
Lane 3:B18.12-1





B18.12 virB4F bes03 2mer M13R bes04

suicide vector inserted
virB4 virB4 F bes bes04
B18.12-1
800bp 700bp 800bp
bes03/bes04: 470bp
M13R/bes04: 1300 bp
virB4 F/bes04: 950bp (wt), or 2500bp after FLP



Figure 1-9: PCR confirmation of Flp-mediated eviction of pBY17.1. Genes specific
primers (bes03, bes04, virB4F and vector based primers were used in
appropriate combinations. B69 and B18.12 (primary transconjugant with
pBY17.1 inserted in virB4) were used as negative and positive control for the
suicide vector integration respectively. B18.1 2-1 is the secondary
transformant resulting from suicide vector eviction from primary
transconjugant B18.12. Bacterial cells from selection plates were used as
template source for PCR. The expected size of each PCR band is indicated in
the figure. The light blue box represents the homology region targeted for
recombination, and is found duplicated after insertion of the suicide plasmid
pBY17.1 in pXcB. FRT sites recognized the FLP recombinase are symbolized
by yellow circles and flank the internal fragment with homology to virB4
cloned in pBY17.1. The green boxes symbolize DNA stretches carried over
during sub-cloning steps.

































(secondary exconjugants, after eviction ofpBY7.1) Picture on the left was
Figure 1-10: Pathogenicity phenotype of primary and secondary exconjugants disrupted
in virB4. B 18.12; primary exconjugant (virB4::pBY17.1). B 18.12-1
(secondary exconjugants, after eviction of pBY17.1) Picture on the left was
taken 7 days post inoculation. The two pictures on the right were taken 15
days after inoculation. Note the delay phenotype of primary transconjugant
B 18.12, and the total loss of pathogenicity of transconjugant B18.12-1.














CHAPTER 2
IDENTIFICATION OF CITRUS GENES SPECIFICALLY RESPONSIVE TO
PATHOGENICITY GENE pthB OF Xanthomonas citri pv. aurantifolii

Introduction

Many studies on plant-pathogen interactions have dealt with incompatible

interactions using model plant systems (for example see Malek et al., 2000). Emphasis

has been on dissecting signaling pathways of resistance mechanisms, with few studies

considering signaling pathways resulting in diseases of crop plants (Kazan et al., 2001).

Therefore, the molecular events at the origin of disease induction by microbial effectors

of pathogens remain obscure.

Many Gram-negative, phytopathogenic bacteria rely on a Type III secretion system

(TTSS) to deliver effector proteins into the plant cells (He et al., 2004). Inactivation of

the TTSS of bacterial species that utilize such a system results in loss of pathogenesis

indicating that the proteins (named type III effectors) delivered by the TTSS are required

for bacterial virulence (Rohmer et al., 2004). Most type III effectors identified to date

were originally discovered and characterized by their avirulence function (Avr), while

only few are recognized pathogenicity factors [PthA from X citri (Swarup at al., 1991),

AvrB6 from X campestris pv. malvacearum (Yang et al,. 1996), AvrXa7 from X oryzae

(Bai et al., 2000) and DspA from Erwinia amylovora (Gaudriault et al., 1997)]. A limited

number of type III effectors have been assigned proven or putative biochemical function

(Collmer et al., 2000; Rohmer et al, 2000; Chang et al., 2004) and for a subset of these

(principally avirulence effectors), a plant protein or cellular process has been identified as









a possible target for pathogenesis (Rohmer et al, 2004 and Chang et al. 2004). In two

cases, a bacterial effector-triggered plant phenotype has been shown to be required for

pathogenesis. In the case of pathogenicity factor DspA, a member of the P. syringae

AvrE family, its induction of reactive oxygen species release by the host cell has been

shown to be required for successful colonization (Venisse et al., 2003). While in the case

of pathogenicity factor PthA, a member of the Xanthomonas AvrBs3/PthA family, its

induction of cell division and /or cell expansion is required for pathogenesis (Swamp et

al., 1991)

In this study the compatible interaction between citrus and Xanthomonas citri (X.

citri pv. aurantifolii syn. X. axonopodis pv. aurantifolii) was examined. Probably

originating in Southeast Asia, citrus canker has now spread to most citrus producing areas

of the world and causes severe economical losses (Civerolo, 1994). All canker strains

induce similar disease phenotypes, including water soaked lesions, formation of large

hyperplastic erumpent pustules (cankers) on all aerial plant parts, and rupture of the

epidermis with accompanying cell death (Swamp et al., 1991 and Duan et al., 1999).

Specific members of the avrBs3/pthA gene family are required by strains of

Xanthomonas citri to cause cankers on citrus (Swarup and Gabriel, 1989; Swarup et al.,

1990, Swarup et al., 1991). Members of the avrBs3/pthA gene family are found in many

xanthomonads (Gabriel, 1999; Vivian and Arnold 2000), and all citrus canker strains

examined carry multiple members of the gene family (Gabriel, 1999). All Xanthomonas

avrBs3/pthA members described to date are 90-97% identical in DNA sequence and are

characterized by 1) a series of 12.5-25.5 almost identical 34 amino acid repeats in the

center of the protein that determines host specificity, pathogenicity and/or avirulence









phenotype (Herbers et al., 1992, Yang et al., 1994, Zhu et al., 1998, and Yang et al.,

2000), 2) three C-terminal nuclear localization signals (Yang and Gabriel, 1995; Van den

Ackerveken et al., 1996 and Szurek et al., 2001) and 3) a C- terminal acidic region

considered to function as an eukaryotic transcriptional activator (Zhu et al., 1998, Zhu et

al., 1999, Yang et al., 2000, Szurek et al, 2001).

Sequence and functional analysis of members of the avrBs3/pthA gene family

showed that these proteins are type III effectors, acting in the plant nucleus potentially as

transcriptional regulators (Yang and Gabriel, 1995, Zhu et al., 1998, Zhu et al., 1999,

Yang et al., 2000, Szurek et al., 2001). When the pathogenicity genepthA from Xcitri

was transiently expressed in susceptible plant cells (by Agrobacterium infection or

particle bombardment delivery), it elicited canker-like pustules, indicating thatpthA alone

was sufficient to trigger canker symptoms (Duan et al., 1999). Unlike pthA and its active

homologues in other X citri pv. citri and X citri pv. aurantifolii strains, avrBs3 is not

required for pathogenicity ofX c. pv. vesicatoria (Bonas, 1989). However, it was found

to induce a subtle hypertrophy in the mesophyll of leaves inoculated with slow-growing

strains ofX. c. vesicatoria, concomitant with the up-regulation of 13 plant genes (Marois

et al., 2002). Taken together these results indicate that members of this gene family are

able to induce transcriptional reprogramming in both susceptible and resistant plant cells.

In this study, the citrus canker system was used to probe the functions ofpthB,

another member of the avrBs3/pthA gene family, that is isofunctional withpthA in

eliciting host-specific symptoms. A comparative analysis of gene expression in citrus

leaves inoculated with the wild type X c. aurantifolli strain B69 (carrying pthB) and an

Xca mutant derivative carrying a defective (marker interrupted) pthB was performed.









Methodological approaches in this analysis included differential display-reverse

transcriptase PCR (Liang and Pardee, 1992), suppressive subtraction hybridization, and

microscopy. Forty-six clones of citrus canker responsive genes belonging to several

broad categories of cellular functions were identified as being specifically regulated by

pthB. These categories included genes identified to be involved in cell wall loosening and

growth, water homeostasis and vesicle trafficking. In addition evidence is presented for

the involvement of hormone signaling in canker disease development.

Material and Methods

Plant and Microbial Material

Bacterial strains and plasmids used in this study are listed in Appendix A. B69 and

BIM2 were grown on PYGM (De Feyter et al. 1990) supplemented with 35 mg/L

spectinomycin and 35 mg/L spectinomycin plus 35 mg/L Chloramphenicol. All citrus

plants (Citrusparadisi 'Duncan', grapefruit) were grown under greenhouse conditions.

Plant inoculations involving all citrus canker strains were carried out under quarantine at

the Division of Plant Industry, Florida Department of Agriculture, Gainesville. Bacterial

cells were harvested from log phase cultures by centrifugation (5,000xg, 10 min.),

washed (IX) and resuspended in sterile tap water or distilled water saturated with

calcium carbonate to an OD600nm of 0.6-0.7, unless stated otherwise. Inoculations were

performed by pressure-infiltration into the abaxial leaf surface of the plants. Experimental

inoculations were repeated at least three times.

For differential display-reverse transcriptase PCR (DD-PCR) experiments and

construction of the suppressive subtractive libraries (SSH), inoculations were performed

following a split leaf model. Strain B69 was inoculated on one side of the mid-vein;

while BIM2 was inoculated on the opposite side of the mid-vein, in order to control for









leaf to leaf variations. Tissue was harvested 0, 2 or 7 days post inoculation (dpi)

depending on the experiment.

Bacterial Counts

B69 and BIM2 bacterial cells were normalized to an OD600 of 0.7 and infiltrated as

described previously. At 0 and 2 dpi, a total of 9 discs (0.28 mm in diameter) from 3

leaves (3 discs per leaves) were harvested for each treatment and ground in lml of tap

water. After serial dilution, the bacterial populations of wild type strain B69 and mutant

strain BIM2 were counted. Bacterial cell count determinations represent the average of

three replicate experiments.

Microscopy

Fresh, tender and half-expanded leaves were inoculated with a high inoculum of

B69 or BIM2. At 0, 2, 7 and 14 dpi, leaf samples of an area of approximately 6 mm2 were

harvested and fixed in 2% glutaraldehyde in phosphate buffer saline (PBS) for 48 hr at 40

C. They were then washed three times for 15 min each and fixed in 1% buffered osmium

tetroxide overnight at 40 C. This was followed by one wash in PBS for 10 mi and by two

washes in distilled water. A stepwise dehydration was conducted after these washes using

ethanol (25%, 50%, 75%, 95% and 100%) for 10 min each step, followed by three

washes in acetone for 15 min each. Samples were then infiltrated at room temperature in

30% acetone/EMbed (Electron microscopy sciences, Pennsylvania) for 1 hr, followed by

50% acetone/EMbed for 1 hr and 70% acetone/EMbed for 2 hr. Samples were

subsequently incubated in 100% EMbed overnight at room temperature to complete the

infiltration and polymerized in fresh 100% EMbed in a 75 C oven overnight.









Differential Display-Reverse Transcriptase PCR

Two and seven days after inoculation, leaf tissue was harvested, pooled and frozen

in liquid nitrogen for total RNA extraction as described (Chang et al., 1993). Potential

canker responsive (CCR) cDNAs were cloned as fragments by differential display-

reverse transcriptase PCR (DD-PCR) of mRNA using 48 primer combinations (Liang and

Pardee, 1992) with the RNAimage kit from Genhunter (Nashville, TN, USA).

Suppressive Subtractive Hybridization (SSH) Library Construction

For polyA mRNA isolation, leaves were frozen in liquid nitrogen and stored at -800

C until extraction. PolyA mRNA was isolated from leaves using the FastTrack mRNA

isolation kit (Invitrogen) according to the manufacturer's protocol. SSH was constructed

using a cDNA subtraction kit (Clontech PCR-Select, Palo Alto, CA). For construction of

the forward subtraction library (FS), the tester was chosen to be the pool of mRNA

isolated from B69 inoculated leaves at 2 dpi while the driver was chosen to be the pool of

mRNA isolated from BIM2 inoculated leaves, and therefore, the FS was enriched in

transcripts up-regulated by pthB. For the reverse subtraction library (RS), transcripts

isolated from BIM2 inoculated leaves (2 dpi) were used as tester, and therefore, while the

driver was chosen to be the pool of mRNA isolated from B69 inoculated leaves, the RS

library was enriched in transcripts up-regulated in the absence ofpthB.

Potential differentially regulated clones were sent for sequencing to the

Interdisciplinary Center for Biotechnology Research (ICBR) core at the University of

Florida. Putative functions were assigned based on annotation derived by BLAST

analysis.









Northern Blots

For RNA sample preparation, NorthernMax Formaldehyde Load Dye was used as

recommended by the manufacturer (Abion Austin, TX) with 5-10 |tg of RNA. Samples

were loaded on a denaturing formaldehyde agarose gel (1%) and electrophoresis was

conducted at 5 V/cm. RNA was blotted on GeneScreen Plus hybridization transfer

membrane (NemTM Life Science Products, MA) using 20X SSC as transfer buffer.

Hybridization and washes were done as recommended by the manufacturer (Ultrahyb,

Ambion Austin, TX). Probes were made with DECA primeTMII (random priming),

(Ambion Austin, TX) as recommended.

Reverse Northern Blots

For reverse northern blots, cDNAs identified by DD-PCR or SSH were amplified

using vector primers and purified using Qiaquick columns in plate format (Qiagen,

Valencia CA). Membrane arrays were made essentially as described by Desprez et al.,

(1998). cDNAs were arrayed onto Hybond N+ membranes (Amersham Biosciences,

Piscataway, NJ) using a 96-pin colony replicator (V&P Scientific, San Diego CA). Six

replicate arrays were generated and used to analyze transcript abundance of a subset of

potential canker responsive genes or CCRs. Each cDNA was spotted in two locations,

and several cDNAs were represented by more than one clone. Three replicate membranes

for each treatment (B69 or BIM2 infection) were used in hybridization experiments (total

of six membranes or 3 pairs). Each membrane was probed with radiolabelled cDNA

synthesized from RNA isolated from one of three split leaf-experiments conducted, 2 dpi.

Each membrane pair was one of three biological replications. Signal intensities were

statistically compared after normalization.









For probe preparation, first strand cDNA probes were prepared from 10 |tg of total

RNA by reverse transcription using MMLV-RT (Gibco-BRL, Gaithersburg MD) in the

presence of 32P-dCTP. Unincorporated nucleotides were separated from first strand

cDNA using Sephadex G-50 columns (Amersham Pharmacia Biotech, Ithaca NY) and

quantified using a liquid scintillation counter (Beckman Coulter, Fullerton CA). Pre-

hybridization, hybridization and low and high stringency washes were carried out at

65C. Membranes were exposed to phosphorimager screen for visualization. Spot

intensities (called volumes) on the membrane arrays were quantified using a BioRad

Molecular Imager FX run with the associated Quantity One software (Bio-Rad

Laboratories, Inc. Hercules, CA). Data were imported into Microsoft Excel (Microsoft

Corp., Redmond, WA, USA) for further analysis.

Statistical Analysis

A mixed model analysis (SAS Proc Mixed) was run on the log base 2 transformed

(normalization) local background adjusted volumes. cDNAs that did not exhibit a mean

value greater than 120 from either treatment were not included in the analysis. The linear

model used included replication (three biological replications), treatment (B69 treated or

BIM2 treated) and gene (CCRs or Citrus Canker responsive clones). Least square means

for the treatment by gene interaction were saved and used to form by-gene contrasts

between treatments. Significance of these contrasts was controlled for an experiment-

wide alpha level.









Results

Macroscopic Disease Phenotype of Citrus Leaves Inoculated with X. c. aurantifolii
B69 and Its Mutant Derivative BIM2 Lacking the Pathogenicity GenepthB

Xanthomonas strains B69 (wt) and its nonpathogenic mutant derivative BIM2,

carrying a marker integration in genepthB (pthB::pUFR004), were inoculated at high

levels (OD = 0.7) on tender half-expanded leaves of new flushes of Duncan grapefruit

and the corresponding induced disease phenotype analyzed. At day two post-inoculation,

no symptoms were visible and no macroscopic differences were observed among leaves

inoculated with tap water, B69 or BIM2. By seven days post-inoculation, leaves that were

mock inoculated showed no symptoms, while leaves inoculated with the wild type strain

B69 showed a whitish canker phenotype, typical of South American canker disease. On

the abaxial side of the leaf, the entire inoculated area became raised, with a soft, velvet-

like appearance, while a few individualized pustules appeared at the margins of

inoculated areas. Pustules possibly corresponded to areas where bacteria were infiltrated

at low density (Figure 2-1, A and B). On the adaxial side of the leaf, no raising was

apparent; instead some yellowing developed. This rapid symptom development is

typically observed when a high inoculum is used on fresh, young expanding leaves.

By contrast, at 7 dpi, no major symptoms were visible on leaves inoculated with

BIM2. Limited raising of the epidermis occurred at the margins of some inoculation

zones, with development of minimal pustule-like structures reminiscent of those seen in

canker (Figure 2-1, C and D). These symptoms were not observed in mock-inoculated

leaves. BIM2 inoculated leaves ultimately displayed attenuated canker phenotypes after

thirty days (Figure 2-2). This is possibly due to the week canker-inducing activity of

pthBO, the second pthA homologue found in the B strain, B69.









PthB-Dependent Transcriptional Reprogramming Induced upon Infection with Xca

A small scale DD-PCR was conducted to compare transcript levels of leaves

inoculated with B69 to those of leaves inoculated with BIM2 at two and seven dpi. To

maximize the homogeneity and the intensity of the response, B69 and BIM2 were

inoculated at high levels (OD600 of 0.7). In order to minimize leaf-to-leaf variation, a

split-leaf inoculation strategy was used. An average of fifteen leaves (from three trees)

were inoculated with B69 on one side of the mid-vein and with BIM2 on the other side.

Two and seven days after inoculation, half-leaves were harvested, pooled into "B69

treated" or "BIM2 treated" samples and RNA extracted from both samples. Since B69

(carrying pthB and pthBO) differs from BIM2 (carrying pthB::pUFR004 and pthBO) only

by the presence of a single effector, PthB, differentially regulated transcripts (named

citrus canker responsive or CCRs) were PthB responsive. Transcripts identified by DD-

PCR appeared differentially regulated as early as two days post-inoculation despite a

complete lack of symptoms. Twenty cDNAs were identified by DD PCR (Table 2-1),

including six with homology to biotic or abiotic stress response genes (CCR20.2 to PR-1

proteins and CCR9.5, CCR15.1 to PR-5 proteins, CCR2.2, CCR17.2 to peroxidases and

CCR12.1 to catalases). One cDNA, CCR6.4 displayed homology to cell wall remodeling

enzymes of the cellulase family. CCR25.1 was homologous to the small ubiquitin like

modifier SUMO.

To remove the possibility that potential changes in transcript level were due to

differences in the number of bacteria present in B69 inoculated leaves compared to BIM2

inoculated leaves, both bacterial populations were monitored at 0 and 2 dpi. B69 and

BIM2 bacterial populations were found to be comparable with almost no growth

observed during the first two days post-inoculation (Figure 2-3). Bacterial growth at 2 dpi









will occur if bacteria are inoculated at lower initial levels (OD600 of 0.3-0.4) (data not

shown). However, when inoculated at lower levels, growth of BIM2 is very poor (data

not shown and discussed later).

Construction of Two Libraries Enriched in pthB Responsive cDNAs

Following the same split leaf scheme as for the DD-PCR experiment, forward and

reverse libraries were constructed by suppressive subtraction hybridization (see Figure 2-

4 for illustration of the methodology), extending the collection of putative CCRs. The

forward subtraction library (FS) was constructed to be enriched in transcripts up-

regulated by PthB while the reverse subtraction library was constructed to be enriched in

transcripts up-regulated in the absence of PthB (see Materials and Methods for design of

the SSH).

Approximately 500 clones were sequenced and annotated using homology based

searches. Figure 2-5 illustrates the distribution of CCRs for each of the FS and RS

libraries according to their putative function. Categories representing genes of unknown

function (8 %) and genes involved in cell growth and division (10%) were found more

frequently in the forward library (up-regulated in the presence ofpthB) compared to the

reverse library (1 % and 2 % respectively), while genes in the category representing

abiotic and biotic stress responses were found more frequently in the reverse library (15%

vs 6% in the FS)

Transcript Analyses of CCRs

cDNAs from each of the forward (131) and reverse (161) libraries, as well as 20

clones identified by DD PCR (total of 312 cDNAs) (Figure 2-6) were chosen for reverse

northern-blot analysis. CCRs homologous to genes of known function were preferentially

selected. Six replicate arrays were generated as described in materials and methods, and









used to analyze transcript abundance of a subset of potential CCRs. Three membranes per

treatment were probed with radiolabelled cDNA synthesized from RNA isolated from

three split leaf-experiments, 2dpi with B69 and BIM2 (three biological replicates). For

each experiment, inoculated leaves were sampled from new and older flushes, were half

to fully expanded and were all tender (minimal cuticle). Signal intensities were

statistically compared after normalization as described in material and methods. Forty-six

clones were identified as differentially regulated at (p < 0.05) (Figure 2-7). Only fifteen

out of forty-six clones were found up-regulated in the absence of PthB, while the

remaining thirty-two were found up-regulated by PthB. Ratios of transcript abundance

were calculated for each cDNA. Ratios ranged from -3.5 to +34.5 (- sign indicating over-

expression of the gene in the absence of PthB and + sign indicating an up-regulation in

the presence of PthB) (Table 2-2).

Identity of cDNAs Identified as Up-Regulated by the Presence ofpthB in X. citri
Genome

Of the forty-six clones identified as differentially regulated, all but four clones

showed significant (e-value >2e-03) matches with sequences in available databases (Table

2-2). Thirty CCRs out of forty-six were found up-regulated by the presence ofpthB in the

bacterial genome i.e. up-regulated in B69 infected leaves compared to BIM2 infected

leaves. These are listed in Table 2-2.

Cell growth. Twelve clones were highly similar to genes involved in cell growth

(cell wall loosening and expansion): CCR339 was similar to cellulases; CCR1511,

CCR113 were similar to expansins; CCR889 was similar to mannanendo-1,4-beta

mannosidases; CCR571 and CCR1453 were similar to pectate lyases and CCR313 was

similar to tonoplast aquaporins (TIP3). Another clone of interest, CCR575, had homology









to the early nodulin gene Enod8 (predicted cell wall localized esterase). An additional

gene represented by CCR 109, CCR959 and CCR501 had homology to a secreted cell-

wall-associated pollen-specific allergen of the ole e 1 family (SAH7).

Giberellic acid pathway. Two CCRs had homology to the GAST1 (GA

responsive genes of unknown function) family of genes.

Vesicle trafficking. Several clones had homology to proteins involved in vesicle

trafficking. For example, CCR673 had homology to a small GTPase of the Rab family

(RAB8B, Vernoud et al. 2003), and CCR1258 had homology to the beta COP protein of

the COPI complex.

Unknown function. Another eight clones found up-regulated had either no

significant homology to any sequences in available databases or had sequence homology

to genes of unknown function.

Identity of cDNAs Identified as Up-Regulated by X citri Lacking pthB

Sixteen CCRs out of forty-six were found up-regulated by X citri lacking pthB

i.e. up-regulated in BIM2 infected leaves as compared to B69 inoculated leaves. These

are listed in Table 2-2.

Cell growth. CCR243, was the only BIM2 up-regulated gene involved in cell

wall metabolism. CCR243 is homologous to caffeic acid methyl transferases and is

involved in phenylpropanoid metabolism.

GA pathway. CCR 237 was homologous to cytP450 ent-keuren oxidase and

CCR105 was homologous to another cytP450 (possibly ent-kautenoic acid oxidase).

Protein modification and stability. For example, CCR409 had homology to

RD21a, a drought responsive cysteine proteinase, and CCR915 had homology to the

small ubiquitin modifiers (SUMO).









Transport. CCR1339 and CCR1435 were homologous to a mitochondrial import

inner membrane translocase and a monosaccharide-H+ symporter, respectively.

Unknown function. Another four clones found-up regulated in BIM2 infected

leaves had either no significant homology to any sequences in available databases or had

sequence homology to genes of unknown function.

Northern Blot Analysis of Representative CCRs

Expression of several candidate CCR genes identified by reverse northern blot

analysis was evaluated by northern blot analysis. Leaf tissue from split-leaf inoculations

using B69 and BIM2 were harvested and processed for RNA extraction. Several labeled

cDNA fragments were used to probe RNA blots (Figure 2-8). As in reverse northern blot

analysis, clones corresponding to expansion, cellulase, SAH7/LAT52, GAST1, Enod8 and

pectate lyase showed high levels of induction.

Microscopic Phenotype of B69 and BIM2 Inoculated Leaves

In order to characterize the microscopic phenotype of B69 and BIM2 infected

leaves, leaf discs mock inoculated and infected with BIM2 or B69 were harvested and

processed for light microscopy analysis (Figure 2-9, 2-10, 2-11 and 2-12). Leaves were

pooled as fast-responding to canker when disease symptoms were fully developed by

seven dpi (see figure 2-1, A and B). Leaves were pooled as slow-responding to canker

when disease symptoms were fully developed by 12 to 14 dpi.

Slow-responding leaves. At 2 dpi B69, BIM2 and mock inoculated leaves looked

identical at both the macroscopic and the microscopic level. At 7 dpi, while mock and

BIM2 inoculated leaves showed no phenotypic signs at both the microscopic or

macroscopic level (data not shown and Figure 2-9, A), the first signs of canker became

visible on B69 inoculated leaves, i.e. regions of darker green color around the veins and









slight swelling. At the microscopic level, B69 leaves showed high levels of cell division

occurring across all the inoculated area (Figure 2-9, compare B, Cto A). Intense cell

expansion and cell division phase resulted in complete filling of the air spaces of the

spongy mesophyll in B69 infected leaves (Figure 2-9, compare B, C to A). The number

of mesophyll cells from the abaxial to the adaxial epidermis more than doubled compared

to the day 0 control or day 7 BIM2 inoculated leaves, while some cells almost tripled in

size (Figure 2-10, compare A, B and D to C, and Figure 2-12). At later stages (14 dpi),

increased raising of the epidermis and whitish coloration with soft or velvety appearance

were observed at the macroscopic level. These phenotypes coincided with a phase of

increased cell expansion (data not shown and Figure 2-11, compare A, B to C and D).

While areas of cell division were still visible, a significant subset of cells became much

larger and the leaf dramatically thickened (twice that of the control leaf, see Figure 2-12).

A critical preliminary conclusion from these analyses indicated that the earliest visible

canker phenotype was mainly due to cell division, with a moderate cell expansion, while

late onset phenotypes were due to scattered but dramatic increases in cell expansion.

Fast-responding leaves. Macroscopic analysis indicated that cell expansion was

the primary phenotype with very little cell division occurring (Figure 2-13, compare B, C

and D to A). Furthermore, several areas of cell lysis, were visible immediately under the

abaxial epidermis.

Bacterial growth in B69 and BIM2 infected leaves. Canker visible symptoms

(cell division, cell expansion and resulting cell death) appeared necessary for B69 growth

as very few bacteria were visible in BIM2 infected tissue 14 dpi while numerous pockets









of bacteria were seen in B69 infected tissue (Figure 2-11, compare B to C and D and data

not shown).

Discussion

In this study, we have used macroscopic and microscopic phenotypic analysis in

combination with targeted gene discovery techniques to understand how the

pathogenicity factorpthB, ofX. c. aurantifolii belonging to the avrBs3/pthA gene family

elicits host-specific citrus canker symptoms in a compatible plant microbe interaction.

The nonpathogenic mutant BIM2, lacking pthB was used in combination with the wild

type strain, Xca B69, to study the specific effects of PthB on the plant cell transcriptome.

A split-leaf inoculation experimental design was used to minimize leaf-to leaf variations

in gene expression. In addition, bacterial cells were inoculated with high inoculum to: (1)

ensure near saturation of infection sites, (2) maximize the synchronicity of the host

response, and (3) artificially normalize the levels of bacterial populations (wild type and

mutant) present during early infection stages of the plant leaves (up to 2dpi). In order to

obtain a collection of genes potentially responsive to PthB, two complementary

techniques, DD-PCR, and forward and reverse SSH, were used to enrich for: (1)

transcript up-regulated when PthB is secreted in plant cells by Xcitri and (2) those up-

regulated in the absence of a functional PthB. Transcript analysis of a subset of 312

clones was conducted using reverse northern blot technique. Statistical analysis was used

to identify a list of forty-nine PthB responsive genes and differential regulation for a

subset of these was verified by northern blot analysis. Northern blot analysis was also

conducted on several CCR that did not show differential regulation by reverse northern

blot analysis (Appendix B). Several of these showed differential regulation when

northern analysis was used suggesting a better sensitivity than with reverse northern









analysis. This implies that the subtraction libraries contain additional CCR that need

identification.

PthB Induces Cell Division and Cell Expansion in Citrus Leaves

When inoculated on citrus leaves, Xanthomonas citri pv. aurantifolii was able to

cause cell division and cell expansion, consistent with previous reports onpthA-induced

phenotypes (Duan et al. 1999). Quantification of the three visible phenotypes of canker

i.e. cell division, cell expansion and the resulting thickening of the leaves was difficult

due to (1) the heterogeneity of the cells in the spongy mesophyll and (2) the

heterogeneity in distribution of the abundant air filled spaces in citrus leaf tissue.

Therefore, as first approximation of the phenotype, quantification measurements were

performed on areas where cellular activity was the most dramatic (areas of intense cell

expansion, cell cycle activity and thicker leaf areas). Analysis of PthB induced symptoms

over time revealed that the earliest visible phenotype associated with canker was cell

division in the infected spongy mesophyll, whereas heterogeneous but massive cell

expansion was observed at later stages of the infection. Interestingly, when canker

developed rapidly, i.e advanced canker symptoms at 7 dpi versus 12 to 14 dpi, symptoms

of cell division were found to be reduced compared to slower developing canker. In

addition, cell expansion was the major phenotype, primarily affecting mesophyll cells

directly under the abaxial epidermis layer. This supports the hypothesis that the primary

cellular mechanism affected by PthB alteration of the plant cell transcriptome is the

integrity of the cell wall and the induction of cell expansion. In turn, cell division could

either be: (1) a consequence of modification associated with cell expansion (e.g. changes

in cell volume) and (2) due to a second and distinct effect of PthB. However, because cell

expansion constituted a major phenotype in both rapid and slow developing canker,









induction of cell expansion may be the primary consequence ofpthB functions in the

plant cell. Furthermore, a specific set of genes with homology to genes involved in cell

growth were identified as responsive to PthB.

PthB Induces the Expression of Cell Wall Remodeling Enzymes

In order to understand PthB-induced phenotypes on citrus leaves, we have

identified a set of forty-six genes (CCRs) specifically regulated by the presence of this

effector in the plant cell. Consistent with the PthB-induced morphological phenotypes,

several CCRs were homologous to genes involved in plant cell wall modifications.

Expansins. Among these PthB-up-regulated plant genes, two were homologous to

ca-expansins. The role of the expansion gene family in wall loosening (polymer creep) and

cell expansion has been widely documented (Cosgrove, 2000). Expansins are

extracellular proteins that facilitate cell wall expansion probably by altering hydrogen

bonds between hemicellulosic wall components and cellulose microfibrils (Coscrove,

1998). These can act alone to induce cell wall extension in vitro, however, in vivo they

act with a suite of enzymes capable of restructuring the plant cell wall (Cosgrove, 1998).

Consistent with this, several CCRs homologous to genes associated with cell wall

remodeling were also identified.

Pectate lyases. Among CCRs associated with cell wall remodeling, CCR571 and

CCR1453 were similar to pectate lyases (PLs). These enzymes are involved in hydrolysis

of wall polymers, via cleavage of de-esterfied pectin, thereby facilitating cell expansion

(Carpita and Gibeaut, 1993 and Domingo et al., 1998). Although the role of bacterial

secreted PLs in cell wall degradation is well known (Collmer and Keen, 1998), the role of

endogenous plant PLs in development has not been extensively examined. In pollen,









plant PLs are thought to initiate the loosening of the cell wall enabling the emergence and

growth of the pollen tube (Cosgrove et al., 1997). PLs also mediate cell wall breakdown

in the style's transmitting tissue, allowing penetration of the pollen (Taniguchi et al.,

1995, Wu et al., 1996). Thus, induction of plant PLs by PthB can help account for aspects

of the disease phenotype.

Cellulases. Another PthB up-regulated CCR was homologous to the cellulase

family, another class of cell wall remodeling enzymes. Cellulases catalyze the cleavage

of internal 1,4 P linkages of cellulose and are involved in several aspects of plant

development involving cell wall modifications, including abscission, fruit softening and

cell expansion (Lewis and Koehler, 1979, and Fisher and Bennet, 1991). Relevant to

PthB induced phenotypes, it has been shown that constitutive expression of a poplar

cellulase in A. thaliana led to a significant increase in cell size (Park et al., 2003).

Beta-endo-mannanase. In addition to CCRs homologous to expansins, PL, and

cellulases, a fourth type of cell wall remodeling enzyme, a mannan endo-1,4-3D

mannosidase (endo-beta-mannanase) was also identified as up-regulated by PthB. This

enzyme catalyzes the hydrolysis of 1-4-pD mannosidic linkages in mannans,

galactomannans, glucomannans and galactoglucoomannans (Matheson and McCleary,

1985 and Matheson, 1986) and has been implicated in cell wall weakening during anther

and pollen development (Filichkin et al., 2004) and in seed ripening where it is involved

in mobilization of the mannan-containing cell walls of the tomato seed endosperm (Mo

and Bewley, 2003).

Caffeic acid methyl transferase. Only one gene involved in cell wall metabolism

was down regulated by PthB, CCR243. This clone was homologous a caffeic acid methyl









transferase (COMT), belonging to the phenylpropanoid pathway that leads to lignin

biosynthesis. Its expression has been shown to be regulated by biotic and abiotic elicitors

including infection by avirulent and virulent bacteria (Toquin et al., 2003). It is possible

that down-regulation of this enzyme relates to down-regulation of defense responses by

down-regulation of lignin deposition. This event could occur due to alterations in the

lignin content or composition. COMT down-regulation is in accord with the cell

expansion induced by PthB since mature walls lack acid-induced extension (Cosgrove,

1989). It is also interesting that fully expanded mature leaves are more resistant to canker,

whereas young leaves (one half to two-third expanded) are the most sensitive ones

(Graham et al., 2004). This is consistent with the hypothesis that PthB targets the cell

wall, inducing cell expansion ultimately resulting in disease progression.

A synthesis of our results indicates that type III effector PthB triggers the up-

regulation of an array of proteins whose combined activities induce cell wall loosening

and cell expansion. The roles of expansins, PLs and cellulases in cell wall loosening have

been shown to be complementary in other systems (Cosgrove et al.,1998; Carpita and

Gibeault; 1993, Domingo et al., 1998, Inouhe and Nevins, 1991).

Enod8 and SAH7/LAT52 are a Link Between Canker Symptoms Development and
Nodule Organogenesis and Pollen Tube Growth Respectively

Two additional classes of CCRs (CCR575 and CCR109, 959 and 501) identified

as up-regulated by PthB also support the theory that this effector targets cellular growth.

The first one, CCR575, was homologous to Enod8, an early nodulin gene associated with

the development of rhizobial nodule structures prior to nitrogen-fixation (Dickstein et al.,

1988, 1993). Enod8 has sequence similarity to exopolygalacturonase and lanatoside 15'-

O-acetylesterase (Pringle and Dickstein, 2003). Intriguingly, the up-regulation of Enod8









in response to X citri and Rhizobium suggests some common steps between nodule

formation and canker pustule formation. This is also supported by the fact that both

infections trigger cellular reprogramming events that lead to cellular growth. The

function of Enod8 is unknown, but in-vitro characterization and sequence analysis predict

that it is a cell wall localized esterase with acetylated oligo- or polysaccharides as

substrates (Pringle and Dickstein, 2004). Thus the enzymatic activity of Enod8, its cell

wall localization and involvement in both nodule and canker pustule formation point to

its involvement in modification of cell wall components during cellular growth.

The second class of CCRs reinforcing the hypothesis that the cell wall is the target

of PthB, displayed homology to SAH7 and LAT52 genes encoding for members of the ole

e I family of proteins. Originally identified as pollen allergens, members of this family

have also been found expressed in other tissues (e.g. SAH7 in leaves). A recent study of

one homologue, LAT52 (tomato), indicates that these genes may be involved in

controlling hydration and pollen tube growth (Tang et al., 2002). LAT52 interaction with

the pollen receptor kinase LePRK2 (LRR kinase) led to the hypothesis that binding of

LAT52 initiates a signal transduction pathway required for pollen germination and pollen

tube growth (Tang et al., 2002 and Johnson and Preuss, 2003). The up-regulation of a

LAT52- like gene in canker might, therefore, be part of a signaling pathway leading to

cell growth (the phenotype of both canker and pollen tube). Interestingly, pollen tube

growth, which occurs by tip extension, involves expansion and deposition of cell wall

precursors at the growing tip and requires the concerted action of endo-beta-mannanase,

expansins and pectate lyases (Marin-Rodriguez et al., 2002, Cosgrove, 1998 and

Filichkin et al., 2004), also found up-regulated during canker symptoms development.









PthB Induces Up-Regulation of a Tonoplast Aquaporin

CCR313, identified as up-regulated by PthB, displayed sequence similarity to a

tonoplast aquaporin of the TIPs family (Maurel, 1997 and 2002, and Hill et al., 2004).

Besides cell wall loosening, expansion requires extensive solute and water uptake

resulting in the formation of a prominent vacuolar compartment. This maintains the

turgor pressure that drives cell expansion (Veytsman and Cosgrove, 1998). Expansion is

thought to require high hydrolic permeability of the tonoplast in order to support water

entry into the vacuole, and tonoplast aquaporins (TIPs) play a critical role in this process

(Ludevid et al., 1992; Chaumont et al., 1998). TIPs are enriched in zones of cell

expansion (Tyerman et al. 2002) as well as in zones of active cell division where their up-

regulation is linked to vacuole biogenesis (Marty, 1997). Whether the identified tonoplast

aquaporin is indeed a marker for cell division and/or is actively involved in driving the

cellular expansion is unknown.

PthB Induces Up-Regulation of Two Components Involved in Vesicle Trafficking

Cell expansion and cell division both require deposition of new wall components

into the extending cell walls (Veytsman and Cosgrove, 1998) or into the cell plate of

dividing cells (Staehlin and Hepler, 1996 and Samuels et al, 1995). This may be achieved

via secretary processes involving vesicle trafficking. However, most genes identified here

suggest that in response to canker, cell walls are mainly extended without the building of

new cell wall components. This would imply that walls become thinner as cells expand.

This indeed has been observed at late stages of canker (Figure 2-10 compare A, B to C

and D). Although plant cell walls generally appear not to become thinner as they extend

(Veytsman and Cosgrove, 1998), expansion without new cell wall deposition could be at

the origin of the cell lysis observed in advanced canker stages.









Several CCRs identified as up-regulated by PthB are involved in vesicle

trafficking. Among these, CCR673 and CCR1258 have homology to RAB8B and beta

COP respectively. RAB8B is a member of the small GTPase gene family. The yeast

homologue of Rab8 (also named RABE see Vernoud et al., 2003) regulates membrane

trafficking to the daughter cell bud site (Salminen and Novick, 1987 and Goud et al.,

1988). Interestingly, in tomato, members of this subfamily appear to be targeted by the

Pseudomonas avirulence factor, AvrPto. This implies that in susceptible plants, AvrPto

may interfere with membrane trafficking pathways (Bogdanove and Martin, 2000). It has

been suggested that RAB8B might be involved in polarized secretion of antimicrobial

compounds (Bogdanove and Martin, 2000).

In mammals, beta COP belongs to a large complex that coats COPI vesicles

(Kreis et al. 1995). COPI vesicles transport membrane proteins and soluble molecules in

a retrograde, and possibly anterograde, direction through mammalian Golgi stacks

(Nickel and Wieland, 1997 and Harter, 1999). In plants little is known about COPI

vesicles. Recent evidence suggests that COPI-like vesicles are functional in plant

secretion and localize mainly to the Golgi apparatus as well as to the cell plate of dividing

cells (Couchy et al, 2003).

Hormone Pathways are Possibly Involved in Canker Symptoms Development

Triggering of cell expansion as well as induction of expansins and pectate lyases

constitutes a common point between the effect ofpthB on citrus (this study) and that of

the avirulence effector avrBs3 on susceptible pepper plants (Marois et al., 2002). Cell

expansion induction by both effectors share similar features; however, several plant

auxin-induced proteins of the SAUR family were found up-regulated by avrBs3 (Marois

et al. 2002). Several clones identified aspthB responsive are regulated by auxin in other









systems. These include the expansins (Catala et al., 2000, Civello et al., 1999, Hutchison

et al., 1999) and the pectate lyases (Domingo et al., 1998). In the pepper model, one of

two identified c-expansins was found up-regulated by exogenous application of auxin;

whereas a second expansion as well as a pectate lyase were not (Marois et al., 2002).

These data suggest that an auxin-independent pathway might operate under certain

conditions leading to cell expansion. In addition to a possible role of auxin in canker

disease, there is evidence for the involvement of the gibberellic acid signaling pathway in

the plant response topthB. CCRs with homology to ent-kaurenoic acid oxidase and

possibly to ent-kaurene oxidase (KO) (of GA biosynthetic pathway) (Oszewski et al.,

2002) and two clones with homology to the GAST1 family (GA induced genes) were

identified. Interestingly the GAST1 homologues were up-regulated by pthB; whereas the

putative KO and KAO were down-regulated. This may be explained by feed-back

regulation of KAO and KO expression by GA. However, feedback regulation of several

enzymes of the GAs biosynthetic pathway has been described in other systems, it has not

been reported to occur in the case of KAO and KO (Olszewski et al., 2002).

GA is known to regulate TIPs (Phillips and Huttly, 1994, Ozga et al., 2002),

expansins (Oka et al., 2001, Vogler et al., 2003, Lee and Kende, 2002, Chen and

Bradford, 2000), GAST1-like genes (Kotilainen et al., 1999 and Aubert et al., 1998),

endo-beta-mannanase (Dutta et al., 1997, Yamaguchi e al., 2001) and cellulases (Litts et

al., 1990). Therefore, PthB may act on regulatory steps upstream of GA biosynthesis. The

involvement of GA does not preclude that auxin is also involved since the latter is able to

regulate the production of the bioactive GA1 in elongating shoots (Ross et al., 2000).









Indeed, these two hormones are known to, in concert, promote cellular division and

elongation (Cleland, 2001 and Davies, 1995).

Conclusions and Future Prospects

The tight relationship between cell division and cell expansion makes it difficult

to address the question of whether cell expansion or cell division are the cellular

pathways that are altered as a downstream consequence of PthB regulating the plant cell

transcriptome. However, the following results presented here support the hypothesis that

cell wall loosening and expansion is the major plant cellular mechanism targeted by

PthB: 1) cell expansion occurs whether canker symptoms develop rapidly or slowly, 2)

genes involved in cell expansion have been identified as responsive to PthB, 3) cell

expansion is triggered by one another member of the avrBs3/pthA gene family and 4)

PthB responsive genes are involved in cell growth.

Microscopic analysis of leaves showing a slow canker symptom development

indicated that cell division is the major visible phenotype in initial infection stages, while

cell expansion remains at a moderate level. During the late infection stage however, cells

dramatically expanded leading to areas of cell lysis. It is possible that PthB induces cell

expansion and cell division by targeting several distinct cellular mechanisms. Another

hypothesis is that PthB targets cell expansion by altering cell wall composition

(loosening). This in-turn leads to a cell autonomous response that mainly involves the

triggering of cell division in the early stages and massive cell wall loosening and

expansion in later stages. The concentration of bacteria surrounding infected cells and,

therefore, the concentration of PthB protein secreted into the plant cells as well as the

physiological state of the infected tissue (for example immature expanding leaves will

readily expand) would modulate this response. When the concentration of PthB is low,









moderate expansion and the subsequent change in cell volume would lead to cell

division, while in later stages, elevated concentrations of PthB would lead to gross cell

expansion and cell lysis (Figure 2-14).

The relationship between cell expansion and cell division in plant growth and

development remains controversial. Whether growth starts by an increase in cell size,

triggering division, or whether division occurs first followed by restoration of the original

cell size (Foard, 1971 and Cleland, 2001) is mainly unknown. Studies on leaf primordial

(LP) initiation may begin to resolve this issue. Initially, since the first visible sign of a

new LP is a periclinal division in the L1 or L2 layer of the shoot apical meristem, it was

suggested that division occurs first (Steeves and Sussex, 1989). However, recent evidence

indicates that cell enlargement is the first step in LP initiation since LPs can be induced

by adding expansins either by microinjection of by up-regulation of expansion transcripts

at the shoot apical meristem (Pien et al., 2001, Fleming et al. 1997). Canker could follow

a similar pattern where cells expand first and then divide in response to expansion.

The canker phenotype is necessary for optimal growth and dispersal of X citri

(Swamp et al., 1991 and this study); therefore, induction of cell division and or expansion

are key steps in canker disease development and, unlike AvrBs3 forXcv, PthA/B confer a

benefit to X citri strains carrying it. The PTHA/B family of pathogenicity effectors may

prove to be a valuable tool in dissecting the molecular events surrounding microbe-

induced diseases since they are required for pathogenesis and can induce canker

symptoms alone. Finally, an understanding of the mechanisms by which PthB induces

canker phenotypes could help unravel the intricate relationship between cell division and

cell expansion that occurs in plant development.







49


Table 2-1: List of putative CCR identified by DD-PCR.
CCR Homology e-value
CCR23.2 Unknown protein [A. thaliana] (NP_196103.1) 2e-37
CCR24.5 Putative protein [A.thaliana] (NP_195874.1) 3e-24
CCR1.1 Putative Transposase [A. thaliana] (NP_189803.1] 4e-33
CCR22.5 3-hydroxyisobutyryl-coA hydrolase [A. thaliana] (NP_193072.1) le-22
CCR27.1 Cytochrome P450 [soybean] (T05942) 5e-41
CCR11.4 Cytochrome f [Nicotiana tabacum] (NP_054512.1) 4e-53
CCR6.2 Phosphoribosyl pyrophosphate synthase [Spinacia oleracea] 6e-25
(CAB43599.1)
CCR28.2 Putative mitochondrial carrier protein [A. thaliana] (NP_181124.1) 4e-34
CCR7.6 Copper Transport Protein [A. thaliana] (NP_200711.1) 4e33
CCR8.2 Receptor-like protein kinase-like (LRR) [A.thaliana] 6.8e-45
CCR6.4 Cellulase [sweet orange] (eC3.2.1.4) le-23
CCR28.4 Peroxidase [A. thaliana] (CAA66035.1) 2e-50
CCR12.1 Catalase [Campylobacterjejuni] (Q59296) 2e-10
CCR2.2 Bacterial-induced peroxidase [Goss hirsutum] (AF155124) 3e-26
CCR17.2 Peroxidase [Nicotiana tabacum] (BAA82306.1) 6e-63
CCR20.2 Pathogenicity-related protein la [barley] (AF245497) 2e-43
CCR15.1 Osmotin-like protein [Fagus sylvatica] (AJ298303) 2e 17
CCR9.5 Osmotin -like protein [Fragaria x ananassa] (AF1999508) 3e-61
CCR21.1 Auxin induced protein, putative [A. thaliana] (NP_176274.1) 1. 9e-3
CCR25.1 Ubiquitin-like protein [A.thaliana] (NP 194414.1) 4e-28







50


Table 2-2: List of CCRs confirmed by reverse northern blot analysis.
CCR Homology e value Ratio*
(aO.05)


Ribosomal protein
1385 30S ribosomal protein S20 (A. thaliana) gi21592469
Unknown function
1065 EST (0. sativa) gi50919279
1243 EST (A. thaliana) gi42569501
497 Putative protein (0. sativa) gi50919279
767 Putative protein (A. thaliana) gi15241855
1111 No significant homology
171 No significant homology
137 No significant homology
809 No significant homology
1139 Ring Finger Protein (A. thaliana) gi26450511
1061 Splicing factor RSZp22 (A. thaliana) gi21554419
475 Zinc finger protein (A. thaliana) gi28416541
1312 LRR receptor kinase (A. thaliana) gi42562316
Metabolism/energy
539 CytoF (N. tabacum) gi11465970
1415 RubisCO activase (malus x domestic) gi415852
1239 FIFO ATPase inhibitor protein (0. sativa) gi 52077175
1057 Hydroxymethyltransferase (A. thaliana) gi21593312
901 UMP-kinase (A. thaliana) gi2497486
1445 UMP-kinase (A. thaliana) gi2497486
33 UDP-galactose epimerase (A. thaliana) gi9758701
Transport
1339 Mitochondrial import inner membrane translocase S.U gi42568553
343 Copper T protein (A. thaliana) gi15237802
1435 Monosaccharide-H+ symporter (D. glomerata) gi30349804
Protein modification/stability
915 Small ubiquitin-like modifier (A. thaliana) gi15236885
1262 Protease inhibitor/seed storage//LTP (A. thaliana) gi42567284
1345 Aminopeptidase (A. thaliana) gi34098848
409 Putative cysteine proteinase RD21A (A. thaliana) gi22136972
GA pathway
1535 GASTI-like protein (A. thaliana) gi25406361
493 GASTI-like protein (A. thaliana) gi25406361
237 Cyt. P450 ent-keuren oxydase (Malus x domestic) gi45551401
1051 Cyt P450 (possibly ent-kaurenoic acid oxidase) (P. sativum) gi27776451
Vesicle trafficking
673 RAB 8B (Lotus corniculatus) gi1370192
1258 beta COP protein (0. sativa) gi50900798
279 Phosphatase (put. membrane trafficking factor) (A. thaliana) gi21553471


le 28

4e-1
2e-40
4e-27
3e-24





5e-07
2e 03
le07
le43

4e 53
9e-58
4e 10
4e-73
3e-38
le-10
4e 21


-3.36

4.79
-3.2
4.89
-2.41
2.95
3.60
3.09
-3.14
3.86
3.16
-2.55
3.25

-3.05
-2.60
-2.91
3.53
2.64
6.92
3.58


le-19 -2.62
4e33 4.92
2e-13 -3.36


4e-28
6e-04
2e-21
5e-32

3e-10
3e-34
7e-44
le 23


-2.53
5.35
3.09
-2.99

11.00
6.10
-3.2
-2.46


le 28 6.68
7e-20 2.38
3e-19 -2.51










Table 2-2. Continued
CCR Homology e value Ratio
(aO.05)
Cell growth (cell wall metabolism and expansion)
889 Mannan endo 1,4 beta mannosidase (0. sativa) gi34912090 4e 15 3.53
113 Alpha expansion (P. cerasus) gi13898655 4e-49 4.06
1511 Alpha expansion (P. cerasus) gi13898655 4e51 4.26
571 Pecate lyase (malus x domestic) gi 34980263 le-54 7.89
1453 Pectate lyase (A. thaliana) gi21593312 12-15 4.69
243 Caffeic acid 0-methyl transferase (C. roses) gi 18025321 6e-59 -2.39
339 Cellulase (sweet orange) gi7488904 le 23 34.53
575 Enod8 (early nodulin 8 like) (A. thaliana) gi26451820 3e-07 4.59
313 Tonoplast aquaporin gamma TIP (TIP3) (A. thaliana) gi3688799 5e-13 4.82
109 SAH7/LAT52 (ole e I allergen family) (L. esculentum) gi 295812 7e-17 3.78
959 SAH7/LAT52 (ole e I allergen family) (L. esculentum) gi 295812 5e-11 3.73
501 SAH7/LAT52 (ole e I allergen family) (L. esculentum) gi 295812 9e-21 2.75
*: A positive ratio indicates up-regulation in B69 infected tissue compared to BIM2
infected tissue. A negative ration indicates up-regulation in BIM2 infected tissue
compared to B69 infected.tissue.






52

























Figure 2-1: Phenotype of B69 and BIM2 infections on grapefruit leaves. BIM2 lacks
PthB and induces formation of very small pustule like structures, reminiscent
of canker pustules, at the edges of some inoculated areas. (A), (C) are BIM2

inoculations. Pictures were taken 7 days post inoculation.
i~ ').'- ." ., :












inoculations. Pictures were taken 7 days post inoculation.







53








.4P



A'






Figure 2-2: Late B69 and BIM2 phenotypes. (A) BIM2 inoculated leaves 30 dpi and (B)
B69 inoculated leaves 30 dpi. Note the much attenuated phenotype of BIM2
infected leaves.












1.E+06


1.E+04


1.E+02


1.E+00


Odpi 2dpi Odpi 2dpi Odpi 2dpi Odpi 2dpi


Expl


Exp2


BIM2

Expl


BIM2

Exp2


Figure 2-3: Quantification of bacterial population two days post inoculation with B69 and
BIM2. (cfu: colony forming unit), Expl: experiment 1, Exp2: experiment 2).















Citrus leaves infected Citrus leaves infected
with BIM2 with B69
Poly A+ RNA Isolation

f
AAAAAAA cDNA Synthesis AAAAAAA
Sby Reverse Transcriptase Y

AAAAAAADouble-stranded cDNA Synthesis T
-AAAAAAA AAAAAAA
Restriction Enzyme Digestion 4

Dnver withRsa I Tester

Adaptor Ligation to the Tester DNA Adaptor tor 2R
Dnver




Firnt Hybridiation
a 68 OC for 8 hrs
b m
c-- c

d Dnver d{


Second Hybridization
68 OC for 16 hrs
aIh



d{

em

SFill in the ends

b
aE-E

d_

e
PCR Amplification
using an Adaptor Pnmer

a and d No amplification
b b b No amphfication
c Linear amphfication
511 1 3'
3 5' e Exponential amphfication
3 ~ 5'

Figure 2-4: Diagram of PCR-Select cDNA subtraction. Type e molecules are formed only

if the sequence is up-regulated in the tester cDNA. Solid lines represent the

Rsa Idigested tester or driver cDNA. Solid boxes represent the outer part of

the Adaptor 1 and 2R longer strands and corresponding PCR primer 1

sequence. Green boxes represent the inner part of Adaptor 1 and the

corresponding Nested PCR primer 1 sequence; red boxes represent the inner

part of Adaptor 2R and the corresponding Nested PCR primer 2R sequence.














unknown n function
8%


metabolism/energy
9%


abiotic and biotic sires-
response
6%

/
/




no homology
24%















B) RS


ribosomal protein
20%


abiotic and biotic stress
response
1 ..







no homology
14%


Iranscription and translation
6%


cell growth and division
10%



secondary metabolism
4%
protein stability and degradation
3%


transport
4%
signaling
5%


hormone metabolism and
signaling
1%


unknown function
1%


metabolism/energy
14%


transcription and translation
6%


cell growth and division
2%

secondary metabolism
6%


protein stability/degradation
4%


ribosomal protein
28%


transport
,. 5%
signaling
3%

hor no:,ne metabolism and
signaling
2%


Figure 2-5: Distribution of potential citrus canker responsive genes.


A)FS











200


150 1

SDD
100 101
131 |SSH

50-


0





Figure 2-6: Distribution and origin of the clones stamped on the nitrocellulose
membranes used in reverse northern blot analysis.








58











539
237
1435
1385
1243
1239
1051
915
767
475
409
279
243
1415
1339
1057
959
809
171
137
113
109
33
1345
1312
1139
1061
1453
1445
1262
1065
673
575
571
497
493
343
339
313
1535
1511
901
889
501
501
1258
1111





Figure 2-7: Cluster analysis of genes differentially regulated by PthB. In green are genes
down-regulated by PthB, and in red are genes up-regulated by PthB










BIM2


B69


BIM2


B69


CCR137


GAST1


BIM2

i-? 1" .-
">0:.o J. .. .


Exp


rRNA


LAT52


S
ar


4m


yTIP



18 S


Figure 2-8: Northern blot analysis of CCR genes found differentially regulated by reverse
northern blot analysis. rRNA was used as control for loading.


2 dpi


Enod8


rRNA


2 dpi

cellulase


2 dpi


B69













































Figure 2-9: Microscopic phenotype of leaves inoculated with B69 (wt) and BIM2
(nonpathogenic mutant lacking PthB). 7dpi BIM2 infected leaves, A; 7dpi
B69 infected leaves, B, C. By In canker-infected tissue, by 7 dpi, air spaces of
the spongy mesophyll are almost inexistent. These spaces are replaced by new
divided cells as well as by cell of larger size, resulting in thickening of the
leaves.






































Figure 2-10: Microscopic phenotype of leaves inoculated with B69 (wt) and BIM2
(nonpathogenic mutant lacking PthB). 7dpi BIM2 infected leaves, C; 7dpi
B69 infected leaves, A, B, D. At 40X magnification, pockets of bacterial cells
are visible surrounding mesophyll cells of B69 infected tissue while almost no
bacteria is present in BIM2 infected tissue. Also not the areas of cell lysis in
B69 infected tissue.





































Figure 2-11: Microscopic phenotype of leaves inoculated with B69 (wt) and BIM2
(nonpathogenic mutant lacking PthB) at 14 dpi. A, B: BIM2, and C, D:B69
infected leaves. Note High levels of bacteria in B69 infected leaves compared
to BIM2 infected leaves, as well as possible wall thinning of cells in B69
infected tissue.







63




1000


C 800 -- B69
---- BIM2
600


400


200


0
0 -----------------------
0 2 7 14
30

25-

S g 20

x 15 -

il 10-
o ~o
5 -

0
0 2 7 14
Days post inoculation



Figure 2-13: Quantification of leaf thickening and cell division during B69 and BIM2
infection on Duncan grapefruit leaves. These measurements where taken on
"slow canker-developing" leaves, i.e. leaves showing high rate of cell division
when inoculated with B69. The number of cells from abaxial epidermis to
adaxial epidermis was calculated by counting the number of cells that a virtual
line perpendicular to the epidermal layers would cross. Ten lanes were used n
the analysis and the number shown are averages.



































Figure 2-13: Microscopic symptoms of rapidly developing canker. 14dpi BIM2 infected
leaves, A; 14dpi B69 infected leaves, B, C, D. Note the highly enlarged cells
the large areas of cell lysis and the absence of high rate of cell division in B69
infected tissue.























Cell division Cell expansion
Effector molecules: Effector molecules:
9 Exps, PLs, cellulase, Mannanase, TIP3,
Enod8, GAST1, RAB8B, BetaCOP



9 4......................... Cell wall loosening








V
Cell Division """"""""""""......................... Cell Expansion





Rapid multiplication ofX. citri and Canker phenotypes


Figure 2-14: Possible model for PthB effects on susceptible citrus cell showing
parallel pathways activating cell division and expansion.














CHAPTER 3
CHANGES IN SUMO CONJUGATION ARE ASSOCIATED WITH CITRUS
CANKER DISEASE

Introduction

Citrus canker is an important disease of citrus worldwide (Civerolo, E., 1984). It is

caused by several pathovars ofXanthomonas citri, which differ mainly in their host range

(Shubert et al, 2001, Verniere et al, 1998). Canker infections cause defoliation, fruit

blemishes, premature fruit drop and tree decline, resulting in severe economical losses

(Shubert et al, 2001). Considerable international regulatory efforts are implemented to

prevent the spreading of the already quarantined pathogen, with negative effects on

national and international trade of citrus (Timmer et al, 1996; Shubert et al, 2002).

Canker symptoms are characterized by erumpent corky lesions that can affect all

aerial parts of citrus trees (Shubert et al, 2002). Microscopy studies showed that canker

lesions result from hyperplasia (cell division) and hypertrophy (cell expansion) in the

spongy mesophyll tissue, where the bacteria contact plant cells (Swarup et al, 1991; Duan

et al, 1999 and Chapter 2). Ultimately, this intense increase in cellular growth ruptures

the epidermis and causes necrosis. The rupture of the epidermis is thought to be crucial

for bacterial dissemination and spread of the disease (Graham and Gottwald, 1991; Duan

et al, 1999).

A crucial step towards understanding citrus canker disease was the identification of

a pathogenicity gene, pthA, required by X citri pv. citri to cause canker on citrus (Swarup

et al., 1991). Since then, all canker-causing strains have been shown to carry at least two









members of the pthA gene family, with one copy sufficient for most or all pathogenicity

(Yang and Gabriel, 1995; Al-Saadi and Gabriel unpublished). pthA, found in X citri pv.

citri (Xcc) of the Asiatic group of strains, and pthB, found in X citri pv. aurantifolii B69

(Xca) of the South American group, have been shown to be interchangeable in their

ability to elicit canker (Yuan and Gabriel, unpublished). As forpthA ofXcc, pthB of Xca

was also shown to be required for pathogenicity on citrus (Yuan and Gabriel,

unpublished, and Chapter 2), and therefore, the B69 derivative mutant strain BIM2

lacking pthB does not elicit the typical macroscopic symptoms associated with canker

disease (Chapter 2). When transferred to other xanthomonads carrying a functional type

III secretion system (TTSS), or transiently expressed in leaf cells, pthA was found to

induce cell division, cell expansion, and rupture of the epidermis the three most

prevalent canker symptoms (Swarup et al, 1991 and 1992; Duan et al, 1999). It was

therefore concluded thatpthA alone was able to cause canker-like symptoms and that its

delivery into the plant cell relies on a functional TTSS.

Members of thepthA gene family are also found in non-canker causing strains of

Xanthomonas. Examples of genes belonging to this gene family include avrBs3 and

avrBs3-2 of Xanthomonas campestris pv. vesicatoria (Bonas et al, 1989, and Bonas et al,

1993), avrXalO and avrXa7 of Xanthomonas oryzae pv. oryzae (Hopkins et al, 1992);

along with avrB4, avrb6, and avrb7 of Xanthomonas campestris pv. malvacearum (De

Feyter and Gabriel, 1991 and 1993). Proteins encoded by members of this gene family

are 90 to 97% similar and are characterized by several structural features essential for

their function in avirulence and/or pathogenicity. Such features include 1) nearly identical

102-bp tandem repeats in their center, 2) C-terminal nuclear localization signals (NLS),









and 3) C-terminal eukaryotic acidic transcriptional activator (Herbers et al, 1992; Yang et

al, 1994; Zhu et al, 1998; Yang et al, 2000; Yang and Gabriel, 1995; Van den

Ackerveken et al, 1996, Szurek et al, 2001).

Little is known about how canker disease is initiated inplanta. In order to

understand the molecular mechanism underlying canker, a differential display PCR

experiment was conducted to identify plant genes potentially responsive to canker

(Chapter 2). At two days post inoculation (dpi), transcripts were compared between

leaves inoculated with B69 and leaves inoculated with BIM2 (B69 derivative carrying a

non-functional pthB). One clone was related to AtSUMO1 from Arabidopsis. SUMO

belongs to the ubiquitin family of proteins that are conjugated to target proteins;

however;its functions are distinct from those of ubiquitin.

SUMO conjugation has been shown to be an important regulatory step in processes

such as protein stability, subcellular localization, and response to various stresses.

SUMOylation is carried out in a ATP-dependant reaction cascade similar to the E1-E2-

E3 reactions responsible for ubiquitin conjugation (Melchior F., 2000; Kim et al, 2002;

Kurepa et al, 2003). In addition, SUMO modification has been shown to be important for

cell cycle progression in yeast. Specifically, temperature-sensitive mutants lacking a

functional SUMO conjugation pathway have been shown to arrest at the G2/M transition

(Seufert et al, 1995; Johnson and Gupta, 2001). Such work is of interest, as Xanthomonas

citri infection triggers division of mesophyll cells contacted by the bacteria.

Recent work has shown that strains of the phytopathogenic bacterium

Xanthomonas campestris pv. vesicatoria encode at least two type III effectors with

demonstrated SUMO protease activity (Hotson et al, 2003; Roden et al, 2004). Though









loss of these SUMO protease-like effectors did not affect pathogenicity on susceptible

plants, it raises the possibility that the plant SUMO conjugation pathway could be

targeted during infection by X c. vesicatoria (Hotson et al, 2003; Roden et al, 2004).

This study indicates that: 1) changes in plant protein SUMOylation profiles

occurred after host infection by Xanthomonas citri pv. aurantifolii, 2) these changes in

SUMOylation profiles were of two types, gene pthB-dependent and independent, and 3)

these changes in SUMOylation profiles did not occur following challenge with a non-

pathogenic mutant strain lacking a TTSS. Together, these data indicate that the TTSS of

Xca delivers one or more effectors that directly, or indirectly, de-conjugate SUMO from

host proteins in vivo.

Materials and Methods

Plant Inoculations

All inoculations were done with needle-less syringes on the abaxial surface of the

leaf. Plants (Citrus paradisi 'Duncan' grapefruit) were grown under greenhouse

conditions. Inoculations involving strains B69 and its derivatives were carried out in BL-

3P level containment (refer to Federal Register Vol.52 no 154, 1987) at the Division of

Plant industry, Florida Department of Agriculture, Gainesville, FL. For inoculation,

bacterial suspensions were standardized in sterile 10mM CaCO3 (mock) to an optical

density of 0.5 and pressure-infiltrated. For phenotypic observation, inoculations were

repeated at least three times. For protein extraction, a split leaf inoculation scheme was

followed to normalize differences due to physiological state of inoculated tissue. For each

combination of treatments (i.e. mock/B69 and mock/BIM2), one treatment was

inoculated on the right side of the mid-vein and the other strain on the left side of the









mid-vein. For each split-leaf experiment three trees were used, with an average of 10

leaves inoculated per tree (approximately 5 leaves per treatment combination).

Bacterial Strains and Culture Media

Bacterial strains and plasmids used in this study are listed in Table 1 Appendix A.

All Xanthomonas strains were cultured in PYGM medium at 300C (De Feyter et al.

1990). Escherichia coli were grown on Luria-Bertani (LB) medium (Sambrook et al.,

1990). For culture on solid media, agar was added at 15 g/L. Antibiotics were used at the

following concentrations: Spectinomycin (Sp), 35 mg/L; Kanamycin (Kn), 12.5 mg/L;

Chloramphenicol (Cm), 35 mg/L; Gentomycin (Gt), 3 mg/L.

Marker Integration Mutagenesis

hrpG gene knock-out mutation was generated by triparental matings (as described

in Chapter 1). Briefly, a 550 bp internal fragment of hrpG was cloned in the suicide

vector pUFR012 [derivative of pUFR004 carrying kanamycin resistance (Gabriel

laboratory, unpublished)] creating pBY23. Transconjugants resulting from E. coli

DH5 /pBY23, DH5 /pRK2013 (helper plasmid) and B69 matings were selected on

spectinomycin to select against E. coli and chloramphenicol and kanamycin to select for

plasmid insertion events. Putative transconjugants were purified to a single colony, and

Southern hybridization was used to confirm the integration of suicide vector pBY23 in

hrpG.

For complementation purposes, a HindIII to KpnI fragment was cloned out of

plasmid pXG8 (REF) and recloned in pUFR053 (Yuan and Gabriel, unpublished)

creating pBY24. DH5ca/pBY24 was used in triparental matings to create B23.5/pBY24

(B23.5c and B23.5cl). Putative exconjugants were purified to a single colony, and









Southern hybridization was used to confirm the presence of the complementation

plasmid. Total DNA extractions were performed as described in Gabriel and De Feyter

(1992). Southern hybridizations were performed as described by Lazo and Gabriel

(1987).

Bioinformatics

Alignments and box shading were carried out using Clustal W

(http://clustalw.genome.jp).

Protein Extraction and Western Blotting

Citrus leaf tissue was harvested at 0, 2 or 7 days post inoculation (dpi), depending

on the experiment, and ground to a fine powder in liquid nitrogen. Soluble proteins were

extracted in two volumes of extraction buffer (50mM Tris, pH = 8.0, 300mM sucrose,

2mM EDTA, 0.3% DIECA, 10mM N-ethylmaleimide, ltg/tl pepstatin, 1 tgg/tl

leupeptin, and 7.5% w/v PVPP). Extracts were vortexed and briefly sonicated, then

clarified by two rounds of centrifugation at 16,000 x g for 10 min at 40C. Soluble

proteins were quantified by the BCA assay (Pierce Biotechnology, Rockford, IL).

Proteins were separated by polyacryalmide electrophoresis on a 15% Tris-Tricine

gel, and transferred to PVDF membrane (Millipore, Bedford, MA). For immunoblot

analysis, membranes were probed with 1:2,500 immunopurified polyclonal PopSUMOl

(gi:23997054) antiserum (Cocalico, Reamstown, PA) diluted in phosphate buffered saline

(137 mM NaC1, 2.7 mM KC1, 1.4 mM K2HPO4, 10.1 mM Na2HPO4, pH 7.4) containing

0.1% Tween 20 (T-PBS) with 1% v/v goat serum (Sigma, St. Louis, MO). The antibodies

were raised against purified PopSUMO1, which also contained an additional N-terminal

hexahistidine tag generated by PCR (Reed, J., Master's Thesis University of Florida,

2005). For secondary antibody, the membranes were probed with 1:25,000 horseradish









peroxidase conjugated donkey anti-rabbit secondary antibodies (Amersham,

Buckinghamshire, England) diluted in IX T-PBS. Chemilluminescence was carried out

according to the manufacturer's instructions using the ECL plus (+) kit (Amersham).

Following chemilluminescence, each membrane was rinsed in IX T-PBS and stained

with Coomassie R250 as a loading control.

Results

SUMO Conjugation Profiles are Altered in X citri-Infected Leaves

The grapefruit partial cDNA, CCR915 was identified by differential display as

being canker responsive. Following reverse northern blot analysis, CCR915 which shows

homology to SUMO, was found up-regulated in leaves inoculated with BIM2 (lacking

pthB) compared to leaves inoculated with B69 (wt) (Chapter 2). To determine if shifts in

SUMO transcript abundance reflected regulation at the protein levels, a split-leaf

experiment was conducted in which Duncan grapefruit leaves were mock inoculated on

one side of the mid-vein, and Xanthomonas citri pv. aurantifolii strain B69 was

inoculated on the other side. Soluble extracts taken from canker or mock -inoculated

leaves were probed for CitSUMO and CitSUMO-conjugated proteins using PopSUMO1

antibodies. The grapefruit sequence was highly similar to poplar SUMO isoform

PopSUMO1 (gi:23997054) (Figure 3-1) and as expected, the grapefruit SUMO and its

protein conjugates cross-reacted with antibodies raised against PopSUMO 1. Using anti

PopSUMOl, it was found that at two days post inoculation, the profile of SUMO

conjugation is noticeably altered (Figure 3-2). The amounts of free CitSUMO and high

molecular weight CitSUMO conjugated proteins were higher in B69-infiltrated leaves as

compared to mock-infiltrated leaves.









SUMO Conjugation Profiles in Infected Leaves are Partially PthB Dependent

To determine if SUMOylation patterns were associated with disease symptom

development, a split leaf inoculation experiment was conducted and the effects of three

separate treatments examined over time. Split-leaves were mock infiltrated, or inoculated

with wild type strain B69, or the non-pathogenic mutant strain BIM2, which lacks pthB.

At 0, 2, and 7 dpi, half-leaves were harvested and soluble proteins examined by western

blot analysis.

SUMO profiles of leaves inoculated with B69 were compared to those of leaves

inoculated with mutant BIM2 at two dpi. There were no changes in the abundance of free

CitSUMO or SUMOylated proteins in BIM2 inoculated leaves (Figure 3-3, lane 4 and 5).

The expected changes were seen in leaves inoculated with B69, i.e. an increase in the

amount of free SUMO and SUMO-conjugated proteins (Figure 3-3, lane 7 and 8).

SUMO profiles at 7 days post inoculation revealed that the majority of the high

molecular weight conjugates seen at 2 dpi in canker infected leaves were lost (Figure 3-3,

lane 8 and 9). Interestingly, this loss of high molecular weight conjugates was also

observed in leaves inoculated with non-pathogenic mutant strain BIM2. Whether the

identities of SUMOylated proteins in canker infected leaves are similar to the ones in

BIM2 infected leaves is unknown; however, in both cases, SUMO de-conjugation

occurred 7 dpi. These findings suggest that the SUMO de-conjugation observed at 7 dpi,

in both BIM2- and B69-inoculated leaves is PthB-independent and is also independent of

the development of the macromolecular disease symptom of canker (Figure 3-4).

Conversely, the increase in the amounts of free SUMO and SUMO-conjugated proteins

seen at 2 dpi with B69 are PthB-dependent.









SUMO De-Conjugation Observed at 7 days Following Infection with B69 and BIM2
is Dependent on a Functional Type III Secretion System

To determine if the SUMO de-conjugation observed at day 7 post inoculation in

both B69- and BIM2-inoculated leaves is dependent on a functional type III secretion

system, a hrpG integrative mutant, B23.5, was generated. B23.5 was no longer

pathogenic on citrus, and the hrpG- phenotype was complemented after transformation of

B23.5 with pUFR057::XcvhrpG (Figure 3-5).

There was no SUMO de-conjugation at day 7 following inoculation with B23.5

(Figure 3-6), indicating that SUMO de-conjugation relies on a functional TTSS. In

addition, B23.5 inoculation stimulated accumulation of a 45kDa SUMO conjugate. A

SUMOylated product of similar size was observed in leaves inoculated with B69 and

BIM2, but did not accumulate (Figure 3-3).

Discussion

A great deal of effort has been directed towards investigating the mechanisms by

which plants mount defense responses towards pathogenic bacteria. Most studied cases

involve incompatible plant microbe interactions that lead to the classical hypersensitive

response or HR (Malek et al., 2000; Kazan et al, 2001). However, far less effort has been

invested in trying to elucidate the mechanisms by which a specific pathogen, or a group

of pathogens elicit a particular disease with specific sets of morphological and molecular

symptoms. In an effort to understand the processes by which different pathovars of

Xanthomonas citri trigger canker symptoms, a canker responsive gene with sequence

similarity to the SUMO gene family was identified by differential display PCR.

The SUMO conjugation pathway in canker disease was investigated using a split-

leaf inoculation experiment to normalize for leaf-to-leaf variations. It was found that at 2









dpi, X citri pv. aurantifolii infection induces an increase in free CitSUMO and an

increase in the number of high molecular weight SUMOylated proteins. These changes

were not observed in mock-inoculated leaves. SUMO conjugation in plants and other

systems has been shown to be up-regulated by various instances of biotic and abiotic

stresses (Kurepa et. al., 2003, Lois et. al., 2003 and O'Donnell et. al., 2003).

In order to test if changes in SUMO conjugation observed were specific to X citri

pv. aurantifolii infection, two mutant strains unable to cause canker on citrus were used in

this study, BIM2 (interrupted in pathogenicity gene pthB) and B23.5 (interrupted in the

TTSS regulatory gene, hrpG). Disruption of hrpG was previously shown to disable the

type III secretion system in Xanthomonas (Wengelnik et al. 1996). Using split leaf

inoculations, it was shown that in BIM2 inoculated leaves, at 2 dpi, there were no

changes in the amount of free SUMO and SUMOylated high molecular weight proteins.

Thus, the increase in free SUMO and in the number of SUMOylated proteins is likely to

be a PthB-specific plant response rather than a general stress response. A large number of

SUMO targets identified in other organisms are cell-cycle related (Melchior, 2000). It has

been shown in yeast (Saccharomyces cerevisiae) that temperature-sensitive mutants

corresponding to SUMO and the enzymes involved in its conjugation pathway arrest the

cell cycle at the G2/M transition, therefore, showing a critical role for SUMO in cell

cycle progression (Johnson and Gupta, 2001). It is possible that the observed up-

regulation of SUMOylated proteins and free SUMO reflects activation of the plant cell

cycle by X c. pv. aurantifolii in the early stages of infection. Remarkably, this increase in

free SUMO and in the amount of high molecular weight SUMOylated proteins is lost 7

dpi, potentially indicating a transition to a second disease phase. The deconjugation









phenotype observed at 7 dpi with B69 is also observable at 7 dpi with BIM2, and

therefore, the triggering factor of de-conjugation is probably independent of PthB.

The possibility that another effector could be the trigger of the de-conjugation

observed at 7 dpi came from the finding that the TTSS mutant B23.5, did not induce de-

conjugation. Therefore it is possible that another type three effector, beside PthB is

responsible for the de-conjugation observed at day 7. Alternatively, it is possible that the

second PthA homologue, PthB0 (not required for canker, Chapter 2), found in B69 and

BIM2 is also able to trigger the de-conjugation observed 7dpi.

It has been proposed that the abundance of SUMO proteases in X campestris pv.

vesicatoria could reflect an important role of theses effectors in Xcv pathogenesis (Hotson

et al. 2003 and Roden et al 2004). However, none of the identified proteases have been

implicated in disease and are, in fact, dispensable. Given the critical role of SUMO

conjugation in cell cycle processes (Melchior, 2000), and the lack of apparent SUMO

proteases encoded by another canker causing strain X citri pv. citri it is possible that the

late de-conjugation phenotype is not directly triggered by a type III effector of a protease

nature, but rather that a type III effector(s) acts to induce endogenous citrus SUMO

protease(s) leading to the de-conjugation observed in late stages of canker infection.

Both hypotheses are not mutually exclusive and characterization of additional X

citri effectors as well as citrus proteins SUMOylated in response to canker are required to

better characterize the involvement of SUMOylation in the infection process of canker

causing xanthomonads.














popSUMO1
GfSUMO/CCR915
AtSUMOI


popSUMO1
GfSUMO/CCR915
AtSUMO1


MSEATGQPQEEDKKPNDQSAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVEIN 60
------------------EFHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVEIN 42
MSAN----QEEDKKPGDGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMN 56


SIAFLFDGRRLRGEQTPDELDMEDGDEIDAMLHQTGGAVKASDYA 105
SIAFLFDGRRLRGEQTPDELDM----------------------- 64
SIAFLFDGRRLRAEQTPDELDMEDGDEIDAMLHQTGGSGGGATA- 100


Figure 3-1: Alignment of grapefruit SUMO (partial sequence) with (PopSUMO1,
gi:23997054, and AtSUMO1, At4g26840).






78





A

206.7\
115.8
98.0-
Cu
54.6
U)
37.4

29.6-

o 20.4-


7.0

B I m




Figure 3-2: SUMO profiles of B69- and mock-challenged grapefruit leaves. 10pg of
crude protein from day 2 of the split leaf experiment was separated by
electrophoresis, blotted to PVDF and (A) probed with purified PopSUMO
antisera. Lane 1, Mock treated leaf; lane 2, B69 inoculated leaf; lane 3, 2 ng
purified recombinant PopSUMO ([]): high molecular weight SUMOylated
proteins. (->): un-conjugated SUMO. (B) The membrane was stained with
Coomassie R250 as a loading control (Shown is the small subunit of Rubisco).











A Mock
027
0 2 7


BIM2
027


=P WP a M ff"
WI, ..


B69
027


lop "
'U
-m


I m - -
29.6 m
20.4
wo e~memu.I


Treatment
DPI


Figure 3-3: SUMO de-conjugation occurs 7 days after infection. Leaves were inoculated
with Mock, BIM2, and B69 strains. 7.5 [g of crude protein from 0, 2, and 7
dpi from each treatment of the split leaf experiment was separated by
electrophoresis, blotted to PVDF and (Upper panel) probed with purified
PopSUMO1 antisera. ([]): high molecular weight SUMOylated proteins. (-):
un-conjugated SUMO. (Lower panel) The membrane was stained with
Coomassie R250 as a loading control (Shown is the small subunit of Rubisco).


206.7
115.8
98.0-
54.6
37.4














i i






Figure 3-4: Split leaf inoculation ofXanthomonas citri pv. aurantifolii (B69) and
derivative BIM2 mutant. Duncan grapefruit leaf 7 dpi with B69 (shown on the
left side of the mid-vein and BIM2 (shown on the right side of the mid-vein).
(A) adaxial side and (B) abaxial side of the leaf. Note the whitish canker
characteristic of the Xca strain and yellowing associated with the day 7 post
inoculation canker phenotype. (C) Advanced B69 canker phenotype.















X Hin dIII


B69 B23.5 B23.5c




d-om


Figure 3-5: B69 mutant derivative B23.5 lacks a functional Type III secretion system. (A)
Southern blot hybridization profiles contrast B69, B23.5 and B23.5c
(B23.5/hrpG). DNA was digested with HindIII and probed with the same
internal fragment of hrpG used as homology region for marker interruption.
(B) B69 and B23.5c inoculation on Duncan grapefruit. hrpG complemented
the hrp phenotype of B23.5










B23.5
0 2 7


B69
0 2 7


206.7 .
115.8-
98.0 &
54.6-

37.4-

29.6-

20.4-


7.0-


J
*


Figure 3-6: SUMO de-conjugation at 7 dpi requires a functional TTSS. Leaves were
inoculated with B23.5 and B69 strains. 7.5[tg of crude protein from 0, 2, and
7 dpi from each split leaf treatment was separated by electrophoresis, blotted
to PVDF and (A) probed with purified PopSUMO1 antisera. ([]): high
molecular weight SUMOylated proteins. (->): un-conjugated SUMO. (*)
novel 70kDa protein unique to B23.5 7 dpi leaves. Equal amounts of protein
was loaded in each lane.


- m




m mM dm
a*4














APPENDIX A
LIST OF PLASMIDS AND STRAINS


Table A-i: List of strains and plasmids used in this study.
Strains or plasmids Relevant characteristics Reference or source
Escherichia coli
DH5a F-, endA1, hsdR17(rk-mk), Gibco BRL, Gaithesburg,
supE44, thi-1, recA1, gyrA, MD
relAl.,80OdlacZAM15,
A(lacZYA-argF)U169
HB 101 supE44, hsdS20(rk-mk), Boyer and Roulland-
recA 13, ara-14, proA2, Dussoix
lacY1, galK2, rpsL20, xyl-5,
mtl-1, SmR
ED8767 supE44, supF58, hsdS3(rkrkr), Murray et al. 1977
recA56, galK2, galT22,
metB1l
Xanthomonas
3213T' X citri pv. citri A Gabriel et al, 1989
3213Sp X citri pv. citri A, SpR Swamp et al., 1991
derivative of 3213
B21.1 pthA::Tn5-gusA, marker Swamp et al., 1991
exchanged mutant of 3213 Sp,
SpRKnR
B69 X axonopodis pv. aurantifolii
69, ATCC, B form citrus
canker type strain
B69Sp Spntaneous SpR derivative of Unpublished
69, SpR
BIM2 pthB::CmR, marker integration Unpublished
mutant of B69Sp, SpRCmR
BIM6 Marker integration mutant of Unpublished
B69Sp, CmR integrated
upstream ofpthB, SpRCmR
B13.2 VirB4::CmR, marker This study
integration mutant of B69Sp,
SpRCmR









Table A-1. Continued
Strains or plasmids Relevant characteristics Reference or source
B 13.1 VirB4o::CmR, marker This study
integration mutant of B69Sp,
SpRCmR
B69.4 Unpublished
pRK2013 ColE1, KmR,Tra helper Figurski and Helinski, 1979
plasmid
pUFR004 ColE1, Mob+, Cmr, lacZ+ De Feyter et al, 1990
pUFR012 Derivative of pUFR004 with Unpublished
Kn resistance. ColE1, Mob+,
KnRCmR, lacZUa+
pBY13 270 bp fragment of virB4 This study
cloned in pUFR004, CmR
pB13.1 virB4::pBY13 of pXcBO, CmR This study
pB13.2, pB13.4, pB13.5 virB4::pBY13 ofpXcB, CmR This study
PXcB Natural plasmid of B69 Unpublished
carrying pthB
pXcBO Natural plasmid of B69 Unpublished
carrying pthBO
pBIM2 pthB::CmR(pYY40.10) of Unpublished
pXcB, CmR
pBIM6 pXcB:: CmR(pQY92. 1), pthB Unpublished
still functional, CmR
pBY23 550 bp fragment of hrpG This study
cloned in pUFR012, KnR
CmR
pBY23c HrpG from pXG8 (REF) This study
cloned in pUFR53
B23.5 hrpG::pBY23 of B69, KnR This study
CmR
B23.5c B23.5/pBY23c This study

















APPENDIX B
NORTHERN BLOT ANALYSIS OF CCRS


Mock BIM2 B69 Mock BIM2 B69
PR1

PR2 lrr

iss f l 'A P* w ***
2 dpi 7 dpi
Mock BIM2 B69 BIM2 B69


2 dpi B69 BIM2
CHI

rRNA


GST


rRNAMC


RD22


rRNA m

2 dpi B69
PR5 5


2 dpi B69
Frap/tor uiAW


V 1


BIM2

is


BIM2

w


rRNA


2dpi 7dpi
CaCO3 BIM2 B69 BI2
TIP mu
Ti U^^^I


rRNA


2 dpi

P Exp


BIM2


rRNA


2 dpi B69 BIM2

pip3

18 S I


Figure B-1: Northern blot analysis of CCR genes not found differentially regulated by
reverse northern blot


e


-I -.Wdk JM& -
















LIST OF REFERENCES


Alfano, J.R. and Collmer, A. 1996. Bacterial pathogens in plants: life up against the wall.
Plant Cell. 8: 1683-1698.

Alfano, J.R. and Collmer, A. 1997. The type III (Hrp) secretion pathway of plant
pathogenic bacteria: trafficking harpins, Avr proteins and death. J. Bacteriol.
179:5655-5662.

Anderson, D.M., Fouts, D.E., Collmer, A. and Schneewind, O. 1999. Reciprocal
secretion of proteins by the bacterial type III machines of plant and animal
pathogens suggests recognition of mRNA targeting signals. Proc. Nat. Acad. Sci.
USA. 96:12839-12843.

Bergey's Mannual of Determinative Bacteriology, 9th Eddition, JG Holt (ed.), Williams
and Wilkins, Baltimore, MD, USA.

Bonas, U., Stall, R.E. and Staskawicz, B. 1989. Genetic and structural characterization of
the avirulence gene, avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol. &
Gen. Genet. 218:127-136.

Boyer, H. W., and Roulland-Dussoix, D. 1969. A complementation analysis of the
restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459-465.

Brunings A.M. and Gabriel D.W. 2003. Xanthomonas citri: breaking the surface. Molec.
Plant Pathol. 4(3):141-157.

Burns, D.L., 1999. Biochemistry of type IV secretion. Curr. Opin. Microbiol. 1999.
2(1):25-29.

Christie, P.J. 1997. Agrobacterium T-Complex transport apparatus: a paradigm for a new
family of multifunctional transporters in Eubacteria. J. Bacteriol. 179:3085-3094.

Christie, P.J. 2001. Type IV secretion: intracellular transfer of macromolecules by
systems ancestrally related to conjugation machines. Mol. Microbiol. 40:294-305.

Christie, P.J. and Vogel, J.P. 2000. Bacterial type IV secretion: conjugation systems
adapted to deliver effector molecules to host cells. Trends Microbiol. 8:354-360.

Cornelis, G.R. and VanGijsegem, F. 2000. Assembly and function of type III secretary
systems. Annu. Rev. Microbiol. 54:735-774.









Cubero, J. and Graham, J.H. 2002. Genetic relationship among worldwide strains of
Xanthomonas causing canker in citrus species and design of new primers for their
identification by PCR. Appl. Environ. Microbiol. 68:1257-1264.

da Silva, A. C., J. A. Ferro, et al. (2002). "Comparison of the genomes of two
Xanthomonas pathogens with differing host specificities." Nature 417(6887): 459-
63.

De Feyter, R., Kado, C. I., and Gabriel, D. W. 1990. Small stable shuttle vectors for use
in Xanthomonas. Gene 88:65-72.

De Feyter, R., and Gabriel, D. W. 1991. At least six avirulence genes are clustered on a
90-kilobase plasmid in Xanthomonas campestris pv. malvacearum. Mol. Plant-
Microbe Interact. 4:423-432.

De Feyter, R., Yang, Y., and Gabriel, D. W. 1993. Gene-for-genes interactions between
cotton R genes and Xanthomonas campestris pv. malvacearum avr genes. Mol.
Plant-Microbe Interact. 6:225-237.

Duan, Y.P., Castaneda, A.L., Zaho, G., Erdos, G. and Gabriel, D.W. 1999. Expression of
a single, host-specific, bacterial pathogenicity gene in plant cells elicits division,
enlargement and cell death. Mol. Plant-Microbe Interact. 12:556-560.

Egel, D.S., Graham, J.H. and Stall, R.E. 1991. Genomic relatedness of Xanthomonas
campestris strains causing diseases of citrus. Appl. Environ. Microbiol.
57:2724-2730.

El Yacoubi, B., Brunings,A., Yuan, Q. and Gabriel, D.W. 2001. A self-transmissible
plasmid isolated from Xanthomonas campestris carries a member of the avr/pth
gene family and additional factors) required for pathogenicity. Abstract of the 10th
International Congress of Molecular Plant-Microbe Interactions, Madison, WI, 10-
14 July 2000, #650.

Falcow, S. 1996. The evolution of pathogenicity in Escherichia, ./ngel//l and Salmonela;
in Cellular and Molecular biology (ed.) F.C. Neidhadz (Washington DC: American
Society for Microbiology). 2723-2729.

Figurski, D. H., and Helinski, D. R. 1979. Replication of an origin-containing derivatives
of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl.
Acad. Sci. USA 76:1648-1652.

Gabriel, D. W., Kingsley, M., Hunter, J. E., and Gottwald, T. R. 1989. Reinstatement of
Xanthomonas citri (ex Hasse) and X phaseoli (ex Smith) and reclassification of all
X campestris pv. citri strains. Int. J. Syst. Bacteriol. 39:14-22.









Gabriel, D. W., and De Feyter, R. 1992. RFLP analyses and gene tagging for bacterial
identification and taxonomy. Pages 51-66 in: Molecular Plant Pathology: A
Practical Approach. Vol. 1. S. J. Gurr, M. J. McPherson, and D. J. Bowles, eds.
IRL Press, Oxford.

Gabriel, D.W. 1999. Why do plant pathogens carry avirulence genes? Physiol. Mol. Plant
Pathol. 55: 205-214.

Gottwald, T.R., Graham, J.H., Schubert, T.S. 2002. Citris canker: the pathogen and its
inpact. Online. Plant health Progress. doi:10.1094/PHP-2002-0812-01-
RV.http://plant managementnetwork. org/pub/php/review/citruscanker/.

Graham, J.H., Gottwald, T.R., Cubero, J., Achor, D.S. 2004. Xanthomonas axonopodis
pv citri factors affecting successful eradication of citrus canker. Molec. Plant
Pathol. 5:1-15.

He, S.Y. 1998. Type III protein secretion system in plant and animal pathogenic bacteria.
Annu. Rev. Phytopathol. 36: 363-392.

Hildebrand, D.C., Palleroni, N.J. and Schroth, M.N. 1990. Deoxyribonucleic acid
relatedness of 24 xanthomonad strains representing 23 Xanthomonas campestris
pathovars and Xanthomonasfragariae. J. Appl. Bacteriol. 68: 263-269.

Jin, Q. and S.Y. He (2001). "Role of the Hrp pilus in type III protein secretion in
Pseudomonas syringae." Science 294(5551): 2556-2558.

Jones, J.B., Bouzar, H., Stall, R.E., Almira, E.C., Roberts, P.D., Bowen, B.W., Subderry,
J., Strickler, P.M., and Chun, J. 2000. Systematic analysis of Xanthomonads
(Xanthomonas spp.) associated with pepper and tomato lesions. Int. J. Syst. Evol.
Microbiol. 50:1211-1219.

Keen N.T. 1990. Gene for gene complementrity in plant-pathogen interactions. Annu.
Rev. Genet. 24:447-63.

Kingsley, M.T., Gabriel, D.W., Marlow G.C. and Roberts, P.D. 1993. The opsXlocus of
Xanthomonas campestris affects host range and biosynthesis of lipopolysaccharide
and extracellular polysaccharide. J. Bacteriol. 175:5839-5850.

Kubori, T. Matsushima, Y., Nakamura, D., Uralil, J., Lara-Tajero, M., Sukhan, A., Galan,
J.E., and Aizawa, S. 1998. Supramolecular structure of the Salmonella typhimurium
type III pretein secretion system. Science. 280:602-605.

Lawrance, J.G. and Roth, J.R. 1996. Selfish operons: horizontal transfer may drive the
evolution of gene clusters. Genetics. 143: 1843-9417.

Lazo, G. R., and Gabriel, D. W. 1987. Conservation of plasmid DNA sequences and
pathovar identification of strains ofXanthomonas campestris. Phytopathology 77:
448-453.