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Identification and Characterization of Genes Unique Genes  Systemic Xanthomonas Pathogens

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Title:
Identification and Characterization of Genes Unique Genes Systemic Xanthomonas Pathogens
Copyright Date:
2008

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Bacteria ( jstor )
DNA ( jstor )
Genes ( jstor )
Genetic mutation ( jstor )
Inoculation ( jstor )
Plasmids ( jstor )
Polymerase chain reaction ( jstor )
Species ( jstor )
Xanthomonas ( jstor )
Xanthomonas campestris ( jstor )

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University of Florida
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University of Florida
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7/30/2007

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IDENTIFICATION AND CHARACTERIZAT ION OF GENES UNIQUE TO SYSTEMIC Xanthomonas PATHOGENS By LUZ ADRIANA CASTAEDA C. 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

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Copyright 2005 by Adriana Castaeda C.

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This document is dedicated to my family and husband

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ACKNOWLEDGMENTS There are so many things and so many people to be thankful about. First I want to thank my parents for their love and for giving me the best education and the best example, my uncle Luis for leading me to follow this area of studies, and Drs. Marcial Pastor-Corrales Talo and Edgar Martnez for encouraging me and, why not, pushing me to go to graduate school. But in order to go to graduate school I also got a lot of help from several people at ICA, first of all from Capt. Jorge Forero, who first brought me to work for ICA and then single-handedly got me the funding and consent from the Institution to go to grad school. I also want to thank the people who helped me in my second round at UF like Drs. Ana Luisa Diaz, Hernando Montenegro, Lilia Amparo Bonilla, and Claudia Marn and all people from continued education (Oficina de Capacitacin). In Gainesville I would like to thank my advisor Dr. Dean W. Gabriel for his support and great ideas, my committee members Dr. Jeff Jones, Dr. Jeff Rollins and Dr. Vallejos who were always available for advice and comments about my research project, and all professors in the department who were also always ready to help. The chairperson Dr. Wisler and the staff in Plant Pathology made my life so much easier with all the help and support they gave me, and also I am grateful to all the friends I made here. I would like to mention Gary Marlow for his great help in the lab and present and past members from Dean Gabriels lab who helped me and taught me so much. iv

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I want to also thank the rest of my family I did not mentioned before, my big brother and sisters, aunt, grandmother, nephews and nieces for their love and support and specially my sister Rosie and her family. They were a big help for me and my husband during the time we lived here. Last but not least, I want to thank my husband for his love, for leaving all behind and to join me here and for putting up with me during these three years and God for blessing me in every single aspect of my life. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES.............................................................................................................x ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 The Crucifera-Xanthomonas Pathosystem...................................................................2 The Host: Cruciferae Family.................................................................................2 Xanthomonas spp. Infecting Crucifer Plants (XCC and XCA).............................3 The Phaseolus vulgaris-Xanthomonas Pathosystem....................................................5 The Host: Common Bean......................................................................................5 Xanthomonas Infecting Phaseolus sp (XAP, XAPF, XAA).................................6 Bacterial Effectors........................................................................................................7 Methods for Cloning and Identification of Bacterial Effectors....................................9 Functional Genomics..................................................................................................10 2 COMPARISON BETWEEN SYSTEMIC AND NON-SYSTEMIC BACTERIA OF BEAN THROUGH SUPPRESSION SUBTRACTIVE HYBRIDIZATION......12 Introduction.................................................................................................................12 Materials and Methods...............................................................................................13 Plasmids, Bacterial Strains and Culture Conditions............................................13 Modified Suppresion Subtractive Hybridization.................................................13 DNA Sequencing and Analyses..........................................................................14 Molecular Biology Techniques...........................................................................15 Plant Assays.........................................................................................................16 Results.........................................................................................................................16 DNA Fragments Obtained by SSH......................................................................16 The Majority of Gene Fragments Cloned were Found Only on CBB Strains.....17 Gene Fragments Categories.................................................................................17 Southern Blot Confirmation................................................................................17 Mutagenesis Analyses.........................................................................................18 vi

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Discussion...................................................................................................................18 3 INTERRUPTION AND TRANSIENT EXPRESSION OF PTHF, AN AVRBS3/PTHA MEMBER CLONED FROM XPF...................................................28 Introduction.................................................................................................................28 Materials and Methods...............................................................................................29 Plasmids, Bacterial Strains and Culture Conditions............................................29 Genomic Library.................................................................................................29 Molecular Biology Techniques...........................................................................31 Plant Assays.........................................................................................................31 Transient Expression Assays...............................................................................32 Thin Sections.......................................................................................................32 Complementation Attempts.................................................................................33 Results.........................................................................................................................33 Cosmid Library....................................................................................................33 Several Cosmid Clones Contained a Copy of Three Homologues......................34 Transient Expression Assays and Thin Sections.................................................34 Attempts to Mutagenize pth Homologues...........................................................35 Some Mutants of pthF Caused a Pathogenicity Reduction.................................35 Attempts to Complement Pathogenicity Deficient Mutants................................35 Discussion...................................................................................................................35 4 SITE DIRECTED MUTAGENESIS IN THE REGIONS IN THE C-TERMINUS OF TWO AVRBS3/PTHA MEMBERS.......................................................................43 Introduction.................................................................................................................43 Materials and Methods...............................................................................................44 Plasmids, Bacterial Strains and Culture Conditions............................................44 Site-Directed Mutagenesis...................................................................................44 Plant Inoculations................................................................................................46 Results.........................................................................................................................46 UDG Cloning.......................................................................................................46 No Change in the Non-Host HR on Bean...........................................................47 No Change in Water-Soaking or the Non-Host HR on Cotton...........................47 Mutations of CK2 or the UCK, but not LZL Region, Resulted in Reduced Canker Symptoms............................................................................................47 Discussion...................................................................................................................48 5 INDIVIDUAL AND SEQUENTIAL MUTAGENESIS OF XCC AVR GENES, IDENTIFICATION OF A FUNCTIONAL AVR GENE, ATTEMPTS TO DEMONSTRATE HR SUPPRESSION BY AVR GENES........................................53 Introduction.................................................................................................................53 Materials and Methods...............................................................................................54 Plasmids, Bacterial Strains and Culture Conditions............................................54 Molecular Biology Techniques...........................................................................54 vii

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Suppression Subtractive Hybridization...............................................................56 DNA Sequencing and Analysis...........................................................................57 Mutagenesis Experiments....................................................................................58 Growth Kinetics...................................................................................................59 Electrolyte Leakage Measurements.....................................................................59 Complementation Assays....................................................................................59 Race Specificity Change......................................................................................60 Plant Assays.........................................................................................................60 Cell Death Suppression Assays...........................................................................61 Results.........................................................................................................................62 Suppression Subtractive Hybridization...............................................................62 No Evidence of Pleiotropic Pathogenicity Function by any Individual XCC avr Gene or XopD............................................................................................62 No Evidence of Collective Pleiotropic Pathogenicity Function by all Annotated XCC avr Genes..............................................................................63 avrXccFM confers avirulence on B. juncea without inducing a typical mesophyl HR...................................................................................................63 avrXccFM Is a Critical Race Determinant of XCC.............................................65 Electrolyte Leakage Assays Showed That No Apparent Cell Death Is Involved in B. juncea Resistance.....................................................................66 HR In B. juncea Appears to be Restricted to Cells Surrounding the Vascular System..............................................................................................................66 Non-HR Resistance Response in Arabidopsis was Not Affected in Any of the Mutants............................................................................................................66 Non-host HR Was Not Altered in Strains Carrying Mutations in avr Genes.....67 HR Induction by a P. syringae Gene Was Not Inhibited by XCC avr Genes.....67 Discussion...................................................................................................................68 6 SUMMARY AND CONCLUSSIONS.......................................................................86 APPENDIX A BACTERIAL STRAINS AND PLASMIDS..............................................................88 B XANTHOMONAS TOTAL DNA EXTRACTION.....................................................93 C PRIMERS USED........................................................................................................95 D PLANTS USED........................................................................................................100 E PLASMID EXTRACTION......................................................................................101 LIST OF REFERENCES.................................................................................................103 BIOGRAPHICAL SKETCH...........................................................................................121 viii

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LIST OF TABLES Table page 2-1 Summary of sequences found by SSH........................................................................26 2-2 Categories of genes found by SSH..............................................................................26 2-3 Candidate genes selected from Southern hybridization..............................................27 5-1 List of genes classified as avr in XCC528T.................................................................84 5-2 Races in XCC..............................................................................................................84 5-3. Race changes due to the presence of avrXccFM.......................................................84 5-4. Actual sizes of four of the avr genes according to our analyses...............................85 5-5. Seedlings assay for Vascular Hypersensitive Response (VHR)................................85 5-6. List of additional putative effectors...........................................................................85 ix

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LIST OF FIGURES Figure page 2-1 Schematic representation of SSH procedure............................................................22 2-2 PCR amplifications for SSH....................................................................................23 2-3 PCR amplification from random pGEMTeasy clones using M13R and M13 primers......................................................................................................................23 2-4 Dot blot hybridization of pGEMT clones................................................................24 2-5 Chart summarizing the categories of genes obtained by SSH..................................24 2-6 Southern blot hybridization of three of the genes identified by SSH.......................25 2-7 Southern hybridization performed on two of the genes identified by SSH.............25 2-8 Southern blot hybridization showing confirmation a hypothetical protein interrupted in Xanthomonas phaseoli (XP) strain G66............................................26 3-1 Diagram showing the domains of a protein belonging to the avrBs3/pthA family..38 3-2 Southern blot of XPF total DNA probed against pthA.............................................38 3-3 Diagram representing cosmid vector preparation....................................................38 3-4 Fractions collected after sucrose gradient of partially digested DNA......................38 3-5 Agarose gel at 0.7% of eighteen cosmid clones DNA digested with EcoRI............39 3-6 Colony hybridization of the cosmid library probed against pthA............................39 3-7 Southern hybridization of three of the hybridizing cosmid clones..........................40 3-8 Transient expression in bean leaves.........................................................................40 3-9 Thin sections performed in leaves inoculated with Agrobacterium tumefaciens for transient expression............................................................................................41 3-10 Southern blot hybridization of of pthF mutants.......................................................42 x

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3-11 Bean leaf inoculated with pthF mutants...................................................................42 4-1 Map of a avrBs3/pthA proteins.................................................................................50 4-2 Schematic representation of UDG cloning...............................................................50 4-3 PCR amplification of the HincII-HindIII region of pthA in pUC19........................51 4-4 Common bean plants inoculated with KX-1 carrying pthA mutations....................51 4-5 Cotton plants inoculated with H2.2S strain with avrb6 mutations..........................51 4-6 Citrus plants inoculated with mutant versions of pthA into B21.2...........................52 5-1 Mutagenesis strategies used.....................................................................................75 5-2 Dot blot of pGEMTeasy clones hybridized against total DNA...............................76 5-3 Distribution of some relevant gene fragments found in XCC-XCA by Suppression Subtractive Hybridization (SSH).........................................................76 5-4 PCR performed in mutant X23.................................................................................77 5-5 Southern blots of XCC528T wild type and X8.8 DNA digested with EcoRI...........77 5-6 Growth of XCC528T and X8.8 in white turnip Hakurei Hybrid..............................78 5-7 Inoculation of 528T in B. juncea...............................................................................78 5-8 Clip inoculation of some mutants inoculated in Florida Mustard............................78 5-9 Clip inoculation of strains carrying single mutation in Xcc2109.............................79 5-10 B. juncea leaf inoculated by syringe infiltration at high concentration....................79 5-11 Codon preference analyses performed on four avr genes by GCG..........................80 5-12 Complementation tests in B. juncea.........................................................................80 5-13 Southern blot of different strains representing three races of XCC probed against avrXccFM.................................................................................................................81 5-14 Race change of two XCC strains in Florida mustard inoculated by leaf clipping...81 5-15 Growth of XCC6181 and X83 (6181/avrXccFM) syringe infiltrated in Florida Mustard plants..........................................................................................................82 5-16 Time course of electrolyte leakage from leaves of Florida Mustard plants inoculated with four different strains of XCC and one of XCA..............................82 xi

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5-17 Seedling assay performed B. juncea plants..............................................................83 5-18 Apparent HR suppression by avr genes from XCC in pepper and tobacco plants..83 xii

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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 IDENTIFICATION AND CHARACTERIZATION OF GENES UNIQUE TO SYSTEMIC Xanthomonas PATHOGENS By Luz Adriana Castaeda C. August 2005 Chair: Dean W. Gabriel Major Department: Plant Pathology Suppression Subtractive Hybridization (SSH) was used to identify genes present in the systemic pathogen Xanthomonas campestris pv. campestris (XCC), and missing from the nonsystemic pathogen Xanthomonas campestris pv. armoraciae (XCA). Among the DNA fragments unique to XCC was Xcc2109, one of eight putative avr genes identified in the XCC528T genome (NC 003902). Individual and sequential deletion and/or insertion mutations of all eight putative XCC avr gene loci were made, but no change in pathogenicity was observed in any combination of avr mutations, including a strain with all eight avr genes deleted. However, insertion or deletion mutants at the Xcc2109 locus lost avirulence (i.e., became virulent) on Brassica juncea, a race-determining differential host. The Xcc2109 ORF as annotated was cloned and found to be nonfunctional. A longer gene, encompassing Xcc2109 and here designated avrXccFM, was defined and found to confer avirulence to Xcc2109 mutant strains and to two additional wild type xiii

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strains, thereby changing their designation. Resistance in B. juncea to XCC528T strains carrying avrXccFM occurs without a typical HR, but with a vascular HR (VHR). A similar but modified SSH was performed between the two systemic pathogens Xanthomonas phaseoli (XP) and Xanthomonas axonopodis pv. phaseoli var. fuscans (XAP), and the non systemic Xanthomonas axonopodis pv. alfalfae (XAA). Among other gene fragments, a large number of transposable elements as well as gene fragments of unknown function were obtained. Five genes also present in other bacterial pathogens were mutagenized in XP. None of the mutant strains obtained exhibited any effect in virulence except for a pthA homolog, a gene earlier identified in XP and included in the functional study. Complementation assays were inconclusive although in detached leaves transient expression of the pthA gene homolog obtained from an XPF cosmid library, induced a phenotype similar to common bacterial blight of bean. xiv

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CHAPTER 1 INTRODUCTION Xanthomonads are Gram-negative, plant-associated bacteria. Cells exist mostly alone or in pairs, but chains and filaments may also occur (Swings and Civerolo 1993). Cell length is variable even within the same strain, and a single polar flagellum is usually observed. Individual bacterial cells are surrounded by the extracellular polysaccharide xanthan gum, and most strains form yellow, water-insoluble pigments (xanthomonadins) when grown in culture media (Shaad 1988). Xanthomonads are biotrophic (derive their nutrition from living cells) and capable of intimate, highly-specific host interactions. Although the host range of the genus is wide, the host range of any one strain is typically quite narrow (the host range of each pathovar is usually limited to few species or genera of the same plant family, (Hayward 1993)). Bacteria belonging to the genus Xanthomonas produce leaf spots, cankers, and systemic blights. Among the Xanthomonas species that cause systemic diseases that are relevant for this study are: Xanthomonas campestris pv campestris (XCC) causing bacterial blight and black rot of crucifers (Alvarez et al. 1994), Xanthomonas pv. phaseoli, (XP) causing common bean blight (Vidaver 1993), and Xanthomonas axonopodis pv. phaseoli var. fuscans (XPF) also causing common bean blight (Schwartz and Pastor-Corrales 1989). Xanthomonas species that cause leaf spot that are relevant to this study are Xanthomonas campestris pv. armoraciae (XCA) which causes a leaf spot of crucifers (Schaad and Alvarez 1993) and Xanthomonas axonopodis pv. alfalfae (XAA) which causes leaf spot of alfalfae (Esnault et al. 1993). 1

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2 Up to this date three xanthomonads species have been sequenced: Xanthomonas citri strain 306 (da Silva et al. 2002), Xanthomonas campestris pv. campestris strains ATCC33913 (synonym 528T), B100 and 8004 (da Silva et al. 2002; Vorhlter et al. 2003; Qian et al. 2005), and Xanthomonas oryzae pv. oryzae strain KACC10331 (Lee et al. 2005). The Crucifera-Xanthomonas Pathosystem The Host: Cruciferae Family The Brassicaceae comprises more than 300 genera and 3000 species of plants (Rubatzky and Yamaguchi 1997). Some of the plant species members of this family are recognized for their contribution to human nutrition, and suggestions have been made that when consumed fresh, these plants possess cancer-preventive attributes (Talalay and Fahey 2001; Mezencev et al. 2003; Keck and Finley 2004). According to the 2004 FAO (FAO statistics, 2004) reports, in the year 2002, oil mainly from rape and mustard seed, provided 32 calories per capita per day. Brassica is the most important genus agriculturally with 40 species, and the majority of cultivated ones occur in six species: three diploid species with 8, 9 and 10 pairs of chromosomes identified as B, C and A genomes of B. nigra, B. oleracea and B. rapa respectively, and three amphidiploid species (B. carinata, B. juncea and B. napus) having the BC, AB and AC genomes with 17, 18 and 19 chromosomes, respectively. In the United States production of broccoli (B. oleracea var. italica), cabbage (B. oleracea var. capitata) and cauliflower (B. oleracea var. botrytis) yielded 916,000, 2,433,110 and 397,850 metric tons respectively. The total market value was $1.2 billion in 2003 with California and Arizona being the major producers. Broccoli was the crucifer most cultivated in 2003 in the U.S., with 136,000 acres harvested, followed by cabbage and cauliflower with 76,850 and 44,000 acres

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3 harvested respectively (USDA, NASS, 2004). According to FAO statistics for 2003, cabbage was the most cultivated fresh market crucifer in the world with production of 65,956,162 metric tons, followed by cauliflower with production of 15,948,166 metric tons. The most cultivated seed crucifers in the world were oil-rape (B. napus), with a total yield of 36,145,663 metric tons, followed by mustard seed (B. nigra) with a 632,354 metric tons yield (FAO statistics 2004). Xanthomonas spp. Infecting Crucifer Plants (XCC and XCA) XCC is a bacterial species that infects a wide range of plant taxa throughout the world (Bradbury 1984). Black rot of crucifers is one of the most destructive diseases (Williams 1980) attacking cauliflower, cabbage, kale, broccoli, brussels sprouts, Chinese cabbage, collard, kohlrabi, mustards, rape, rutabaga and turnip. During periods of warm, wet weather, black rot may reduce yield by more than 50% (RPD 924 1999). The bacterium can overwinter in plant debris, on seeds from diseased plants, and on weeds. The pathogen may survive in diseased crop residue buried in soil for up to two years, but not for more than sixty days when free of plant material in the soil. Crucifer seed is the primary means of dissemination (Miller et al. 2002). XCC enters the plant primarily through hydathodes, and then colonizes and moves through the vascular system (Williams 1980). The systemic pathogen can spread throughout the plant by seed, root or leaf infections (Cook et al. 1952). In natural infections, mechanical or insect wounds are also a major route of entry (Kucharek and Strandberg 1981), but natural openings become most important in the absence of such wounds (Shaw and Kado 1988). Extensive yellowing of leaf margins is a common early symptom of black rot. The affected tissue may become necrotic and advanced systemic infections can cause darkened leaf veins and vascular tissue within the stem, expanded

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4 leaf yellowing, leaf necrosis and leaf wilting. By contrast, XCA enters the plant mainly through stomata (Hugouvieux et al. 1998) and remains localized in the mesophyl tissue (McCulloch 1929). XCA lesions occur near the original point of entry and lesions are limited, appearing as circular spots. As with other Xanthomonads, XCC requires a hrp system for both pathogenicity on host plants and the ability to elicit the hypersensitive response on non-host plants (Kamoun and Kado 1990). The virulence of XCC also depends upon a number of other factors including the synthesis of enzymes like proteases (Dow et al. 1990), and the extracellular polysaccharide (EPS) xanthan (Crossman and Dow, 2004). The rpf gene cluster (for r egulation of p athogenicity f actors) positively controls both the production of these compounds and the virulence in plants (Tang et al. 1991). Genes within the rpf cluster encode elements of a regulatory system involving the diffusible signal factor, (DSF), which has been implicated in regulation of biofilm dispersal (Barber et al. 1997). rpf mutation studies show coordinated regulation of endoglucanases, proteases, polygalacturosate lyases and EPS xanthan. rpfB and rpfF control the production of DSF which is most probably a lipid or a lipid derivative (Barber et al. 1997). DSF does not accumulate in later growth phases, and it does not function as an auto-inducer (Wilson et al. 1998; Dow et al. 2000; Crossman and Dow 2004). Other enzymatic proteins that are involved in pathogenicity include a zinc metalloprotease which has been shown to degrade proline-rich glycoproteins in Brassica species (Dow et al. 1998), and an endo -(1,4)-mannanase that is required for full virulence in Chinese radish. The later is probably involved in bacterial release from the xanthan-based biofilm to planktonic state in order to promote colonization of the vascular system (Dow et al. 2003).

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5 XCC was initially shown to be comprised of five races, based on disease reactions on different plant species: Brassica oleracea, Brassica rapa and Brassica juncea (Kamoun et al. 1992). Later a new race system was developed which included an extended series of Brassica species (B. oleracea var. botrytis and B. carinata (Vicente et al. 2001)) and as a result six races were renamed accordingly. Three major resistance genes designated R1, R3 and R4 appear to be present in the host differentials used to identify races. B. juncea has at least one resistance gene (R1) that probably originated from the B genome of B. nigra. The A genome also has one resistance gene (R4), and R3 likely originated in the C genome (Taylor et al. 2002). The complete genomic DNA sequence of the XCC type species ATCC 33913 (synonym to 528T) was recently published (da Silva et al. 2002). The genome carries a single chromosome of about 5.0 Mb, no plasmids, a GC content of 65% and a total protein coding region of 84%. It also possesses one copy of a type IV secretion system, has a conserved type III secretion machine and contains 8 putative avirulence genes. Analyses indicate that numerous events of horizontal gene transfer probably occurred in this species, based in part on the appearance of 109 different types of transposable elements. A total of 285 genes present in XCC are suggested to have been acquired horizontally (Garcia-Vallv et al. 2003), including three of the eight avirulence/pathogenicity effector genes identified. The Phaseolus vulgaris-Xanthomonas Pathosystem The Host: Common Bean Common dry bean, Phaseolus vulgaris, is the most important food legume for direct human consumption in the world (Schwartz and Pastor-Corrales 1989, Broughton, 2003). The genus Phaseolus comprises 55 species, five of which have been

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6 domesticated: P. coccineus, P. acutifolius, P. lunatus, P. polyanthus and P. vulgaris (Broughton et al. 2003). In 2003, 19,038,458 metric tons of dry bean and 5,933,264 metric tons of green beans were produced in the world. Of these, 7,000,000 metric tons were produced in Latin America and Africa. In the US, 1,021,260 of dry beans were produced on 545,000 hectares and 127,500 metric tons of green beans were produced on 21,400 hectares during 2003 (FAOSTAT, 2004). During the same period, the production of green beans in the US was valued at $267,762,000 (USDA, NASS, 2004). Xanthomonas Infecting Phaseolus sp (XAP, XAPF, XAA) X. phaseoli (XP) and X. axonopodis pv phaseoli var. fuscans (XPF) are two bacterial species that cause common bacterial blight of bean (CBB). Both bacteria induce identical symptoms on leaves, stems, pods and seeds (Schwartz and Pastor-Corrales, 1989). The two bacteria are frequently found in the same field and even in the same plant. Leaf symptoms initially appear as water-soaked spots which enlarge and frequently coalesce with adjacent lesions, often surrounded by a zone of lemon-yellow tissue. CBB bacteria enter leaves through natural openings such as stomata and hydathodes and wounds. Cells invade intercellular spaces causing a gradual dissolution of the middle lamella. Colonization of the xylem tissue may cause wilting as a result of plugging vessels or disintegrating cell walls. By contrast X. axonopodis pv. alfalfae (XAA) produces only a leaf spot in bean similar to the disease caused by XCA in crucifers. Yield losses due to CBB can be very high. For example, in 1967 the disease affected at least 75% of 265,000 hectares of Navy bean in Michigan causing between 10 and 20% losses (Saettler in Schwartz 1989). In 1975, Wallen and Galway reported yield losses of 38% in Ontario, Canada. In Colombia, natural and artificial infections resulted

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7 in yield losses of 22 and 45%, respectively (Yosshi et al. 1976). In Florida, it is the most frequently found bacterial disease on both snap beans and dry beans. XP can survive and multiply as an epiphyte on weed hosts, especially from the legume family (Pernezny and Jones 2002). There is little molecular information available regarding either organism causing common bacterial blight of bean. The chromosome size of an XPF strain was estimated to be 3938 kb based on Pulse-Field Gel Electrophoresis (PFGE) and Southern blots (Chan and Goodwin 1999). Genes involved in the oxidative metabolism of XP have been identified (Chauvatcharin et al. 2003; Vattanaviboon et al. 2002, 2003); however, no pathogenicity or avirulence genes have been identified. Bacterial Effectors Most pathogenic xanthomonads deliver effectors through a type three secretion system (T3SS), which allows them to inject proteins directly into the plant cytoplasm that are crucial for eliciting disease and hypersensitive responses (Galan and Collmer 1999; Gauthier et al. 2003; Alfano and Collmer 2004; He S.Y. et al. 2004). Avirulence (avr) genes, considered as a class of effectors, have been identified in both bacteria and fungi based on their ability to confer incompatibility in otherwise compatible interactions (Ellingboe 1976). In gene-for-gene systems, dominant avr genes in the pathogen interact with specific resistance (R) genes in the host and are known to determine pathogenic races and the range of host cultivars attacked (Flor 1956). Recent publications (Rohmer et al. 2004) suggest that pathogenic bacteria may secrete as many as fifty effectors through its T3SS. Besides their major role in race-cultivar specificity, avr genes have pleiotropic functions. AvrPtoB from Pseudomonas syringae pv. tomato inhibits programmed cell death in tobacco and induces a

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8 hypersensitive response in tomato plants carrying Pto (Abramovitch et al. 2003); avrE confers avirulence to Pseudomonas syringae pv. glycinea, adds pathogenicity to some strains of Pseudomonas syringae pv. tomato and it also complements dsp mutants in Erwinia amylovora (Bogdanove et al. 1998). The avrBs2 gene of X. vesicatoria, which is widespread in Xanthomonas, has been shown to contribute to the fitness of the pathogen by increasing the in planta growth (Kearney & Staskawicz 1990). pthA, a member of the avrBs3/pthA family is present in X. citri and is necessary for production of symptoms on citrus plants, and also for eliciting a hypersensitive response in non-hosts (Swarup et al. 1992). Spontaneous and marker exchanged avrb6 mutants of X. axonopodis pv. malvacearum, also a member of the avrBs3 family had reduced pathogenicity as shown by a decrease in water-soaking ability in cotton. Enzymatic activity has been demonstrated for nine effectors and plant defense suppression phenotype for ten effector genes (Alfano and Collmer 2004). Pleiotropic fitness effects are a common feature of several avirulence/effector genes and have been reported in several other pathogen species (Lorang et al. 1994; Yang et al. 1996; Badel et al. 2003; Wichmann et al. 2004). However, there appears to be no associated fitness benefit for some avr effector genes. Indeed avrBs1 has been shown to produce a fitness cost when present in the pathogen, since when present in X. vesicatoria (XV), it decreases the ability of the pathogen to survive in soil and dead plant material (OGarro et al. 1997). Horizontal gene transfer could account for the lack of a fitness advantage of some avr genes. However, it is unlikely that effector genes lack a beneficial function at least in some contexts since selection is not expected to maintain genes that incur a high cost with no counteracting benefit (Frank et al. 1992). In fact, Wichmann (2004) found that

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9 wild type XV was better fit than all mutant effector genotypes, showing greater ability to develop disease lesions. Different combinations of avr mutations have also given different results in transmissibility, demonstrating that complex interaction exist among effector genes. Additionally only after mutation of several effector genes were fitness costs observed in terms of lesion development and in planta growth. These mutations contributed to a drastically reduced ability to be transmitted in the field. Methods for Cloning and Identification of Bacterial Effectors Diverse molecular and genomic approaches have been used to identify potential bacterial effectors (Buttner et al. 2003; Nomura and He, 2005). Among those, the complementation of non-pathogenic strains with a genomic library of a pathogenic strain, or the transfer of an avirulent strain genomic library into a virulent strain of the same pathogen have been widely used (Ronald and Staskawicz 1988; DeFeyter et al. 1991; Dong et al. 1991; Whalen et al. 1991 and 1993; Ronald et al. 1992; Chen et al. 1994). Recently, reporter fusion systems have been used in which the gene of interest was disrupted and fused to a reporter gene; this fusion is measured or detected by RT-PCR, -Galactosidase activity, or calmodulin-dependent adenylate cyclase assays (Cunnac et al. 2004; Losada et al. 2004). Other gene fusions have also been attempted in which known HR inducing domains of genes are fused with the gene of interest in order to obtain a hypersensitive response when effector genes are injected. These include fusions to P. syringae pv. tomato AvrRpt2, which were screened in Arabidopsis thaliana carrying RPS2 gene (Guttman et al. 2002), or fusions to AvrBs2 from XV screened in pepper plants carrying Bs2 (Roden et al. 2004). Effector induction or co-regulation also can be screened in planta by cDNA-AFLP (Noel et al. 2001), or detected by a modified in vivo expression technology (Boch et al. 2002). Finally, another technique that has been used

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10 is the Agrosuppresion assay in which the target gene is introduced into a tumorogenic Agrobacterium strain in order to detect HR or otherwise to obtain a tumor (Kamoun et al. 2003). In silico identification of genes using common sequences features present on effector genes such as PIP and hrp boxes, signatures of horizontal acquisition and an N-termini with characteristics such as a high percentage of serine residues, aliphatic amino acids or proline in the third or fourth position, and the lack of negatively charged amino acids in the first two residues and structural motifs for eukaryotic activity (Lloyd et al. 2002; Petnicki-Ocwieja et al. 2002; Schechter et al. 2004) are also widely exploted for bacteria with genome sequence data. Functional Genomics In the pre-genomics era (McKusik et al. 1993), mutations to observe a loss of function were randomly introduced by physical (X-ray irradiation or UV light), chemical (N-methyl-Nnitro-N-nitrosoguanidine (NG) or ethyl methanosulfonate EMS-) agents, or biological agents (transposable elements (Turner et al. 1984)). In addition, genes that are differentially represented in two cDNA populations (Harakava and Gabriel 2003) can be isolated by a simplified subtractive hybridization procedure called suppression subtractive hybridization (SSH) developed by Diatchenko et al. (1996). With the outcome of complete sequences from diverse plant pathogenic bacteria (Simpson et al. 2000; Woods et al. 2001; da Silva et al. 2002; Salanoubat et al. 2002, Buell et al. 2003; Vorhlter et al. 2003), efforts have been focused to investigate the function of genes found in each of the pathogens and their role in disease elicitation. This can be done by inactivation of the gene product(s) of interest and analyzing the phenotype (Snyder and Gerstein 2003). Homologous recombination in bacteria has been

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11 used to create such directed mutations. Techniques based on PCR to introduce mutations in the gene of interest include UDG cloning and replacement (Rashtchian et al. 1992), marker disruption (OReilly et al. 1986), Spliceoverlap PCR (Ho et al. 1989) and FLP recombinase (Hoang et al. 1998). The latter two have been used to induce deletions of the target gene allowing multiple rounds of mutations to study the function of several genes simultaneously.

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CHAPTER 2 COMPARISON BETWEEN SYSTEMIC AND NON-SYSTEMIC BACTERIA OF BEAN THROUGH SUPPRESSION SUBTRACTIVE HYBRIDIZATION Introduction Xanthomonas phaseoli (XP) and Xanthomonas axonopodis pv. phaseoli var. fuscans (XPF) are two different bacterial species that systemically infect bean (Phaseolus vulgaris) to cause common bacterial blight (CBB). Both bacteria induce indistinguishable symptoms on leaves, stems, pods and seeds (Schwartz and Pastor-Corrales 1989), even though RFLP and DNA-DNA hybridization studies have shown that they are genetically diverse (Gabriel et al. 1989; Vauterin et al. 1995). The two bacteria are frequently found in the same field and even in the same plant. Growth in most culture media (any that contain tyrosine) will differentiate the two bacteria, since XPF produces a brown diffusible melanin-like compound, and like most xanthomonads, XP does not (Basu 1974). At the beginning of the CBB infection process, XP and XPF enter leaves through natural openings such as stomata, hydathodes and wounds. Water-soaked spots appear on leaves about 7 days later, which enlarge and frequently coalesce with adjacent lesions. As the disease advances, the lesions usually show a zone of lemon-yellow tissue around the edges (Schwartz and Pastor-Corrales 1989). The bacteria then invade intercellular spaces, causing a gradual dissolution of the middle lamella that allows them to reach the xylem tissue (Schwartz and Pastor-Corrales 1989). In contrast, a related but a non-systemic pathogen of bean, X. axonopodis pv. alfalfae (XAA), infects primarily through 12

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13 stomata, causing a water-soaked spot that later turns dark and usually does not exceed 3 mm in diameter (Moffett and Irwin 1975). A simplified subtractive hybridization procedure, called suppression subtractive hybridization (SSH), was developed by Diatchenko (1996) to isolate genes that are differentially represented in two cDNA populations. Because bacterial genomes are smaller and less complex than most eukaryotic cDNA populations, SSH can be applied to find genomic differences between closely related bacterial strains (Akopyants et al. 1998; Bogush et al. 1999, Harakava and Gabriel 2003). A modified SSH experiment was performed here in order to find unique sequences present in both XP and XPF and absent from XAA. Materials and Methods Plasmids, Bacterial Strains and Culture Conditions Bacterial strains and plasmids used or constructed in this study are listed in Appendix A. Xanthomonas strains were grown in PYGM (Gabriel et al. 1989) at 30C and E. coli strains were grown at 37C in Luria-Bertani (LB) medium (Maniatis et al. 1982). When necessary, appropriate antibiotics were used at the following concentrations: ampicillin (Amp), 100 g/ml; gentamicin (Gm), 3 g/ml; kanamycin (Kn), 20 g/ml; rifampicin (Rif), 75 g/ml; chloramphenicol (Cm), 35 g/ml, and spectinomycin (Sp), 50 g/ml. Modified Suppresion Subtractive Hybridization DNA from XP and XPF was extracted according to protocol in Appendix B. XP and XPF DNAs was first digested with RsaI and they were each ligated to one of the specific SSH adaptors, and XAA DNA was used as driver DNA. The rest of the procedure, illustrated in Figure 2-1, was performed according to the instructions of the

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14 commercially available SSH kit (PCR-Select Bacterial Genome Subtraction Kit-Clontech Laboratories, Inc) with minor changes as follows. Two micrograms of DNA from each of three strains (XP strain G66, XPF strain 203B and XAA strain KX-1) were extracted using the protocol in Appendix B. The DNA was digested with 15 units of RsaI for 16 h at 37C. The DNA from XP was ligated to adaptor 1 and XPF DNA ligated to adaptor 2R (Appendix C). Two microliters of XAA DNA was diluted with 1l of 4x hybridization buffer and mixed with 1 l of DNA from XP and 1 l of DNA from XPF in separate 0.5 ml tubes. The samples were heated at 98C for 1.5 m, followed by incubation at 63C for 1.5 h. The XP/XAA and XPF/XAA samples were then combined and an additional 1 l of XAA DNA melted at 98C for 1.5 m was added. The mixture was heated at 63C overnight and then PCR amplified with primer 1 at four annealing temperatures (57C, 59C, 61C and 63C) for 35 cycles. A second round of PCR amplification using the product from the first PCR at 61C was performed with nested primers 1R and 2R at 58C, 60C, 62C and 64C annealing temperatures for 25 cycles. Three microliters of the DNA from the second PCR amplification at 64C annealing temperature was ligated into pGEMTeasy according to the manufacturer, transformed into DH5 cells, and selected on LB plus Amp plates. DNA Sequencing and Analyses Primers used in this study were ordered from Integrated DNA technologies, Inc. (Coralville, Iowa) and are listed in Appendix C. All DNA clones resulting from SSH were sequenced at the University of Florida ICBR (Interdisciplinary center for biotechnology research) DNA Sequencing Core facility. To confirm that the cloned DNA fragments were unique to CBB, all SSH clones were colony PCR amplified with

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15 vector-based primers M13R (-48) and M13 (-47). Two microliters of DNA from the PCR amplification were spotted in duplicate on a nylon membrane with a replicator (VP 408S2a, V & P Scientific Inc. San Diego, CA) and also run on a 1% agarose gel to confirm that the amplified products were uniform. Five hundred nanograms of total RsaI-digested DNA from all strains were separately labeled with 32P with the Random Primer kit II (Stratagene) according to the manufacturers protocol. The labeled DNA was probed against each of the corresponding membranes. The DNA fragments of interest were further confirmed as CBB-specific by Southern hybridization analysis (Maniatis et al. 1982). Probes used were labeled PCR products amplified with vector-based primers, or with primers specific for internal regions of the sequences (Appendix C). Molecular Biology Techniques For marker interruption, an internal region of the target gene was PCR amplified with appropriate primers. A single bacterial colony taken with a toothpick and swirled into the PCR reaction mix, and it was used as template DNA. PCR reactions were performed using Taq polymerase (Invitrogen Corporation, Carlsbad, CA) with the Invitrogen PCR buffer, using magnesium chloride, primers and nucleotide concentrations as recommended by the manufacturer. The mix was initially denaturated at 95C for 3 m to release DNA from the cells, and then 35 PCR cycles were performed (30 s at 95C, 30 s at the specified annealing temperature, 70C for the specified extension time) with a final 10 m extension at 70C. One microliter of the PCR reaction was used for cloning into the TOPO vector (Invitrogen Corporation, Carlsbad, CA) and transformed in E. coli cells. Approximately 250 ng of DNA from the TOPO clones containing the insert were

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16 electroporated into 40 l of electrocompetent Xanthomonas phaseoli at 1.8 kV/cm in an Eppendorf 2510 electroporator. PYGM liquid medium was added to the bacteria to bring the volume to 1 ml. The cultures were grown at 30C for 2 h at 120 rpm and then the whole ml was spread onto PYGM plates containing Rif and Kn. Single colonies growing on the plates were restreaked and later confirmed to be transformed with the appropriate cloned DNA by PCR and Southern hybridization analysis. Plant Assays Xanthomonas cultures grown overnight at 30Cin liquid PYGM, were centrifuged at 5000 rpm for 5 m, washed with sterile tap water and adjusted to 0.3 OD600 (approximately 108 CFU/ml). A 1:1000 dilution of each suspension was made for low concentration inoculations (approximately 105 CFU/ml) of Phaseolus vulgaris plants cultivar California Redloud Kidney (Appendix D). For each inoculation, 2 cm at each side of the trifoliate leaves from 4 week old plants were cut with scissors and dipped in the bacterial suspension. Scissors were flame sterilized between inoculations. The plants were kept in a growth chamber at 27C with a 14 h light, 10 h dark cycle. Symptoms were scored daily for up to 3 weeks. Results DNA Fragments Obtained by SSH Since the Clontech procedure for SSH was standardized based on E. coli DNA, different temperatures were tested for Xanthomonas DNA. Figure 2-2 illustrates first and second round PCR products from reactions performed at different annealing temperatures. Four temperatures were tested, and the amplified products obtained at 61C in the first PCR round and at 64C in the second PCR round were selected for cloning into pGEMTeasy. Agarose gels were run with the PCR amplified products

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17 obtained from 348 colonies (Figure 2-3) to confirm uniformity of concentration prior to the dot blots. The Majority of Gene Fragments Cloned were Found Only on CBB Strains Dot blot analyses (Figure 2-4) showed that the majority of gene fragments cloned were present exclusively in CBB. Only one of the 96 clones spotted on the membrane pictured in Figure 2-4 hybridized with XAA labeled total DNA. Similar results were obtained with all plates analyzed (data not shown). Gene Fragments Categories Three hundred and eighty eight putative CBB-specific pGEMTeasy clones were sequenced. The average size of the sequence reads was 492 bp. Forty-two of the insert sequences were eliminated due to the low quality of the sequence read and 24 of the clones contained no insert. Three hundred and twenty two sequences were retained for further analysis (Table 2-1), and Blast analysis demonstrated that the sequences showed similarity to: transposable elements (104) (Table 2-2, Figure 2-5), transcriptional regulators (58), S-receptor kinases (34), hypothetical proteins (26), pectate lyases (23) and ABC transporters (16) among others. Forty of the 322 sequences (12.4%) were obtained only once. One avirulence/pathogenicity gene homolog belonging to the avrBs3/pthA gene family was found. Southern Blot Confirmation Twenty-five genes that appeared to be CBB exclusive by dot blot hybridization were analyzed by Southern blot hybridization. Three of the 25 genes tested were not CBB unique by Southern hybridization (Figure 2-6, some data not shown). All others were confirmed by Southern hybridization to be present only in the CBB strains (XP and XPF) (3 representative Southern hybridization results are shown in Figure 2-7). Some of

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18 the analyses also showed that few of the CBB-specific clones hybridized multiple times to different probes, suggesting that the CBB specific DNA could represent portions of transposable elements (not shown). Table 2-3 shows the gene fragments selected after Southern hybridization as candidates for mutagenesis, based on their presence only in the two CBB strains. Mutagenesis Analyses Five genes from XP (Table 2-3) were marker interrupted with internal regions cloned into TOPO, including XAC2373, a pectate lyase, RSp1239, a hypothetical protein, XACa0025, a hypothetical protein, XCC3132, a conserved hypothetical protein, and XAC2620, a VirB4 homolog. Figure 2-8 shows confirmation of one of the mutations (homolog of XACa0025) performed in XP strain G66. None of the mutational inserts appeared to affect pathogenicity, morphology or growth. Discussion Three hundred and twenty two gene sequences putatively conserved in both CBB strains of X. phaseoli and X. phaseoli var. fuscans, but not found in a leaf spot strain of X. axonopodis pv. alfalfae were identified by SSH. The modifications used (two tester strains instead of one, and different hybridization temperatures) appear to have increased the efficiency of the method and resulted in a reduced background, since about 88% of the sequences analyzed by Southern hybridizattion were demonstrated to be present only in CBB strains. Analyses between two E. coli strains have shown that the test efficiency is close to 50% (clontech manual). The appearance of many phage-related sequences in the library is surprising, since these elements tend to be strain specific (Winstanley 2002). However, the fact that XP and XPF are frequently found together in the same plant in the field would allow

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19 development of a common phage. Bacteriophages could be important components of the evolution of CBB strains, as these elements have been shown to be mediators of horizontal gene transfer. Bacteriophages could bring new advantageous genetic material to its host and/or interrupt host genes upon integration (Hendrix et al. 1999). Some gene sequences obtained through SSH and confirmed as exclusively present in CBB strains were not considered for functional analyses since they were present in high copy number (>10 copies, data not shown), and elaborate methods would be needed to accomplish mutagenesis of such a large number of copies. Those include ABC transporter proteins and S-receptor kinases that are usually involved in a variety of basic cellular processes and also in virulence in animal pathogens (Davidson and Chen 2004). In the case of pectate lyase, a Southern blot showed that two hybridizing gene fragments were present in both CBB strains (Figure 2-6 B). Nevertheless, an attempt was made to mutate one of these by marker interruption, but the mutation had no evident effect on virulence. In order to mutate both copies, a different method, such as splice overlap PCR (Horton et al. 1989) or FLP recombinase (Hoang et al. 1998) would need to be used to sequentially mutate the additional copy and determine the role of these genes if any, in CBB-specific pathogenicity. Several copies of the type IV secretion system genes VirB9-VirB10 were found only in CBB strains, and a copy of the VirB4 homolog was chosen to knock out the entire system, since this gene was also found present in single copy in both CBB strains and not in XAA (data not shown). VirB4 plays a major role in the type IV secretion/transfer system as an ATPase (Remaut and Waskman 2004) and plays an essential role in pathogenicity of Agrobacterium tumefaciens (Sagulenko et al. 2001). However, mutation

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20 of the VirB4 locus did not produce a reduction in pathogenicity. In XP and XPF, the type IV system could be involved solely in plasmid conjugation. It is also possible that XP could be carrying an additional but functional type IV system that is divergent at the DNA level, but able to functionally complement the mutation (Xanthomonas ONSA FAPESP network. 2001/2002). Some genes originally identified as CBB unique and potential pathogenicity factors (including HrpD5, pthA, and a conserved hypothetical protein (Figure 2-6), some data not shown) were not considered for knock-out experiments after Southern hybridization revealed their presence in all three bacterial species. Traditional techniques such as complementation of non-pathogenic strains with a genomic library of a pathogenic strain (Chen et al.1994), reporter fusion systems (Cunnac et al. 2004; Guttman et al. 2002; Roden et al. 2004a), screening of genes induced in planta (Noel et al. 2001), and promoter-trap assays (Losada et al. 2004) have been successfully used to identify pathogenicity genes present in a given pathogenic strain. However, none of these techniques has ever resulted in discovery of genes involved in systemic pathogenicity vs. mesophyll-limited pathogenicity. This study was designed to find genes common to two different systemic strains and absent from a non-sytemic. This is the first report of an attempt to use the SSH technique with two tester strains and one driver. SSH followed by limited knock-out mutagenesis did not reveal any genes critical for systemic pathogenicity among 322 examined. There are several possible explanations for this: first, the putative gene or genes involved in pathogenicity or systemic movement could be present in all three bacteria species but they could be either regulated

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21 differently, or they could carry mutations when present in the non-systemic pathogen (frame shifts, or short deletions or insertions); such differences would be undetectable by SSH. Second, even though the two CBB species apparently cause the same disease, they could potentially utilize different effectors to cause symptoms. If this were the case, SSH would not be useful to detect the genes involved in pathogenicity in each CBB strain, since each strain would carry different genes. Third, Pomati and Neilan (2004) suggested that SSH-based techniques preferentially select for genomic regions with high GC content. Since virulence genes are often found in pathogenicity islands that have atypical GC content than the rest of the genome (Schmidt and Hensel 2004), it is possible that the candidate genes could be present in regions with low GC. Fourth, since CBB strains were used as testers and XAA as driver, the presence of a negative factor present in XAA could be responsible for its inability to cause systemic symptoms. A reverse analysis (i.e. by using XAA as tester and CBB as drivers) might be used to obtain this type of gene. Some modifications in SSH could improve the results obtained by this technique. DNA shearing instead of a RsaI digest, different hybridization temperatures and a titration of PCR annealing temperatures, could have help reduce background and obtain a more representative group of gene sequences possibly involved in pathogenicity.

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22 X. phaseoliX. alfalfae X. fuscansC)D)A) B) E) *1*1*2*2*3*4 Figure 2-1 Schematic representation of SSH procedure. A) Genomic DNA from the three strains is digested with RsaI. B) The two tester DNAs (XP and XPF) are each ligated to a different adaptor. C) A hybridization step is performed separately for both adaptor-ligated tester DNA samples using an excess of driver DNA D). A second hybridization step is performed with fresh denatured driver DNA and the two samples from the previous step. E) A fill-in reaction is performed followed by two PCR reactions. The first PCR with a primer which sequence is found in both adaptors (primer 1), and the second one with two primers, indicated in the figure (Nested primer 1 and Nester primer 2R). The molecules type *1 will amplify linearly, since they carry only one adaptor. Molecules type *2 will likely form a panhandle-like structure due to the suppression effect. Type *3 molecules will not amplify due to the lack of adaptors. Only the molecules type *4 that have two different adaptors at their ends will be amplified exponentially. The amplified products obtained are then cloned in a PCR cloning vector.

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23 BAM C 58 C 60 C 62 C 64 M C 57 C 59 C 61 C 63 Figure 2-2 PCR amplifications. Five microliters from the 25 l reactions were run on a 1% Agarose gel. Products of first (A), and second round PCR (B) performed at different hybridization temperatures are shown. From left to right, M, 1 kb ladder, C, control unsubracted DNA, and SSH mix at the different temperatures shown on top of the gels. M Figure 2-3. PCR amplification from 18 random pGEMTeasy colonies using M13R and M13 primers (Appendix C) were performed and 5 l of the 25-l reactions were run in a 1% agarose gel. From left to right M, 1 kb ladder, and PCR products from 18 clones.

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24 A B C Figure 2-4. Dot blot hybridization of clone inserts obtained by SSH. DNA was PCR amplified with M13R and M13 primers, spotted twice on each of the membranes on each of the three membranes and each one was hybridized against 500 ng of labeled DNA from A) Xanthomonas phaseoli strain G66, B) Xanthomonas axonopodis pv. phaseoli var. fuscans strain 203B, and C) Xanthomonas axonopodis pv. alfalfae strain KX-1. Transposase Transcriptional Regualtor S-receptor kinase Hypotetical protein Pectate Lyase ABC Transporter Unknown VirB9 HrpD5 Integrase Recombinase pthA Others Figure 2-5 Chart summarizing the categories of genes obtained by SSH

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25 ABXPXAAXPFXPXAAXPF Figure 2-6 Southern hybridization performed with two of the genes found by SSH. From left to right /HindIII, Xanthomoas phaseoli XP (strain G66), Xanthomonas axonopodis pv. alfalfae XAA (strain KX-1), and Xanthomonas axonopodis pv. phaseoli var. fuscans XPF (strain 203B) DNA. Total genomic DNA was extracted with the Amersham total DNA kit (A) or with protocol in Appendix B, (B), digested with EcoRI and hybridized with pthA gene (pUFY 14.5) in A, or with a sequence encoding an unknown protein in B. XPXAAXPF/HindIIIXPXAAXPFXC XAAXPFXCABCXCCXCCXCAXCA/HindIII/HindIII Figure 2-7 Southern blot hybridization of three of the genes found by SSH. Total DNA from Xanthomonas axonopodis pv. phaseoli var. fuscans XPF (strain 203B), Xanthomoas phaseoli XP(strain G66), Xanthomonas axonopodis pv. alfalfae XAA (strain KX-1), Xanthomonas citri (strain 3213), Xanthomonas campestris pv. campestris (strain 528T) and Xanthomonas campestris pv. armoraciae (417T) was extracted with the Amersahm total DNA kit, EcoRI digested and run in a 0.7% gel. Blots were probed against PCR amplified internal regions of: A. A gene encoding a hypothetical protein, B. Pectate Lyase and C. A gene encoding a conserved hypothetical protein.

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26 XP M XP M Figure 2-8. Southern blot hybridization showing confirmation a gene encoding a hypothetical protein (XACa0025 homolog) interrupted in Xanthomonas phaseoli (XP) strain G66. From left to right: Marker (/HindIII), XP (G66) and interrupted mutant (M) digested with BglII, XP (G66) and interrupted mutant (M) digested with MluI. Table 2-1 Summary of sequences found by SSH Number of clones obtained 348 Zero trimmed 8.05% Empty vector 6.90% Passing sequences 85% Average bases reading 492 Table 2-2 Categories of genes found by SSH Gene fragment Number Percentage Transposases (different classes) 104 32.3 Transcriptional Regulator 58 18.0 S-receptor Kinase 34 10.6 Hypotetical protein 26 8.1 Pectate Lyase 23 7.1 ABC Transporter 16 5.0 Unknown 7 2.2 VirB9 5 1.6 H rpD5 3 0.9 Integrase 2 0.6 Recombinase 2 0.6 p thA 1 0.3 Others 41 12.4

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27 Table 2-3 Candidate genes selected from Southern hybridization Gene Organism Gene Number Pectate Lyase X anthomonas citri XAC2373 Hypothetical protein X ylella/Xanthomonas citri XACa0025, XfasA1573 Hypothetical protein R alstonia solanacearum RSp1239 Conserved hypothetical p rotein X campestris pv. campestris XCC 3132 VirB9, VirB10 X anthomonas citri XAC2620 Hypothetical protein R alstonia solanacearum RSp0593* *This gene was not interrupted

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CHAPTER 3 INTERRUPTION AND TRANSIENT EXPRESSION OF PTHF, AN AVRBS3/PTHA MEMBER CLONED FROM XPF Introduction All members of the Xanthomonas avrBs3/pthA gene family share nearly identical DNA sequence and are important in the pathogenicity of some Xanthomonas spp. and pathovars (De Feyter et al. 1993; Leach and White 1996). All of the predicted AvrBs3/PthA proteins have tandem leucine-rich repeats that are almost always 34 aa in length. In addition, the predicted proteins (Figure 3-1) encode nuclear localization sequences, and a C-terminal eukaryotic transcriptional activation domain (Zhu et al. 1998; Gabriel 1999). Most members of the gene family have been shown to be avirulence (avr) genes that act in a gene-for-gene fashion to elicit a hypersensitive response (HR) on plants that carry cognate resistance genes. pthA was the first member of the gene family shown to be required for pathogenicity (Swarup et al. 1991). Since then, many, but not all (three pthA homologues in X. citri have been shown to be non-functional, (Al-Saadi 2005)) gene family members have been shown to contribute to pathogenicity. For example, avrb6 allows X. c. pv. malvacearum to release many times more bacteria from infected leaves and contributes to water soaking on cotton (Yang et al. 1994), and avrXa7 has been shown to be a pathogenicity determinant of bacterial blight of rice caused by X. oryzae, (Bai et al. 2000, Yang and White 2004). At least 27 members of the avrBs3/pthA family have been identified in diverse xanthomonads (Gabriel 1999; Leach and White 1996), including Xanthomonas phaseoli 28

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29 (XP) and X. a. pv. phaseoli var. fuscans (XPF) (De Feyter et al. 1993, Figure 3-2). In order to clone potential avrBs3/pthA homologues from XPF and determine their possible role in common bacterial blight of beans, an XPF cosmid library was constructed. Cosmid clones containing three pthA homologues were obtained and used for complementation tests, transient expression assays and site directed mutagenesis. Materials and Methods Plasmids, Bacterial Strains and Culture Conditions Bacterial strains and plasmids used or constructed in this study are listed in Appendix A. Xanthomonas strains were grown in PYGM (Gabriel et al. 1989) at 30C, E. coli strains in Luria-Bertani (LB) medium (Maniatis et al. 1982) at 37C, and Agrobacterium strains at 30C in YEB media (Kapila et al. 1997). When appropriate, antibiotics were used at the following concentrations: ampicillin, 50 g/ml (Amp); gentamicin, 3 g/ml (Gm); rifampicin, 75 g/ml (Rif); chloramphenicol, 35 g/ml (Cm) and kanamycin (Kn), 20 g/ml. Genomic Library The XPF genomic cosmid library was constructed according to standard procedures (Maniatis et al. 1992) with some modifications. Total XPF DNA was extracted (Appendix B) and purified by cesium chloride-ethidium bromide density centrifugation. The purified DNA was partially digested with Sau3A and size-fractionated by sucrose density gradient centrifugation at 26,000 rpm for 16-18 h, and the fraction containing the appropriate fragment sizes was used for ligation into the vector. Cosmid vector pUFR043 (DeFeyter and Gabriel 1991) was used for cloning. The vector was cut with EcoRI and SalI to produce two arms and treated with alkaline phosphatase (USB

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30 Corporation Cleveland, Ohio) to prevent self-concatamerization. The treated vector arms were then cut with BamHI to create common cloning ends. DNA fractionated in the sucrose gradient within the size range of 30-50 kb was used for overnight ligations with T4 DNA ligase (Invitrogen Corporation, Carlsbad, CA) with the pretreated cloning vector. The recombinant DNA was packaged with packing mix using GIGAPACK Gold packaging extracts (Strategene, La Jolla, CA), and introduced into E. coli strain DH5 via transfection, according to the manufacturers protocol. Six hundred and fifty eight individual colonies were isolated on selective LB agar medium containing Gm and Kn and stored in LB broth with 14% glycerol plus antibiotics in 96 microtiter well plates and also patched onto agar plates and a nylon membrane using a replicator fork. A modified alkaline lysis procedure was used to prepare the plasmid DNA of 18 randomly picked cosmid clones (Appendix E). The plasmid DNA was digested with EcoRI and the resultant DNA fragments were separated using 0.7% agarose gel electrophoresis to determine average insert size and test for randomness of the inserts. The probe used to screen the clones was an internal BamHI fragment of pthA from pZit45 that was gel purified using QIAquick gel extraction kit according to manufacturers instructions (Qiagen, Valencia, CA). The probe was labeled with 32P by primer extension according to the Prime II kit instructions (Stratagene, La Jolla, CA). Colony and Southern hybridization (Sambrook et al 1989) were performed using GeneScreen Plus nylon membranes according to the manufacturers recommendation (Bio-Rad laboratories, Richmond, CA).

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31 Molecular Biology Techniques For marker interruption of the pthF homologues, pYY40.10, a clone containing a StuI-HincII internal region from pthA cloned into pUFR004 was used (Appendix A). Approximately 250 ng of DNA were electroporated into 40 l of electrocompetent X. phaseoli strain G66 and X. axonopodis pv. phaseoli var. fuscans strain 203B at 1.8 kV/cm in an Eppendorf 2510 electroporator. An additional 960 l of liquid PYGM were added to the bacteria, the culture was grown at 30C for 3 h at 120 rpm and then the whole culture was spread onto PYGM plates containing Rif and Cm at the appropriate concentrations. Colonies were purified, the DNA extracted, digested with EcoRI and BamHI then screened by Southern hybridization. For transient expression assays, a 7 kb BamHI fragment obtained from cosmid CC1 showing a hybridization signal against the pthA probe, was used to replace the BamHI fragment from pGZ6.4 (Agrobacterium vector carrying avrb6 (Duan et al. 1999)). The DNA was transferred to Agrobacterium GV2260 via triparental mating (De Feyter and Gabriel 1991). Plant Assays Inoculations were performed on Phaseolus vulgaris California Red Light Kidney bean cultivar (Appendix D). Overnight cultures were grown in liquid PYGM, centrifuged at 5,000 rpm for 5 min, washed with tap water and adjusted to an OD600 of 0.3 (approximately 108 CFU/ml). A 1:1000 dilution of this suspension was made for low concentration inoculations (approximately 105 CFU/ml). For each inoculation, 2 cm on each side of the trifoliate leaves from 4 week old plants were cut with scissors after dipping them in the bacterial suspension. Scissors were flame sterilized between

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32 inoculations. The plants were kept in a growth chamber at 27C with a 16 h light, 8 h dark cycle. Symptoms were scored daily for up to 3 weeks. Transient Expression Assays Agrobacterium tumefaciens strains (Appendix A) were grown overnight in YEB (Kapila et al. 1997) liquid medium supplemented with antibiotics and transferred to induction medium (YEB plus 10 mM 2-N-morpholino ethasulfonic acid [MES] pH adjusted to 5.6 and 20M of acetosyringone). The culture was grown overnight, centrifuged and resuspended in MMA medum (MS salts, 10 mM MES, 20 g/l sucrose, 200 M acetosyringone, pH 5.6) at an OD600 of 0.8 (Kapila et al. 1997). The suspension was kept at 4C for 1 h. Trifoliate leaves from California red light kidney bean plants (Appendix D) were detached and submerged in the inoculum in a Petri dish. Vacuum infiltration was applied at 27 mmHg for 10 m and then rapidly released to ensure maximum tissue infiltration by the inoculum. The leaves were washed in distilled water and the abaxial side was placed down on a Petri dish containing wet filter paper. The plates were kept in the laboratory at 24C under constant light. Thin Sections Tissue subjected to transient expression was cut into 0.5 x 0.5 cm pieces 24 h after Agrobacterium inoculation. Immediately afterwards, the tissue was infiltrated with fixation solution (Formaldehyde 10%, acetic acid 5%, ethanol 5%) for 20 m and then left shaking in the same solution at 4C overnight. The tissue was washed once with PBS at 30C, and then successively with 30, 40, 50, 60, 70, and 85% ethanol at 4C for 60 m. The last wash was performed in 95% ethanol overnight. The tissue was then washed ten times with agitation at room temperature as follows: two washes with 100% ethanol for 30 m, then two washes in 100% ethanol for 60 m, then a wash in 25% histoclear (R.A.

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33 Lamb, NC)-75% ethanol for 60 m, then a wash in 50% histoclear-50% ethanol for 60 m, then a wash in 75% histoclear-25% ethanol for 60 m, then two washes with 100% histoclear for 60 m and finally a wash with 100% histoclear and paraplast chips (Sigma-Aldrich, St. Louis, MO) overnight. On day four, the mix was heated at 42C to melt the chips, more chips were added until they melted and then the temperature was increased to 60C. The preparation was left for several hours at 60C, the wax was poured off and new wax was added and melted. On days 5, 6 and 7, two wax changes were performed per day and on day 8, the tissue was placed in 2 cm thick molds. On day 9 the tissue was cut into squares of about 1 x 1 x 0.5 cm with a scalpel, fixed on a piece of wood and cut with a microtome into 10 M sections. The sections were placed on slides containing 1ml of water and left at 60C for 10 m, and then at 42C overnight. The slides were stained with safranin and fast green according to Ruzin (1999). Complementation Attempts The cosmid clones that hybridized to pthA were analyzed by restriction enzyme digest profiling. Three cosmid clones that appeared to contain divergent pthA homologues (CC1, CD2 and KB4) were extracted with the high-speed plasmid maxi kit (Qiagen, Valencia, CA) according to the manufacturers instructions. Approximately 250 ng of cosmid DNA were electroporated into the mutant strains and selected as before. Colonies growing in Gm PYGM were single colony purified and used for plant inoculations. Results Cosmid Library Ten microliters of every other 0.5 ml fraction collected from the sucrose gradient were run in a 0.7% gel to select fractions with the most suitable fragment size. Fraction

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34 number 34 (Figure 3-3) was used for ligation into pUFR043 and 658 colonies were obtained after transfection. Eighteen of the colonies were randomly chosen for cosmid DNA extraction and EcoRI digestion. Results shown in Figure 3-4 confirmed that the library was representative. The average size of the inserts was estimated to be 41 Kb. Several Cosmid Clones Contained a Copy of Three Homologues Colony blot hybridizations performed on all 658 colonies indicated that six cosmids contained at least one copy of a potential avr/pth homolog (Figure 3-5). Southern hybridization analysis showed that at least three diverse XPF homologues were present in the library based on restriction profile with different enzymes (CC1, CD2 and KB4, Figure 3-6) and the rest of the hybridizing cosmid clones were duplicates of the same three putative genes (data not shown). The three putative genes were named pthF, pthF1 and pthF2 and the BamHI internal fragment sizes of these genes were approximately 7, 3.5 and 3.6 respectively. The cosmid clones were hybridized with probes containing the 5 and 3 ends of the pthA and were sequenced with vector-based primers to determine if the cosmids contained the entire gene homologues. Transient Expression Assays and Thin Sections A. tumefaciens strain GV2260 carrying pthA (pYD40.1) and an empty vector (pYD40.2) were used as controls (Duan et al. 1999). As shown in Figure 3-7, strain GV2260 containing pthA induced an HR after 48 hours and the empty control showed a slight chlorosis in bean leaves starting at 72 h. A. tumefaciens containing pthF (pLZ1.7) showed a blight-like phenotype, apparently different from pthA or empty vector at 72 h. Thin section experiments showed that pthF induced necrosis to a lesser extent than pthA, but pthF expression caused collapse to an extent similar to that seen with pthA (Figure 3-8).

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35 Attempts to Mutagenize pth Homologues Several XP and XPF colonies grew on Rf-Cm PYGM plates after attempts to marker-interrupt pthA homologues with PYY40.10. Southerns hybridization patterns similar to wild type using pthA as a probe indicated that about half of the colonies were spontaneous Cm resistant mutants (data not shown). Hybridization analyses also showed that three mutants had apparently lost an entire plasmid (F3 and F6 from XP and FF19 from XPF) and two others (F1 and F2) showed interruption of one of the small BamHI fragments (Figure 3-9, some data not shown). Some Mutants of pthF Caused a Pathogenicity Reduction None of the marker-interrupted mutants were affected in growth, in planta, pathogenicity or any other obvious characteristic. Three mutants that apparently had lost a plasmid, had also lost the ability to grow in Cm plates, and also exhibited reduced pathogenicity on bean plants (F3, F6 and FF19, Figure 3-10, some data not shown). The two mutants showing an interruption in one of the pthF genes (pthF1 or pthF2) showed no alteration of pathogenicity. Attempts to Complement Pathogenicity Deficient Mutants Three cosmid clones from the library were used for complementation assays: CC1, CD2 and KB4 carrying pthF, pthF1 and pthF2 respectively. None of the cosmid clones complemented the pathogenicity deficiency in any of the mutants (data not shown). Discussion Diverse members of the avrBs3/pthA gene family have been shown to be involved in pathogenicity in several Xanthomonas spp. They have been associated with elicitation of diverse plant phenotypes, including water soaking, hypertrophy, hyperplasia, epidermal necrosis, hypersensitive response and blight (Yang et al. 1994; Duan et al.

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36 1999; Yang and White 2004). In the transient assays reported here, one of the pthA homologues from XPF, pthF, consistently elicited a blight-like symptom when expressed in bean leaves. Since empty vector in Agrobacterium elicits a slight chlorosis in bean leaves, thin sections were examined to confirm the pthF-elicited phenotype observed. The analyses showed that pthF induced symptoms similar to those elicited by pthA in bean, although less necrosis was observed with pthF. In both cases the intercellular spaces were greatly reduced when compared with empty vector alone. However, pthF knock-out mutations were not obtained and therefore the potential role of pthF in pathogenicity of XPF or XP was not determined. Since fragments that hybridize to pthA have been found in all examined CBB bacteria, a role for pthF and other avrBs3/pthA genes in bean blight is likely. Marker interruption mutations in pthA homologues were all equally pathogenic to the wild type. Plasmid cured strains revealed reduced virulence when compared to wild-type strains, but these were not complemented by any of the three XPF pthA homologs. None of the mutants obtained occurred in pthF that was used for transient expression. There are several reasons that might explain why complementation of a plasmid cured strain failed when using single genes. First it is possible that additional genes present in that plasmid besides the pthF gene could also be needed for full pathogenicity. Second, it is possible that the clone chosen for complementation was not functional. Third the plasmid vector may have been unstable in XPF even though repW has been demonstrated to be highly stable in all other Xanthomonas tested, including X. citri, X. malvacearum, X. albilineans and X. campestris (De Feyter et al. 1990).

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37 A new study using different techniques for interruption of homologous genes is needed since Southern hybridizations showed that the plasmid containing the pthF gene was apparently lost after incorporation of the suicide vector. A different and more quantitative inoculation method also needs to be developed in order to detect possible subtle differences in pathogenicity between mutant and the wild type strains.

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38 | | | | | | | | | | | | | | | | NH2Repeat regionTACOOHLZ CK| | | | | | | | | | | | | | | | | | NH2Repeat regionTACOOHLZ CK| | SHABB Ss Figure 3-1 Diagram of pthF showing restriction sites and domains of the predicted protein: Leucine zipperlike area (LZ), casein kinase 2 (CK), nuclear localization signals (NLS) and transcriptional activator (TA). Letters appearing on top of the figure represent the following enzymes: BamHI (B), StuI (S), HincII (H), AatII (A) and SstI (Ss). XPF 23 Kb9 Kb6 Kb pthFpthF1pthF2 Figure 3-2 Southern blot of total DNA from XPF probed with an internal fragment from pthA. DNA from XPF strain 203B was cut with BamHI, run in a 0.7% agarose gel. Arrows indicate the locations of bands corresponding to pthF, pthF1 and pthF2. 20222426283032343623 kb_9 kb_6 kb_4.5 kb_2.2 kb_2 kb_ Figure 3-3 Fractions collected after sucrose density gradient centrifugation of XPF, partially digested Sau3A DNA were run on a 0.7% agarose gel. Fraction 34 was used for constructing the cosmid library.

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39 23 kb_9 kb_6 kb_4.5 kb_2.2 kb_2 kb__23 kb_9 kb_6 kb_4.5 kb_2.2 kb_2 kb Figure 3-4 Eighteen XPF cosmid clones, digested with EcoRI, and run on a 0.7% gel. Cosmid DNA from randomly chosen colonies was extracted and digested with EcoRI. The outside lanes carry marker DNA (/HindIII). A B C D E F 12345678 Figure 3-5 Colony hybridization of 48 clones from the XPF cosmid library probed with pthA. Three hybridizing clones are shown.

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40 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 BamHIBamHI+KpnIBamHI+PstIBamHI+SstIPstI+KpnI23 Kb9 Kb6 Kb2.2 Kb2.0 Kb Figure 3-6 Southern hybridization of the three different cosmid clones. Clones CC1 (1), CD2 (2) and KB4 (3) were extracted, digested with the indicated enzymes, run on a 0.7% gel and probed with pthA. pthFEmptyvectorpthA Figure 3-7 Transient expression of pthF and pthA in bean leaves. From left to right LZ 1.7 (pthF), pYD 40.2 (empty vector) and pYD40.1 (pthA).

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41 A B C Figure 3-8 Transient expression of pthF in detached bean leaves. Thin sections of A, LZ 1.7 (pthF); B, pYD 40.2 (empty vector), and C, pYD40.1 (pthA).

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42 23 Kb XP F3 F6 F1 F2A9 Kb6 KbBXP F3 F6 F1 F2 23 Kb9 Kb6 Kb XP F3 F6 F1 F2~15 Kb~4 KbC DE23 kb_23 kb_9 kb_9 kb_6 kb_6 kb_XPXPF3F3F6F6F1F1F2F2 Figure 3-9 Southern blot hybridization of four putative pthF mutants (F3, F6, F1 and F2) and X. phaseoli (XP) strain G66. In panels A and C the total DNA was probed with pthA and in B, with pUFR004 vector DNA. Gel D was used for blots A and B and E used for blot C. In A, B, and D total DNA was digested with EcoRI and in C and E with BamHI. The asterisk in panel C indicates pthF band. Figure 3-10 Bean leaf inoculated with F6 mutant on the right side and with the wild type XP (G66) on the left side of the leaf. The picture was taken 6 days after inoculation.

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CHAPTER 4 SITE DIRECTED MUTAGENESIS IN THE REGIONS IN THE C-TERMINUS OF TWO AVRBS3/PTHA MEMBERS Introduction Xanthomonas campestris pv. malvacearum (XCM) causes cotton blight and Xanthomonas citri (XC) causes citrus canker disease. Avrb6 from XCM and PthA from XC are effector genes that function in avirulence and pathogenicity and belong to the Xanthomonas AvrBs3/PthA family. These genes have been shown to contribute significantly to cotton blight and citrus canker diseases respectively (Yang et al. 1994; Swarup et al. 1991). All members of the AvrBs3/PthA family are very similar in sequence and all carry three nuclear localization sequences (NLS), a C-terminal eukaryotic transcriptional activation domain (TA), and a 34 amino acid, tandem direct repeat region (Figure 4-1, Gabriel 1999; Zhu et al. 1998). NLS, TA and repeat region domains of the protein family have been shown to be required for the specific pathogenicity and/or avirulence phenotypes that are determined by the proteins. The one notable exception is avrBs3-2 (synonym avrBsP), which elicits gene-for-gene avirulence in tomato without the NLS and/or TA domains (Canteros et al. 1991). The three NLSs function additively in avirulence and pathogenicity (Van den Ackerveken et al. 1996, Duan et al. unpublished). The repeat region is essential for host-pathogenic specificity and for gene-for-gene avirulence specificity (Yang et al.1994); the repeat region is also essential for homo-dimerization of the proteins before entering the 43

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44 nucleus (Gurlebeck et al. 2005). The eukaryotic transcriptional activation domain (TA) is also required for pathogenicity and avirulence (Yang et al. 2000; Zhu et al. 1998). The purpose of this study was to examine the potential role of three additional regions in the function of pthA and avrb6: a leucine zipper-like region (LZL), a casein kinase 2 (CK2) site and a randomly selected region between the LZL and CK2 (U pstream of C asein K inase (UCK)). Materials and Methods Plasmids, Bacterial Strains and Culture Conditions Bacterial strains and plasmids used or constructed in this study are listed in Appendix A. Xanthomonas strains were grown in liquid PYGM at 30C and E. coli strains were grown at 37C in Luria-Bertani (LB) medium (Maniatis et al. 1982). When appropriate, antibiotics were used at the following concentrations: ampicillin, 50 g/ml (Amp); gentamicin, 3 g/ml (Gm); kanamycin, 20 g/ml (Kn); rifampicin, 75 g/ml (Rf); spectinomycin, 50 g/ml (Sp) and tetracycline, 50 g/ml (Tc). DNA constructs were introduced by triparental mating into B21.2, and KX-1 strains (De Feyter and Gabriel 1991) and inoculated into their hosts Citrus paradise (grapefruit) and Phaseolus vulgaris (common bean), respectively (Appendix D). For introduction into HM2.2S, the DNA was first methylated as described (Yang et al. 1996), introduced into the strain by triparental mating and then inoculated on cotton (Gossypium) plants (Appendix D). Site-Directed Mutagenesis UDG cloning (Rashtchian et al. 1992, Figure 4-2) was used to introduce specific, site-directed mutations. The respective primers were synthesized by Gibco BRL (Carlsbad, CA), and are listed on Appendix C. The regions of interest were amplified using pQY107.1 as target DNA (3 end of pthA extending from the HincII site to HindIII,

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45 refer to Figure 4-1). For PCR standardization, 1, 10 and 100 ng of DNA template, and 0.5 mM, 1 mM and 2 mM of magnesium chloride were used with Taq Polymerase (Gibco, Carlsbad, CA) and the remaining components at concentrations recommended by the manufacturer (Gibco, Carlsbad, CA) in a 50 l PCR mix. Denaturation was at 95C for 4 m, followed by 25 cycles at 94C for 45 s, 62C for 30 s and 72C for 90 s, with a final extension of 5 m at 72C. Following amplification, 10 l of the PCR product was treated with DpnI for 30 m at 37C to degrade methylated residues in the template, and then the enzyme was heat inactivated for 30 m at 65C. The mix was then incubated with 1.5 Units of UDG at 37C for 30 m to degrade the deoxy-uracyl residues contained in the primers, followed by incubation for 10 m at 65C to both inactivate the enzyme and to melt the remaining oligonucleotides (12 bp) adjacent to the uracils. The mix was annealed at room temperature for 1 h and then transformed in DH5 without ligation. Plasmid DNA from transformed cells was extracted with the alkaline lysis protocol in Appendix E, cut with NcoI, and plasmids showing the expected restriction profile (pLZ7.1 for LZL, pUCK for UCK and pCK2.1 for CK2) were sequenced at the ICBR (Interdisciplinary center for biotechnology research) core facility (http://www.biotech.ufl.edu/Genomics/). Plasmids with the correctly mutated sequence were cut with AatII and SstI and the mutated region was substituted for the wild type region in pthA using pYD9.4 cut with SstI and then partially cut with AatII. The desired bands from pYD9.4 and pLZ7.1, pUCK1 or pCK2.1 were gel purified using Qiagen columns (Qiagen, Valencia, CA) and ligated. The resulting plasmids were cut with EcoRI-HindIII and the fragment carrying the modified pthA gene was recloned into shuttle vector pUFR047 to form pAC1.16, pAC14.1 and

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46 pAC6.1, respectively. The internal wild type StuI-HincII region from avrb6 was swapped with the same region in pAC1.16, pAC14.1 and pAC6.1 to form pAY8.1, pUCK3 and pALZ4, respectively. In the LZL region replaced in pAC1.16 (pthA) and pAY8.1 (avrb6), two residues were changed from leucine to methionine (L918 to M918 and L925 to M925 in pthA, and L790 to M790 and L797 to M797 in avrb6). In the UCK region replaced in pAC14.1 (pthA) and pUCK3 (avrb6), one residue was changed from arginine to proline (R955 to P955 in pthA and R827 to P827 in avrb6). In the CK2 region replaced in pAC6.1 (pthA) and pALZ4 (avrb6), one residue was changed from glutamic acid to valine (E997 to V997 in pthA, and E867 to V867 in avrb6). Plant Inoculations All citrus plants (Appendix D) were grown under greenhouse conditions and inoculated in the quarantine facility at the Division of Plant Industry, Florida Department of Agriculture in Gainesville, Florida. Cotton (Gossypium hirsutum) and common bean plants (Phaseolus vulgaris) (Appendix D) were grown under greenhouse conditions and after inoculations they were kept in growth chamber conditions with a 16 h light 8 h dark cycle at 30C. Bacterial cultures were grown in PYGM at 30C, suspended in sterile tap water to 109 CFU/ml, and inoculated into newly expanded leaves by pressure inoculation with the blunt end of a tuberculin syringe as described by Swarup et al. (1991). Results UDG Cloning Different PCR conditions were tested for the three site directed mutations performed. Figure 4-3 shows a 1% agarose gel with the products obtained at different conditions for mutation of the leucine zipper-like region. In this case, the PCR product

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47 obtained with 0.5 mM of magnesium chloride using 10 ng of DNA as template was used for treatment with UDG and then transformed into E. coli cells. No Change in the Non-Host HR on Bean When pZit45 (pthA) was introduced into X. axonopodis pv. alfalfae strain KX-1, it elicited a strong HR on common bean 24 h after inoculation. When the different mutated versions of pthA were introduced into KX-1 and inoculated on bean, none showed a change in the timing or intensity of the HR when compared to strains carrying pZit45, (Figure 4-4, some data not shown). No Change in Water-Soaking or the Non-Host HR on Cotton HM2.2S is an X. c. pv. malvacearum strain in which six of the twelve avr genes have been mutated, including avrb6; this strain releases approximately 1600 times less bacteria than the wild type and is nearly asymptomatic on cotton, including resistant lines (Yang et al. 1996). When avrb6 is re-introduced into HM2.2S, water soaking symptoms were restored on resistant cotton line AcalaB6. When the different mutated versions of avrb6 were introduced into HM2.2S strain, all conferred the same phenotype as the wild type gene. Mutations of CK2 or the UCK, but not LZL Region, Resulted in Reduced Canker Symptoms B21.2 carries a Tn5 mutation in pthA and is unable to induce canker on citrus; when pZit45 carrying pthA is introduced in this strain, the canker phenotype is restored (Swarup et al. 1991). B21.2 strains carrying pthA mutated in the LZL region exhibited canker symptoms comparable to that of wild type strains. However, pthA mutated in the CK2 or the UCK regions was and introduced into B21.2, reduced canker symptoms compared to strains carrying pZit45 (Figure 4-6).

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48 Discussion AvrBs3/PthA proteins are unique effectors with eukaryotic features. They apparently function as transcriptional activating factors, which often contain leucine zipper motifs (Jakoby et al. 2002). A detailed analysis of the amino acids present in Avrb6 and PthA showed that they do not have leucine zipper motifs, but a region very similar to leucine zippers with four hydrophobic amino acids for every seven residues. However, instead of forming an helix, which is necessary for the structure of this type of transcriptional factor, two proline residues are present in the LZL region that would break the potential helix. No difference between wild type pthA or mutated avrb6 LZL was observed in the non host-HR on bean, host HR on cotton or pathogenicity on cotton and citrus plants. The methionines used to replace the leucines in these mutants have the same neutral and hydrophobic characteristics as the leucines. It is possible that charged amino acid substitutions would have revealed a different phenotype. Some bacterial effectors are known to induce phosphorylation of host proteins that in turn activate resistance signal response cascades in plants (Mackey et al. 2002). No studies have been conducted to demonstrate phosphorylation in AvrBs3/PthA proteins, but CK2 regions are known to be important in nuclear localization (Jensen et al. 1998). Individual mutations in the CK2 and the UCK region demonstrated their importance in canker elicitation by pthA on citrus, but not in blight or water soaking elicitation by avrb6 in cotton, and not in host on non-host HR responses by either gene. The difference between the two pathogenicity phenotypes could be explained by the nature of the two diseases. While transient expression assays (canker vs. water soaking on blight) have demonstrated that pthA alone induces canker lesions in citrus leaves, similar experiments

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49 in cotton expressing avrb6 were unsuccessful in eliciting blight or water soaking in cotton (Gabriel lab unpublished). In addition, knock-out mutations of pthA (as in B21.2) cause reduction of growth in planta, whereas knock-out mutations of avrb6 cause no such loss of in planta growth (Yang et al. 1996). X. malvacearum carries at least six other members of the gene family, and it is likely that some other members are functional such as a partial loss of nuclear localization ability would not be detected. It is also possible that through hetero-dimerization, a partial loss of nuclear localization ability would not be detected, although hetero-dimerization has not been reported. Yang et al. (1996) reported that there are 2 CK2 sites (at positions 994-997 and 1068-1071 in pthA) and one of them was mutated in this study, possibly a non-functional one. Reanalysis of the gene family for CK2 sites also reveals the possibility that two additional sites (at positions 892-895 and 1120-1123 in pthA) are also present in the gene family.

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50 | | | | | | | | | | | | | | | | Repeat regionNLSTALZ CK2| | | | | | | | | | | | | | | | | | Repeat regionNLSTALZL SHABBATG Ss TGAHi Figure 4-1 Map of typical avrBs3/pthA gene. Leucine zipperlike area (LZL), casein kinase 2 (CK2), nuclear localization signals (NLS) and transcriptional activator (TA). Coding region extends from ATG to TGA and letters appearing on top of the figure represent the following enzymes: BamHI (B), StuI (S), HincII (H), AatII (A), SstI (Ss), and HindIII (Hi). A. Synthesize primersB. PCR amplifyC.Treat with UDGD. Transform withoutin vitroligation Figure 4-2 Schematic representation of the UDG cloning technique. A. Primers with uracil (U) replacing thimidine (T) are synthesized. B. PCR amplification is performed. C. Treatment with Uracyl DNA Glycosilase (UDG) D. The mix is incubated at room temperature and then transformed directly into E.coli.

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51 M 1 2 3 4 Figure 4-3 PCR amplification of the HincII-HindIII region of pthA in pUC19. Five microliters from of the 25-l reaction were run in a 1% agarose. From left to right: M, /HindIII marker; 1, control plasmid cut with EcoRI; 2, 2 mM MgCl2; 3, 1 mM, and 4, 0.5 mM of magnesium chloride used with 10 ng of DNA as template. Figure 4-4 California Light Red Kidney bean plant inoculated with KX-1 carrying pUFR047 (KX-1) and mutants in the leucine rich area (KX-1/pAC1.16 and 1.19). The picture was taken 36 hours after inoculation. AB123461234556 Figure 4-5 Acala 44 cotton plants (A) and AcalaB6 cotton plants (B) inoculated with: 1, HM2.2S; 2, HM2.2S containing pUFR047; 3, HM2.2S containing avrb6, 4, HM2.2S containing avrb6 mutated in Leucine zipper-like area; 5, HM2.2S containing avrb6 mutated in casein kinase 2, and 6, HM2.2S containing avrb6 mutated and upstream casein kinase (6).

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52 ABCDEF Figure 4-6. Citrus leaf inoculated with mutant versions of pthA in B21.2: A, F, pAC6.1 (CK2 mutated); B, pZit45 (pthA wild type); C, pUFR047 (empty vector), and D, E, pAC14.1 (UCK mutated).

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CHAPTER 5 INDIVIDUAL AND SEQUENTIAL MUTAGENESIS OF XCC AVR GENES, IDENTIFICATION OF A FUNCTIONAL AVR GENE, ATTEMPTS TO DEMONSTRATE HR SUPPRESSION BY AVR GENES Introduction Xanthomonas campestris pv. campestris (XCC) causes black rot of crucifers, a systemic bacterial infection that generates serious economic losses worldwide. Xanthomonas campestris pv. armoraciae (McCulloch 1929) Dye 1978b (XCA), also infects crucifer plants, but causes only localized leaf spots and hydathode necrosis (Black and Machmud 1983). Genes determining systemic movement of XCC have not yet been identified. Like most other phytopathogenic xanthomonads, hypersensitive response and pathogenicity (hrp) gene mutations in XCC result in a loss of the ability to induce a hypersensitive response (HR) on nonhosts and pathogenicity on hosts (Kamoun and Kado 1990), indicating that type III secretion is critical for pathogenicity of XCC. Diverse methods have been used to identify bacterial effectors that are secreted by the type III system (Buttner et al. 2003, Nomura and He 2005). These methods include: 1) complementation of non-pathogenic strains with a genomic library of a pathogenic strain (Chen et al.1994), 2) reporter fusion systems (Cunnac et al. 2004; Guttman et al. 2002; Roden et al. 2004a), 3) screening of genes induced in planta or co-regulated with hrp genes (Noel et al. 2001), 4) promoter-trap assays (Losada et al. 2004) and 5) suppression subtractive hybridization or SSH (Harakava and Gabriel 2003). Functional genomics techniques have become increasingly effective for elucidating pathogenicity mechanisms as more genomic DNA sequences have become available. 53

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54 This approach allows rapid in silico identification of potential homologous effectors. In this study, when SSH revealed that a putative avirulence (avr) effector was present in XCC but not XCA, a search of the published genome of XCC528T (synonym: ATCC33913, Xanthomonas-ONSA FAPESP network. 2001/2002) revealed a total of seven additional putative avr genes (Table 5-1). In order to determine the role of the avr genes in pathogenicity of XCC on crucifers, individual and sequential mutations were created by marker interruption in all eight avr genes. Since several studies have shown additive effects of avr genes as pathogenic effectors (Lorang et al. 1994; Yang et al. 1996; Badel et al. 2003; Wichmann and Bergelson 2004; Lin and Martin 2005), cumulative targeted mutagenesis of all eight putative avr genes was performed by using splice overlap PCR and marker interruption techniques. Materials and Methods Plasmids, Bacterial Strains and Culture Conditions Bacterial strains and plasmids used or constructed in this study are listed in Appendix A. Xanthomonas and Pseudomonas strains were grown in PYGM at 30 C and E. coli strains were grown at 37 C in Luria-Bertani (LB) medium (Maniatis et al, 1982). When appropriate, antibiotics were used at the following concentrations: ampicillin (Amp) 50 g/ml; gentamicin (Gm) 3 g/ml; kanamycin (Kn) either at 12.5 g/ml or 20 g/ml; rifampicin (Rif) at 75 g/ml, chloramphenicol (Cm) at 35 g/ml and tetracycline (Tc) at 10 g/ml. When needed, sucrose was added to a final concentration of 5%. Molecular Biology Techniques Primers used in this study were synthesized by Integrated DNA technologies, Inc. (Coralville, Iowa) and are listed in Appendix C, and enzymes used were purchased from Invitrogen Technologies (Carlsbad, California). A more suitable suicide vector was

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55 constructed for marker interruption experiments. pAC3.1 was constructed from pUFR004, a suicide vector, in three steps. First pUFR004 was digested with XbaI, and the recessed termini were filled with Klenow. Then it was digested with EcoRI and ligated to the kanamycin gene from pKLN66 that had been cut with BamHI, filled in with Klenow, and subsequently digested with EcoRI. The resultant plasmid was called pAC7. pUC118 was digested with BsmBI, Klenow filled, digested with EcoRI to obtain the polylinker, which was ligated to pAC7 that had been cut with EcoRI and SmaI. The resultant vector was named pAC2, a suicide vector with Cm and Kn resistance. In order to eliminate the extra BamHI site present at the 3 of the kanamycin resistance marker, pAC2 was partially digested with BamHI, filled in with Klenow and religated to form pAC3.1. For marker interruption, an internal region of the target gene was amplified with appropriate primers from a single bacterial colony touched with a toothpick and added into the PCR mix. Twenty-five microliter reactions were performed using Invitrogen Taq polymerase (Invitrogen Corporation, Carlsbad, CA) with its PCR buffer, magnesium chloride and nucleotides at concentrations recommended by the manufacturers, and with 0.4 M each primer. In order to lyse the cells and obtain the target DNA, an initial denaturation was performed at 95C for 3 m, followed by 35 cycles of 30 s at 95C, 30 s at the specified annealing temperature, 70C for the specified extension time, with a final 10 m extension at 70C. One microliter of the PCR reaction was used for cloning into TOPO vector (Invitrogen Corporation, Carlsbad, CA) using the manufacturers protocol. Except for the XopD homolog, in which the internal region was cloned in TOPO and used

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56 directly for marker interruption, the inserts obtained from all eight avr genes were recloned into pAC7, pAC3.1 or pUFR12 (Figure 5-1 A). For splice overlap PCR, two regions of approximately 1 kb long, upstream and downstream of the target gene were PCR amplified independently with their respective primers (primers a, b and c, d in Figure 5-1 C) in the Fail Safe PCR premix D from Epicenter (Epicenter Technologies, Madison, WI). The primers b and c (Figure 5-1 C) of each amplicon have complementary stretches in the proximal termini that will be used for annealing of the amplicon in the next step. Both products were diluted 1:25 and 1 l of each was mixed and a fill-in reaction was done in the premix D at 95C for 3 m, 50C for 1 m and 70C for 10 m in presence of Taq. After that, the external primers (a and d, Figure 5-1 C) were added at 0.5 M to the reaction and 35 cycles of PCR was performed as before. One microliter was cloned directly into TOPO, or the desired band was gel purified using Qiagen columns (Qiagen, Valencia, CA), cloned into TOPO and transformed in DH5 cells. The insert was then recloned in pUFR080 and sequenced. For the FLP recombinase excision experiments, an internal region of Xcc3731 was PCR amplified using primers AC-19 and AC-20, cloned into TOPO as described, and recloned into BY17.1 using BamHI-SstI restriction enzymes (Figure 5-1 B). Suppression Subtractive Hybridization The procedure was performed using the PCR Select Bacterial Genome Subtraction kit, (Clontech Laboratories, Inc. Palo Alto, California) according to the manufacturers instructions. XCC and XCA genomic DNA were extracted using the protocol in Appendix B. Approximately 2 g of both XCC and XCA genomic DNA

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57 were each cut with 15 units of RsaI for 16 hours at 37C. Half of the DNA from XCC was ligated to adaptor 1 and the other half to adaptor 2R as recommended. Digested XCA DNA was diluted and mixed with each of the XCC adaptor-ligated DNAs in separate tubes, and the samples hybridized according to Clontech instructions except the hybridization temperature which was set at 63C. The final molar ration between tester (XCC) and driver (XCA) DNAs was approximately 1 to 2.5. Amplification of SSH product was also according to Clontech instructions except that the first round of PCR amplification was performed with Nested primer 1 at 61C for 35 cycles and the second round of amplification with Nested primer 2R was at 64C for 25 cycles. Three microliters of DNA from the second PCR amplification mix was ligated into pGEMTeasy (Promega Co, Madison WI) according to the manufacturers instructions, transformed into DH5 cells and plated on LB plus Amp. DNA Sequencing and Analysis All XCC DNA clones resulting from SSH were sequenced at the ICBR (Interdisciplinary center for biotechnology research) DNA Sequencing Core Facility of the University of Florida. To confirm that the cloned DNA fragments were unique to XCC, the SSH clones were PCR amplified with vector based primers M13R (-48) and M13 (-47). Two microliters of the product from the PCR amplification was spotted twice with a VP408S2a replicator (V & P Scientific Inc., San Diego, CA) on two different nylon membranes, and also run in a 1% agarose gel to confirm that the amplified products were uniform in DNA concentration. Five hundred nanograms of total RsaI digested DNA from all strains were separately labeled with 32P with the Random Primer kit II (Stratagene) according to manufacturers protocol. The labeled DNA was used as a probe against each of the

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58 corresponding membranes (Figure 5-2). The DNA fragments of interest were further confirmed as XCC-specific by Southern hybridization analyses. Mutagenesis Experiments For single mutations, approximately 250 ng of the plasmid DNA was electroporated into 40 l of electrocompetent XCC strain 528T at 1.8 kV/cm in an Eppendorf 2510 electroporator. Liquid PYGM to complete 1 ml was added to the bacteria, the culture grown at 30C for 2 to 3 hours at 5000 rpm and then the entire volume was spread onto a PYGM plate containing Rif and Kn at the appropriate concentrations. The colonies growing on Kn plates were transferred to Cm plates, except for the mutant strain interrupted with a TOPO vector, which was PCR tested immediately (XopD mutant) and confirmed by Southern hybridization analysis (not shown). For splice overlap mutagenesis, colonies were also confirmed on Cm plates and then grown in liquid PYGM overnight, the OD adjusted to 0.13, and 20 ml were spread onto PYGM containing sucrose and Rif. Colonies that grew on sucrose were replica plated onto PYGM plus Kn and sucrose plates, and the colonies growing only on sucrose plates were analyzed by PCR and Southern hybridization, and selected for successive rounds of mutation. Probes used were PCR products and/or PCR products cloned in TOPO or pUFR080. For FLP recombinase eviction assays, the mutant strain was selected also on Cm plates, screened by PCR and then electroporated with 50 ng of pJR4 (Appendix A) to evict the plasmid, and selected in Gm plates. The colonies growing on Gm plates were replica plated onto Kn and plain plates. The colonies growing only on plain plates were confirmed by PCR and Southern hybridization, grown in plane PYGM overnight, and transferred to sucrose as before to evict pJR4. Colonies growing in sucrose were replica

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59 plated on Gm and sucrose, and the colonies sensitive to Gm were selected to perform an additional round of mutation. Growth Kinetics The abaxial leaf surface of fully expanded leaves from white turnip and Florida mustard plants (Appendix D) were syringe infiltrated with 106 CFU/ml of inoculum. Each inoculation site was flooded to achieve a zone of ca. 0.5 cm in diameter. Samples were taken in triplicate with a core-borer number 14 (22 mm diameter) to encompass the entire inoculation site starting at day 0 and every two days until day 8. The leaf tissue samples were macerated in 1 ml of 100 l of Silwett L-77 (Osi Specialties Inc., Friendly, WV) per 500 ml and CaCO3 added to saturation. They were serially diluted and plated on PYGM plates, and the colonies were counted two days after plating. The experiment was repeated twice. Electrolyte Leakage Measurements For electrolyte leakage studies, bacterial suspensions were diluted to a concentration of 106 CFU/ml in sterile tap water, inoculated by syringe infiltration and measurements were carried out as previously described (Hibberd et al. 1987). For each sample, six leaf disks were removed with a 0.5 cm cork borer number 7, submerged in 3 ml distilled water, vacuum infiltrated, shaken at 28C at low speed and after 1 h the net leakage was measured with a conductivity meter (YSI Model 31). Two samples were taken for each measurement in each experiment; the experiments were repeated three times. Complementation Assays Primers AC-35 and AC-45 (Appendix C) were used to amplify the ORF Xcc2109 (Xanthomonas-ONSA FAPESP network. 2001/2002). Primers 09-04 and AC-45 were

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60 used to amplify a longer ORF that includes Xcc2109 and 327 bp upstream. The two DNA sequences were cloned into TOPO PCR vector (Appendix A) and recloned into pUFR070 to form pAC10 and pAC19 respectively. Correct orientation of both clones for expression from the lacZ promoter on pUFR070 was confirmed by sequencing, and approximately 50 ng of DNA was electroporated into the individual Xcc2109 knockout mutants X48 and X109. Colonies appearing on appropriate selection media were grown in liquid PYGM supplemented with antibiotics, the cultures were centrifuged, washed with tap water, the concentration adjusted to 106 CFU/ml and the suspension was inoculated into Florida Mustard leaves by scissors clipping described in the plant assays below. Plants were scored one week after inoculation. Race Specificity Change XCC strains 6181 and 3849A, representatives of races 0 and 2, respectively (Kamoun et al. 1992, Table 5-2), were electroporated with pAC19 and pAC10 and selected on the appropriate antibiotics. Cultures were grown and inoculated in B. juncea, B. oleracea var capitata, and B. rapa plants as described for other strains of XCC. At least three plants were inoculated in each experiment, and each experiment was repeated at least once. Plant Assays All plants were grown under greenhouse conditions and are listed in Appendix D. Leaves from 3-week-old crucifers, were inoculated at two concentrations. Overnight cultures were grown in liquid PYGM, centrifuged at 5000 g for 5 m, washed with tap water and adjusted to 0.3 OD (approximately 109 CFU/ml). A 1:1000 dilution of this suspension was made for low concentration inoculation (Approximately 106 CFU/ml). For each inoculation, approximately one third of the leaves were cut with scissors after

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61 dipping them in the bacterial suspension. Scissors were flame sterilized between inoculations. After inoculation, the plants were maintained in a growth chamber for 14 days with a daily regimen of 16 h at 28C in the light, followed by 8 h of dark at 27C. Plants were scored every day up to 14 days. For seedling assays, B. juncea and B. olareceae var. capitata seeds were grown under continuous light at 29C. Seven-day-old plants were inoculated by pricking the hypocotyls with a 25-gauge syringe needle that was dipped into a bacterial colony. Plants were observed starting at 16 hours and for up to five days. For the non-host HR, pepper plants ECW, ECW-10R, ECW-20R and ECW-30R (Capsicum annum), were used. They were inoculated by pressure infiltration using the blunt end of a tuberculin syringe into the abaxial leaf surface at four (109, 108, 107 and 106 CFU/ml) serially diluted concentrations, and observed at 18, 24, 36 and 48 h. At least three plants were inoculated in each experiment, and each experiment was repeated at least three times. HR was observed every h starting at 12 h and for the next 36 h. Cell Death Suppression Assays pAC10, pAC19, pAC99 and pAC31 were electroporated into P. fluorescens strains 55 carrying pHIR11 or pLN18 and selected with the appropriate antibiotics. Colonies were then grown overnight in liquid PYGM suplemented with Gm, resuspended in sterile tap water at the desired concentration and inoculated in tobacco and ECW pepper plants with a syringe by pressure infiltration. Initially 55/pHIR11 carrying each one of the constructs were inoculated starting at 0.2 OD600 and at 1:5 dilutions up to 1:625. Later, 55/pLN18 transconjugants were inoculated at 0.2 OD600 and after 2 h 55/pHIR11 was co-inoculated at 1:100 dilution from the 0.2 OD600 suspension. As an expression control,

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62 pZit45 was introduced in 55/pLN18, selected in PYGM Gm plates, and inoculated in tomato plants at 0.3 OD600. pAC19, pAC10, pAC99 and pAC31 were electropotated into 95-2 and selected in the appropriate antibiotics. Colonies were then grown overnight in liquid PYGM suplemented with Gm, and inoculated in 30R pepper plants as before. All strains were inoculated starting at 0.3 OD600 and at 1:5 dilutions up to 1:625. Results Suppression Subtractive Hybridization A total of 226 XCC clones were obtained and subjected to dot blot analysis. This analysis showed that about half of the clones obtained were represented in both XCC and XCA (Figure 5-2). The XCC clones resulting from SSH were sequenced and the genes of interest confirmed or discarded as XCC-specific after Southern hybridization analysis (not shown). On average, the XCC fragment readings were 375 bp long. As shown in Figure 5-3, more than half of the sequences (119) corresponded to hypothetical proteins, 11 to enzymatic proteins, 2 to proteins involved in two-component system, and one to an the avirulence gene Xcc2109 (NC_003902.). No other potential plant effectors were identified. Twenty-one transposable elements and related genes were also found. Except for six of the genes found once, all gene fragments were obtained two or more times through the SSH analysis. No Evidence of Pleiotropic Pathogenicity Function by any Individual XCC avr Gene or XopD To test if the putative avr effectors identified by SSH (one) or by sequence homology (seven) (Xanthomonas ONSA FAPESP network. 2001/2002) had a function in pathogenicity, single mutations were performed by marker interruption. Mutations were

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63 confirmed by PCR and southern hybridization. Figure 5-4 illustrates confirmation of one of the avr mutants (X23, Appendix A). Individual mutants of the eight avr genes and the XopD homolog inoculated on cabbage at the two concentrations descibed in the materials and methods section, showed no difference when compared with wild type strain 528T at either of the concentrations used (not shown). When inoculated in other susceptible hosts: white turnip Hakurei hybrid, radish Sparkler or Seven Top turnip (Appendix D), none of the mutants showed a change in the in planta phenotype (not shown). No Evidence of Collective Pleiotropic Pathogenicity Function by all Annotated XCC avr Genes Sequential mutagenesis of all eight avr genes was performed using splice overlap PCR, FLP Recombinase and marker interruption. Two different series of additive mutations in the avr genes were constructed using X21 and X37.2 as a base to perform several rounds of mutations (Appendix A) and a mutant for the eight avr genes was obtained (Figure 5-5). None of the mutants inoculated in other susceptible hosts: white turnip Hakurei hybrid, radish Sparkler or Seven Top turnip (Appendix D), showed any difference in virulence when compared with the wild type. To further confirm that there was no effect on pathogenicity, growth curves were performed with wild type 528T and strain X8.8. Growth kinetic studies in planta using wild type 528T and strain X8.8 revealed that population dynamics in white turnip of the two strains was not significantly different up to eight days post inoculation (Figure 5-6). avrXccFM confers avirulence on B. juncea without inducing a typical mesophyl HR Strain 528T is a race 1 XCC strain that is not pathogenic on Florida Indian mustard (Florida Mustard, B. juncea, Kamoun et al. 1992). There is no plant response following low concentration inoculation (approx. 106 CFU/ml). At high (109 CFU/ml)

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64 inoculum concentration, a cell collapse was observed starting 15 h after inoculation (Figure 5-7). When all 528T mutant strains were inoculated at high and low concentrations by both syringe infiltration and scissor clipping, some mutants showed systemic symptoms of progressive chlorosis and necrosis outside of the immediate inoculation zone(s) as early as 4 days after inoculation (Figure 5-8), indicating a gain of virulence on this host differential. Strains carrying single mutations of Xcc2109, whether obtained by marker interruption (eg., X48) or by splice overlap PCR (eg., X109) caused systemic infection symptoms when inoculated on Florida Mustard at both high and low concentration, indicating that Xcc2109 was in fact a functional avr gene on Florida Mustard plants (Figure 5-9). The resulting mutations created a race change from the wild type 528T race 1 to race 0 (Table 5-3). No change in the tissue collapse phenotype at high inoculum concentration typical of the wild type strain was observed in any of the mutants, however the ones carrying a mutation in Xcc2109 were able to systemically produce blight symptoms (Figure 5-10). Xcc2109, a gene earlier identified by SSH, is 72% identical to avrC and 45% to avrB, both avirulence genes from Pseudomonas syringae pv. glycinea. da Silva (2002), annotated the Xcc2109 ORF as avrXccC, 996 nucleotides long and encoding 332 amino acids. pAC10 carrying this ORF fused to a 5 amino acid LacZ leader failed to complement X48 and X109. Further analyses indicated that the functional gene was more likely 1323 bp and 441 amino acids long, and started 327 bp upstream from the previously annotated start site, and additional analyses of the rest of the avr genes from 528T strain by codon preference of GCG program (Genetics Computer, Madison, WI.) showed that four of the eight genes appear to be longer than

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65 annotated (Figure 5-11, Table 5-4). The longer Xcc2109 gene, here designated avrXccFM (avirulence on F lorida M ustard), contains a Shine-Dalgarno region at -11 from the start site (2493758 in NC 003902) and a putative PIP -plant inducible promoterbox in the promoter region at -67 and -83 from the start site (TTCGN16TTCG). The 1323 bp avrXccFM cloned in pUFR070 conferred avirulence to X48 and X109 making the mutants, like the wild type, unable to produce disease in B. juncea (Figure 5-12). avrXccFM Is a Critical Race Determinant of XCC Strain 6181 belongs to race 0 and 3849A belongs to race 2, based on the host differentials proposed by Kamoun et al. (1992). Based on Southern blot analyses, neither strain carries a copy of avrXccFM (Fig 5-13). When pAC19 containing avrXccFM was introduced into strain 6181 (Figure 5-13), the transconjugant became less aggressive in Brassica juncea, induced the formation of smaller lesions (Figure 5-14) and bacterial titer in planta was up to 100 times lower than the wild type (Figure 5-15). Introduction of pAC19 caused a change from race 0 to a new and previously uncharacterized race (Table 5-3). When pAC10, carrying the truncated avrXccFM (Xcc2109) was introduced into either strain 6181 or 3849A, no effect on virulence was observed on the set of host differentials. When pAC19 was introduced into strain 3849A (Figure 5-13), the bacterium became avirulent on Florida Mustard plants at both high and low concentrations, causing a change from race 2 to yet another new, and previously uncharacterized race (Figure 5-14, Table 5-3). These results confirmed that avrXccFM is an active avr gene recognized by B. juncea plants

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66 Electrolyte Leakage Assays Showed That No Apparent Cell Death Is Involved in B. juncea Resistance No significant differences in electrolyte leakage over the first 72 h after inoculation were observed between plants inoculated with the wild type XCC strain and the mutants X48 and X7.8 (Figure 5-16). The HR eliciting strain XCA417T induced electrolyte leakage beginning 48 h after inoculation. According to electrolyte leakage experiments, no measurable cell death occurred in any of the strains tested, including the ones carrying avrXccFM (XCC528T, X52, Figure 5-16). Virulent strains with avrXccFM deleted, started to induce plant cell death 60 h after inoculation, indicative of the necrosis produced by pathogenic strains. Preliminary results also indicated no induction of electrolyte leakage when avrXccFM was present in strain 6181 (not shown). HR In B. juncea Appears to be Restricted to Cells Surrounding the Vascular System Incompatible XCC strains have been shown to induce a vascular HR (VHR) in Brassica plants (Kamoun et al. 1992). Seedling assays experiments performed in B. juncea showed that the incompatible 528T consistently induced a VHR starting at 24 h after inoculation. In contrast, the compatible mutants X48 and X8.10 failed to induce a necrotic response and resulted in a null reaction (Figure 5-17, Table 5-5), confirming that a localized HR occurs, and appears to be restricted to cell layers surrounding the vascular system (Bretschneider et al. 1989) Non-HR Resistance Response in Arabidopsis was Not Affected in Any of the Mutants X. campestris pv. campestris is a pathogen of A. thaliana plants and four general phenotypes induced by different XCC strains have been identified in different Arabidopsis ecotypes: susceptibility, tolerance, HR-resistance and non-HR resistance (Buell 2002). Ecotypes Col-1 and Co-i showed a non-HR resistance against XCC 528T

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67 strain. Some of the avr mutant strains obtained including X8.8 were inoculated in this plant species and none of them had any apparent effect in the resistance phenotype (not shown). Non-host HR Was Not Altered in Strains Carrying Mutations in avr Genes Inoculation of pepper plant cultivars with all mutant strains at different concentrations showed no change in the non-host hypersensitive response (HR) after 48 hours in any of the cultivars tested. All strains elicited an HR typical of the wild type at bacterial concentrations above approximately 107 CFU/ml (not shown). HR Induction by a P. syringae Gene Was Not Inhibited by XCC avr Genes P. fluorescens 55 carrying pHIR11 cosmid induces a HR in tobacco and pepper due to the presence of a functional hrp system and HopPsyA and its chaperon protein (SchA) from P. syringae pv. syringae (Appendix A, Jamir et al. 2004). Strain 55/pHIR11 also carrying three of the avr genes (Xcc2100, avrXccFM and Xcc3731) showed a reduction in the HR response elicited in tobacco and pepper at 107 and 106 CFU/ml (Figure 5-18). Coinoculations of a non-HR inducing P. fluorescens with a functional hrp system (55/pLN18) carrying Xcc2100, avrXccFM and Xcc3731 genes, and later with 55/pHIR11 did not show any reduction in HR at any of the concentrations tested (not shown). As control, pZit45 that contains pthA gene was electroporated into 55/pLN18 since X. strains induce HR in tomato cultivars when carrying this clone. However, when pZit45 was introduced into P. fluorescens as an expression control, no HR was observed in tomato (not shown). As another attempt to detect HR suppression mediated by avr genes, a X. vesicatoria strain (95-2 carrying avrbs3) that induces an HR response in 30-R pepper plants, was used. Strains 95-2 also carrying three avr genes (Xcc2100, avrXccFM and

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68 Xcc3731) showed no HR reduction or change at any of the concentrations tested (not shown). Discussion Genomic analyses of XCC528T indicate that numerous horizontal gene transfer events probably occurred (da Silva et al. 2002). At least 109 genes related to transposable elements were identified, as well as extended regions with low GC content and uncommon codon usage (da Silva et al. 2002) and 285 genes present in XCC528T appear to have been acquired horizontally (Garcia-Vallv et al. 2003), including three of the eight putative avr gene homologs investigated in this study. It is often assumed that when avr genes are acquired by bacteria through horizontal gene transfer, they become fixed in the genome because they confer a selective advantage to the pathogen as effectors by increasing their pathogenicity and expanding their host range (Alfano and Collmer 2004; Rohmer et al. 2004). Pleiotropic pathogenicity effects are a common feature of avr genes and have been reported in several pathogen species (Abramovitch et al. 2003; Alfano and Collmer 2004; Badel et al. 2003; Bogdanove et al. 1998; Kearney and Staskawicz 1990; Lorang et al. 1994; Swarup et al. 1992; Yang et al. 1996; Wichmann and Bergelson 2004). Once fixed in the genome, the % G+C content of an acquired gene with selective value may gradually evolve to match that of the recipient (Lawrence and Ochman, 1997), and other characteristic features indicative of horizontal transfer may be lost as well (Hacker and Kaper 2000). Since five of the eight putative XCC528T avr genes have not been identified as being horizontally transferred (Garcia-Vallv et al. 2003), a selective value in terms of pathogenicity for these five would seem particularly likely.

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69 Nevertheless, none of the eight putative avr gene loci, when deleted or interrupted in XCC528T, affected pathogenicity on any of the six cruciferous host differential species tested. Indeed, they did not appear to confer any pathogenic effect even in an additive way, as inoculations of strain X8.8 (all eight putative avr gene loci mutated) was as pathogenic as the wild type when inoculated onto five susceptible Brassica species. In addition, growth curves showed no difference between wild type and X8.8 in white turnip plants. Some effector genes found in Pseudomonas spp., including avr genes, are known to suppress host defenses as indicated by suppression of the HR (Abramovith et al. 2003; Alfano and Collmer 2004). However, none of the avr gene mutations, singly or in any combination, affected the non-host HR elicited by XCC strains on pepper plants (data not shown). In addition, three putative XCC avr genes (Xcc2100, Xcc2109 and Xcc3731), cloned and expressed in X. axonopodis pv. vesicatoria strain 95-2, failed to suppress the HR elicited by this strain on Capsicum annum 30R plants carrying the Bs3 gene (data not shown). The initial results of apparent HR inhibition by avr genes in P. fluorescens inoculated in tobacco and pepper were not considered, since a P. fluorescens transconjugant control carrying an XCC gene did not elicit the expected response in tomato. The reasons for this phenomenon could be explained by earlier observations that some effectors can block the type three-secretion system (Jamir et al. 2004) inhibiting delivery of HR-inducing genes. Other explanation could be that Xanthomonas proteins are either not expressed or not delivered by Pseudomonas host, or that the vector used (pUFR070) could be unstable in Pseudomonas.

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70 A few putative non-avr effector genes have been identified in XCC based on sequence similarity to Ralstonia, Pseudomonas and Xanthomonas type III secreted proteins, but only one has some experimental support as an effector in Xanthomonas. This ORF, classified as psvA (Xcc2896) by da Silva (2002), is 71% identical to xopD from X. c. vesicatoria and since it was shown to be secreted through the hrp system and to have s mall u biquitin-like mo difier (SUMO) proteolytic activity (Noel et al. 2002, Roden et al. 2004b), this gene was included in the present study. As with many effector genes, it was proposed as to have been horizontally acquired (Garcia-Vallv et al. 2003). However, interruption of the homologue in XCC alone or in combination with others (not shown) did not show any effect on XCC pathogenicity on any of the hosts tested. Among the one putative xopD effector gene and the eight putative XCC528T avr genes experimentally investigated here, only avrXccFM appeared to function non-redundantly as an effector; since avrXccFM was found to confer avirulence on Florida Mustard. Deletion and insertion mutations of this gene in strain XCC528T, which is avirulent on Florida Mustard, enables the mutant bacterium to become virulent on this differential host. The truncated, annotated version of avrXccFM, Xcc2109, was identified as likely acquired by horizontal gene transfer (Garcia-Vallv et al. 2003). When avrXccFM was transferred to other races of XCC pathogenic in B. juncea, the bacteria became either avirulent in the case of race 2 strain 3849A, or much less virulent in the case of race 0 strain 6181 (Table 5-4, Figure 5-14, Figure 5-15). The visible although smaller lesions observed in the latter case might be due to: 1) poor gene expression in this strain; 2) a defense suppression mechanism specific to this strain; 3) lack of a chaperone-like protein or an imperfectly functioning chaperone-like protein

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71 required for delivering into the host. We favor the latter explanation because chaperone-like proteins have been identified as important secretion co-factors for some avr genes (Alfano and Collmer 2004) and in particular, lack of an avrBs3 chaperone in X. vesicatoria resulted in a reduced HR elicited by avrBs3 on pepper (Buttner et al. 2004). Despite the avirulence phenotype (eg., no disease development) elicited by avrXccFM in XCC strains on Florida Mustard, repeated attempts to detect a leaf HR elicited by avrXccFM failed. In all comparisons of avrXccFM mutants (or transconjugants carrying avrXccFM) to the wild type, a tissue collapse identical to that elicited by the wild type was observed, starting at 15 h after inoculation with 109 CFU/ml (not shown). However, in the avrXccFM mutants, systemic disease symptoms develop starting after 4 days. In order to help confirm resistance to XCC in the absence of a leaf HR with Florida Mustard, electrolyte leakage experiments were performed. When inoculated at low bacteria concentration (106 CFU/ml) Florida Mustard is capable of an HR response to at least some xanthomonads such as wild type XCA, beginning at 48 h after inoculation, that was coincident with electrolyte leakage (refer Figure 5-16). However, there was no evidence of an HR response to any XCC strain carrying avrXccFM, and an increase in electrolyte leakage was first detected only beginning at 72 h after inoculation in strains not carrying avrXccFM. Attempts to detect a VHR (Kamoun et al. 1992) were successful and a seedling assay showed a localized necrosis around the inoculation site starting at 24 h after inoculation when avrXccFM was present, while the compatible responses in the absence of avrXccFM resulted in a null reaction (Figure 5-18, Table 5-5) and later developed in disease (not shown). Earlier reports also

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72 associated resistance in crucifers with this phenotype and showed that only the cells surrounding the vascular system appear to be affected (Bretschneider et al. 1989). XCC was proposed to be comprised of five races, based on disease reactions using three differential Brassica species: B. oleracea, B. rapa and B. juncea (Kamoun et al. 1992). We based our race analyses on this system. Vicente et al. (2001) proposed a new race defined by the additional host differentials B. carinata PI199947 and B. oleracea Miracle F1; however, we could not reproduce the race reactions on these additional differentials as proposed. It is possible that there is significant genetic variation within these varieties. A report by Ignatov et al. (2003) suggested that in addition to the HR response in pepper plant cultivar 20R, an avrBs2 homologue was involved in avirulence in Brassica plants with the B genome, which includes Brassica juncea and Brassica carinata. However, our analysis showed that a deletion of the entire putative avrBs2 homolog, Xcc0052, alone or in combination with other avr gene deletions (Figure 5-5), did not have any effect on either pathogenicity or avirulence phenotypes in any host or non-host plants tested (not shown). Besides the putative avr effectors and xopD examined in this study, eight additional putative XCC pathogenicity effectors identified in silico at present remain to be experimentally tested for function (Table 5-6). These include two leucine-rich proteins having PIP boxes (Xcc2565 and Xcc4186) identified by da Silva et al. (2002); two genes identified as type III secreted (Xcc1072 and Xcc1247) by Roden et al. (2004a), and four genes identified as type III secreted proteins (Xcc1246, Xcc3258, Xcc1089, and Xcc3600) by Rohmer et al. (2004) and Genin et al. (2004).

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73 With the exception of avrXccFM, which functioned for avirulence, there was no evidence found that any of the other eight putative effector genes tested were functional as plant effectors. There are a number of possible explanations for the result. First, it is possible that the putative avr effectors are pseudogenes or poorly expressed. Second, these genes may encode proteins with defective secretion signals, and fail to be recognized by the Type III secretion system or by a required chaperone; at least some effectors appear to require chaperones for secretion (Alfano and Collmer, 2004). Third, it is also possible that some of these genes were horizontally acquired without their chaperones, which are usually closely linked to the effector (Alfano and Collmer 2004), and so fail to be secreted. However, these explanations seem generally unlikely for all eight of the putative effectors. A more likely possibility is that the assays for pathogenicity were not sensitive enough in growth chambers or not done in the right context to detect a change in pathogenicity phenotype (Chang et al. 2004). Wichmann and Belgerson (2004) showed that the mutation of three avr genes in Xanthomonas axonopodis pv. vesicatoria had no effect on pathogenicity in experiments performed in a growth chamber, but when the same mutants were inoculated in the field, in combination with an additional mutation in a avrBs2, they showed a large additive effect on pathogenicity. The four avr genes aided in epiphytic survival, in planta growth and lesion development, and apparently interacted with each other in different ways. Field experiments would need to be conducted with the XCC mutants to determine if these putative homologs affect pathogenicity under field conditions. In these experiments, all of the putative avr genes present in the genome

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74 were disrupted or removed. However, it seems unlikely that additive effects on pathogenicity would not be detected in a growth chamber, if all avr genes were deleted. We favor the idea that horizontal transfer of an effector gene to a recipient that does not colonize the same host as the donor should result in the effector being secreted into plant cells for which the effector is unadapted and without conferring any selective value. This idea is based on three facts. First, the type III secretion machine is indiscriminate, secreting all available effectors with a type III signal into both host and non-host cells alike (Silhavy 1997; Jin et al. 2003; Rossier et al. 1999). Second, horizontal gene transfer is by nature a random, stochastic event. Third, effectors are in most cases host-specific. For most putative effector genes, with or without evidence of horizontal transfer, no associated fitness benefit has been found so far (Wichmann and Bergelson, 2004, Chang et al. 2004). Effectors may or may not be recognized as avr genes, depending entirely on the existence of an appropriate indicator plant with a cognate R gene. Horizontal transfer of effector genes should therefore result in the presence in any given pathogenic strain of genes annotated as effector homologs, but which are gratuitous or even detrimental in terms of pathogenicity or other fitness function.

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75 A B pBY18.1KnCm+ FLP recombinase abcdadC Cm pUFR080KnSacB pAC3CmKn Figure 5-1. Mutagenesis strategies used. A) In marker insertion, a duplication of the homology region occurs and the gene gets interrupted by the suicide vector. B) FLP recombinase. The vector gets integrated as in A) and then the FLP recombinase evicts DNA regions that are between FRT sequences (Hoang et al. 1998). C) Splice overlap PCR (Horton et al. 1989). One region upstream and one downstream of the target gene are PCR amplified and then mixed to produce a region lacking the target gene. The PCR product is cloned into a suicide vector containing the SacB gene (pUFR080). Double recombination occurs and as a result, a deletion in the target gene is obtained. Arrows represent primers. Filled arrows represent FRT (Flip recognition target) sites. Regions of homology are shown in boxes with vertical lines.

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76 A B Figure 5-2. Dot blot of SSH clones hybridized against total DNA from. A. XCC B. XCA digested with RsaI. 3%3%3%3%3%70%6%3%6% Hypothetical protein Pil Glycosiltransferases Two component Sensor kinase Regulatory proteins Kinase Avirulence gene(Xcc2109) Anticodon nuclease Figure 5-3. Functional categories of gene fragments found in XCC-XCA (Xanthomonas campestris pv. campestris-Xanthomonas campestris pv. armoraciae) following Suppression Subtractive Hybridization (SSH) and identified in NC_003902

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77 BCA D M 1 2 3 4 5M 1 2 3 4 51 2 3 4 5 Figure 5-4. PCR performed in mutant X23. A. Map of Xcc2396 gene interrupted. Blue arrows represent vector-based primers, black arrows represent external primers, dashed box represent duplicated region, blue region between them represents integrated vector. B. PCR with external primers. M is /HindIII C. PCR with vector based-upstream primers. M is 1kb ladder D. PCR with vector based-downstream primers 1 is wild type and 2 to 5 show individual mutant colonies. Figure 5-5. Southern blots of XCC528T wild type and X8.8 (mutated in eight putative avr genes) DNAs digested with EcoRI. On each blot, from left to right: HindIII marker, wild type 528T, and the mutant strain X8.8. Except for Xcc1629 that was interrupted and Xcc3731 that was flipped, the avr genes were deleted by splice-overlap PCR. Probes used were DNA fragments amplified with the following PCR primer sets or DNA fragments amplified and cloned in TOPO or pUFR080: A) 52C-52D* to detect Xcc0052 deletion. Xcc0052 has an EcoRI site that is lost and consequently an 11 kb band becomes a 16.2 kb band. B) AC5-AC-6 to detect Xcc1629 interruption. The integration of the suicide vector introduces a new EcoRI site and a 2.1 kb band becomes two 5.8 kb and 2.1 kb bands, C) 2099A-2099D to detect both Xcc2099 and Xcc2100 deletion. A 11.3 kb band becomes a 8.4 kb band, D) AC35-AC45 to detect Xcc2109 deletion. A 10.5 kb becomes a 9.4 kb band, E) 2396A*-2396D to detect Xcc2396 deletion. A 12.4 kb band becomes a 10.3 kb band, F) AC32-AC48 to detect Xcc3731 interruption by FLP recombinase. A 7.5 Kb band turns to one 9 kb band due to an internal region duplication and the remains of pAC3.1 vector after flipping the vector out. G) 4229A-4229D to detect Xcc4229 deletion, the fragment deleted contained an EcoRI site. In the mutant, two bands of 1.6 and 2.8 kb become a 2.5 kb band.

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78 012345678902468Days after inoculationLog 10 (CFU/4 cm2) 528 8.8 Figure 5-6. Growth of XCC528T and X8.8 (all eight avr genes interrupted or deleted) in white turnip Hakurei Hybrid. AB Figure 5-7. Inoculation of 528T in B. juncea by A. Infiltration at high concentration 24 h after inoculation and B. Scissorsinoculated at low concentration shown at 7 days after inoculation. A B Figure 5-8. Clipping inoculation of some of the mutants inoculated in Florida Mustard in an initial screen. Wild type strain was inoculated on the left side of the leaf and X2.1 (A) and X5.5 (B) on the right of the leaf.

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79 AB Figure5-9. Strains carrying single mutation in Xcc2109. On the left inoculated with wt strain and on the right single mutation in Xcc2109 obtained by splice overlap PCR X48 (A) and marker interruption X109 (B). Figure 5-10. B. juncea leaf inoculated by syringe infiltration at high concentration inoculum. On the right infiltrated with XCC528T and on the right with X48. Picture taken one week after inoculation.

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80 C A BD Figure 5-11. Codon preference analyses performed on four avr genes by GCG. A. Xcc1629, B. Xcc2099, C. Xcc2109 and D.Xcc3731. The Y axis on the left represents codon preference and the Y axis on the right represents third position bias. AB Figure 5-12. Complementation tests in B. juncea. Mustard plants inoculated with X48 (A) and X109 (B) on the left side of the leaves and in the right side with complementing strains X52 and X113 respectively.

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81 M1 2 3 4 5 6 7 Figure 5-13. Different strains representing three races of XCC probed against avrXccFM. Total DNA was digested with EcoRI and in the gel loaded in the following order: from left to right M: /HindIII, 1: 528T, 2: X48, 3:X52, 4: 6181, 5: 6181/avrXccFM, 6: 3849A, 7: 3849A/avrXccFM. Figure 5-14. Race change of two XCC strains in Florida mustard inoculated by clipping. The wild type strain was inoculated on the left and the transconjugants carrying avrXccFM on the right. A. 6181, B. 3849A. Inoculated areas are shown with asterisks.

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82 0246810027Days after inoculationLog10 (CFU/4 cm2) 6181 6181/avrXccFM Figure 5-15. Growth of XCC6181 and X83 (6181/avrXccFM) syringe infiltrated in Florida Mustard plants. 050100150200250300350400024487296120Hours after inoculationuMHOS 528 48 52 7.8 417 Figure 5-16. Time course of electrolyte leakage from leaves of Florida Mustard plants inoculated with four different strains of XCC (XCC528T, X48, X52, X7.8) and one of XCA (417T).

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83 Figure 5-17. Seedling assay performed B. juncea plants. The two plants on the left were prick inoculated with 528T and the two plants on the right with strain X48. The sites of inoculation are circled. ABCDEABCDE Figure 5-18. Apparent HR suppression by avr genes from XCC in pepper (left) and tobacco plants inoculated with Pseudomonas fluorescens at 107 CFU/ml. A. pHIR11, B. pHIR11/pAC99, C. pHIR11/pAC10, D. pHIR11/pAC19, E. pHIR11/pAC31.

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84 Table 5-1 List of genes classified as avr in XCC528T Gene Number avr homolog Identity Bacterial species Xcc0052 avrBs2 76% X. vesicatoria Xcc1629 avrPphE 26% P. syringae pv. syringae Xcc2099 avrBs1 100% X. vesicatoria Xcc2100 avrBs1 99% X. vesicatoria Xcc2109 avrB 73% P. syringae Xcc2396 avrxcA 25% Pectobacterium carotovorum Xcc3731 avrBsT 20% X. vesicatoria Xcc4229 avrXca 88% X. armoraciae From Xanthomonas ONSA FAPESP network. 2001/2002 Table 5-2 Races in XCC Races Hosts 0 1 2 3 4 Genome Cabbage (B. oleraceae var. capitata) C + + + + + Just right turnip (B. rapa) A + + + + Seven Top turnip (B. rapa) A + + Florida Broadleaf Indian mustard (B. juncea) AB + + From Kamoun et al. 1992 + Black rot symptoms Null phenotype Table 5-3. Race changes due to the presence of avrXccFM Differentials Strains Early Jersey Wakefield B. oleracea Just Right Turnip B. rapa Seven Top Turnip B.rapa Florida Mustard B.juncea Race 528T(avrXccFM) 1 + + + X48 (528TavrXccFM) 0 + + + + X52 (528T avrXccFM/avrXccFM) 1 + + + 6181 0 + + + + X83 (6181/avrXccFM) ? + + + (+) 3849A 2 + + + X54 (3849A/avrXccFM) ? + + + Black rot symptoms Null phenotype (+) Reduced black rot symptoms ? Not characterized

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85 Table 5-4. Actual sizes of four of the avr genes according to our analyses. avr gene Annotated Actual size Position annotated Actual position Xcc1629 1068bp-356aa 1353bp-451aa 1899569-1900636 1899287-1900640 Xcc2099 315 bp-105aa 1068-356aa 2480823-2481137 2480067-2481135 Xcc2109 996bp-332 1323bp-441aa 2492436-2493431 2493758-2493431 Xcc3731 708bp-236 aa 1086bp-361aa 4438603-4439310 4438603-4439686 Table 5-5. Seedlings assay for Vascular Hypersensitive Response (VHR) Xanthomonas strain B. juncea VHR Percentage B. oleracea VHR Percentage 417T 92 85 528T 73 0 X48 15 0 X52 67 ND X8.10 0 0 Table 5-6. List of additional putative effectors Name Homolog genes Number HGT PIPbox Hypothetical protein XopQ-HolPtoQ Xcc1072 NO NO Transducer protein car HolPtoR Xcc1089 NO NO Conserved hypothetical protein Hrp Box protein-avrPphE Xcc1246 YES NO Conserved hypothetical protein XopP Xcc1247 NO NO Leucine rich protein None Xcc2565 NO YES Virulence protein PsvA-XopD Xcc2896 YES NO Conserved hypothetical protein HopPtoH Xcc3258 YES YES Hypothetical protein HopPtoG Xcc3600 NO YES Leucine rich protein None Xcc4186 YES YES HGT Predicted to be horizontally transferred PIP Plant inducible promoter

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CHAPTER 6 SUMMARY AND CONCLUSSIONS Xanthomonads cause devastating diseases worldwide, in crops that are important for human nutrition. As many more animal and plant pathogens, they carry a diverse array of secretion systems that allow them to inject and secrete proteins into their environment, in order to obtain their nutrition. Among them, type III secreted proteins have been proven to be involved in pathogenicity and hypersensitive response in plants. The battery of proteins secreted through this system has been recently proposed to be up to 40 or 50 in Pseudomonas species. By using different techniques, several candidates, including few type III secreted proteins, were identified and by functional analyses we concluded: 1. avrXccFM identified by Suppression Subtractive Hybridization in Xanthomonas campestris pv. campestris, confers avirulence against Florida broadleaf Indian mustard, a differential host. 2. Complementation tests confirmed that the functional gene was 327 bp and 109 aa longer than the annotated one. 3. The avr genes showed no effect in pathogenicity either individually or additively, in any of the susceptible hosts tested. 4. Mutation of a pthA homolog gene (pthF) on both common bacterial blight of bean complex (Xanthomonas phaseoli and Xanthomonas axonopodis pv. phaseoli var. fuscans) showed pathogenicity reduction. However complementation attempts failed. 86

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87 5. Transient expression assays of pthF isolated from Xanthomonas axonopodis pv. phaseoli var. fuscans in detached bean leaves induced a blight-like phenotype. Additional analyses need to be done in order to characterize the rest of effectors identified by homology in Xanthomonas campestris pv. campestris and to confirm the role of pthF in blight phenotype caused by the common bacterial blight complex.

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APPENDIX A BACTERIAL STRAINS AND PLASMIDS Strains Relevant Characteristics Reference or Source E. coli E. coli DH5 F-, endA1, hsdR17 (rk-mk-), supE44, thi-1, recA1 gyrA, relA1, f80dlacZDM15, D (lacZYA-argF)U169 Invitrogen Corporation Carlsbad, California E. coli DH5-T1 F80dlacZM15 (lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 Invitrogen Corporation Carlsbad, California Xanthomonas 95-2 Xanthomonas axonopodis pv. vesicatoria, wild type RifR avrBs3+ Minsavage, personal communication HM2.2S Xanthomonas axonopodis pv. malvacearum avrB4-, avrB5avrB6-, avrBIn-, avrB101-, avr102-SpR Yang et al. 1996 B21.2 pthA::Tn5-gusA, marker exchanged mutant of 3213 SpRKnR Swarup et al 1991 KX-1 X. axonopodis pv. alfalfae wild type, SpR Lazo et al 1987 203B Xanthomonas axonopodis pv. phaseoli var. fuscans wild type RifR DeFeyter et al 1991 G66 Xanthomonas phaseoli wild type RifR DeFeyter et al 1991 F3 G66 pthF::pUFR004 RifR CmR This work F6 G66 pthF::pUFR004 RifR CmR This work FF19 203B pthF::pUFR004 RifR CmR This work 3849A Xanthomonas campestris pv. campestris wild type Race 2 Kamoun and Kado 1992 6181 Xanthomonas campestris pv. campestris wild type Race 0 Vicente et al. 2001 417 Xanthomonas campestris pv. armoraciae wild type RifR Alvarez et al. 1994 528T derived strains 528T Xanthomonas campestris pv. campestris wild Type Rif R Race 1 Alvarez et al. 1994 X05 Xcc0052::pAC7 RifR KnR This work 88

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89 Strains Relevant Characteristics Reference or Source X16 Xcc1629::pUFR12 RifR KnR This work X99 Xcc2099::pAC7 RifR KnR This work X100 Xcc2100::pAC7 RifR KnR This work X109 Xcc2109::pAC7 RifR KnR This work X23 Xcc2396::pAC7 RifR KnR This work X37.1 Xcc3731::pAC7 RifR KnR This work X42 Xcc4229::pAC7 RifR KnR This work X48 Xcc2109 RifR This work X37.2 Xcc3731::FRT RifR KnR This work X44 Xcc2896::TOPO RifR KnR This work X49 Xcc2099, Xcc3731::pAC3 RifR KnR This work X21 Xcc2099, Xcc2100 RifR This work X4.4 Xcc0052, Xcc3731::FRT RifR This work X4.1 Xcc3731::FRT, Xcc2099, Xcc2100 RifR This work X5.1 Xcc2099, Xcc2100, Xcc2109 RifR This work X6.1 Xcc0052, Xcc2099, Xcc2100 RifR This work X12.1 Xcc3731::FRT, Xcc2099, Xcc2100, Xcc2109 RifR This work X2.1 Xcc0052, Xcc2099, Xcc2100, Xcc2109 RifR This work X1.1 Xcc0052, Xcc2099, Xcc2100, Xcc2109, Xcc3731::FRT RifR This work X5.5 Xcc0052, Xcc2099, Xcc2100, Xcc2109, Xcc2396 RifR This work X6.6 Xcc0052, Xcc2099, Xcc2100, Xcc2109, Xcc2396, Xcc3731::FRT RifR This work X6.7 Xcc0052, Xcc2099, Xcc2100, Xcc2109, Xcc2396, Xcc4229 RifR This work X7.7 Xcc0052, Xcc2099, Xcc2100, Xcc2109, Xcc2396, Xcc3731::FRT, Xcc4229 RifR This work X7.8 Xcc0052, Xcc2099, Xcc2100, Xcc2109, Xcc2396, Xcc3731 Xcc4229 RifR This work X8.8 Xcc0052, Xcc1629::pUFR12, Xcc2099, Xcc2100, Xcc2109, Xcc2396, Xcc3731:FRT, Xcc4229 RifR KnR This work X8.10 Xcc0052, Xcc2099, Xcc2100, Xcc2109, Xcc2396, Xcc2896::TOPO, Xcc3731, Xcc4229 RifR KnR This work X52 X48/pAC19 RifR GmR This work X113 X109/pAC19 RifR KnR GmR This work

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90 Strains Relevant Characteristics Reference or Source Pseudomonas fluorescens 55 Wild type, NmR Jamir et al. 2004 Agrobacterium tumefaciens GV2260 RifR Duan et al. 1999 Plasmids 124.4 IncP, Mob+ containing methylases XmaI and XmaIII TcR De Feyter et al. 1991 138.22 avrb6 in pUFR047 AmpR GmR De Feyter et al. 1993 FLP2 ColE1, AmpR, SacB, FLP under Cro promoter GmR Hoang et al. 1998 pAC1.16 Change L918 to M918 and L925 to M925 in pZit45 AmpR GmR This work pAC3.1 pAC7 was cut with EcoRI and SmaI and the poly linker from pUC118 was cut out with BsmBI, Klenow filled, cut with EcoRI and ligated together. To eliminate an extra BamHI site it was partially cut with BamHI, treated with Klenow and religated to form pAC3.1. ColE1, Mob+, lacZ+, CmR KnR This work pAC6.1 Change from E997 to V997 in pZit45 AmpR GmR This work pAC7 pUFR004 cut with XbaI, filled with Klenow. Cut with EcoRI and ligated to the kanamycin gene from pKLN66 that was cut with BamHI, filled with Klenow, and cut with EcoRI. ColE1, Mob+, CmR KnR This work pAC10 996 bp Xcc2109 region into pUFR070 as described in NC_003902 CmR GmR This work pAC14.1 Change from R955 to P955 in pZit45 This work pAC19 1323 bp region encompassing Xcc2109 (avrXccFM) into pUFR070 CmR GmR This work pAC31 Xcc3731 into pUFR070 as described in NC_003902 CmR GmR This work pAC99 Xcc2100 into pUFR070 as described in NC_003902 CmR GmR This work pAY8.1 Change from L790 to M790 and L797 to M797 in avrb6 in pUFR047 AmpR GmR This work pAY12.1 Change from R827 to P827 in pthA in pUC19 AmpR This work pAYLZ4 Change from E867 to V867 138.22 AmpR GmR This work Plasmid Relevant Characteristics Reference or Source

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91 pBY17.1 pAC3.1 with FRTs from sites in the in the cloning site KnR CmR Castaeda et al. unpublished pGEMTeasy PCR cloning vector. AmpR Promega Corporation, Madison, WI pGZ6.4 avrb6 in binary vector pB48.212, double 35S promoter KnR Duan et al. 1999 pHIR11 pLAFR3 derivative containing 25-kb P. syringae pv. syringae 61 hrc-hrp cluster including shcA and hopPsyA TcR Jamir et al. 2004 pJR4 IncW, Mob+, lacZ+, Par+, SacB, FLP under Cro promoter, GmR AmpR Castaeda et al. unpublished pKLN66 Kn gene in pGEMTe derivative of pKLN56 Newman et al. 2003 pLN18 pLAFR3 derivative containing 25-kb P. syringae pv. syringae 61 hrc-hrp cluster with shcA and hopPsyA replaced by an nptII cassette, TcR KmR Jamir et al. 2004 pLZ1.7 L to M in LZL region included in AatII-StuI band of pthA gene pUC19 AmpR This work pLZ7.1 BamHI fragment from pthF into BamHI site of Agrobacterium vector pYD 40.2 KnR This work pQY 107.1 HincII-HindIII region of pthA in pUC19 AmpR Unpublished Duan et al. pQY 113 EcoRI-HincII region from avrb6 in pUC19 AmpR Yang et al. 1996 pRK2013 ColE1,Tra+, helper plasmid KnR Figurski and Helinski 1979 pUC118 ColE1, M13, lacZ+, AmpR Yanisch-Perron et al. 1985 pUCK3 Change from R827 to P827 in avrb6 in pUFR047 AmpR GmR This work pUFR004 ColE1, Mob+, lacZ+, CmR De Feyter et al. 1990 pUFR012 ColE1, Mob+, lacZ+ CmR KnR El Yacoubi 2005 pUFR047 IncW, Mob+, lacZ+, Par+, GmR AmpR De Feyter et al. 1993 pUFR053 IncW, Mob+, lacZ+, Par+ GmR CmR El Yacoubi 2005 pUFR070 IncW, Mob+, lacZ+, Par+, CmR, GmR Castaeda et al. in press pUFR080 ColE1, Mob+, lacZ+, SacB, CmR KnR Castaeda et al. in press pUFY14.5 4.1 SalI region from pthA in pZit45 was recloned into pGEMTeasy AmpR Yang and Gabriel 1995b pYD9.4 pthA complete gene in pUC118 AmpR Duan unpublished data pYD40.1 BamHI fragment from pthA cloned into Agrobacterium vector pYD40.2 KnR Duan et al. 1999

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92 Plasmid Relevant Characteristics Reference or Source pYD40.2 BamHI region was cut out from vector pGZ6.4 and religated KnR Duan et al. 1999 pYY40.10 2.0 Kb internal StuI-HincII fragment of pthA from pZit45 cloned into pUFR004 CmR Yang and Gabriel, unpublished data pZit45 pthA in pUFR047 Swarup et al. 1991 TOPO PCR cloning vector. Version P. AmpR KnR Invitrogen Corporation, Carlsbad, CA Amp=ampicillin, Gm=getamicin, Cm=chloramphenicol, Kn=kanamycin, Rif=rifampicin, ORF=open reading frame

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APPENDIX B XANTHOMONAS TOTAL DNA EXTRACTION For extraction of total DNA from Xanthomonas, 12 mL PYGM (De Feyter et al. 1990) cultures were grown in 25 mL flasks at 30C over night at 120 rpm. Cells were harvested at 8000 g and washed twice with 12 mL and 1.5 mL of 50 mM Tris-HCl, 50 mM EDTA, 0.15 mM NaCl, pH 8.0. The cells were resuspended in 627 L of TES buffer (10 mM Tris-HCl, 10 mM EDTA, 0.5% SDS, pH 7.8). Afterwards 33 L of protease stock solution was added, the tubes were inverted several times to mix well, and the cell suspension was incubated at 37C for 3 hours. The protease stock solution consists of 20 mg/mL protease in 10 mM Tris-HCl, 10 mM NaCl, pH7.5; the protease was predigested at 37C for 1 hour and stored at -20C. The mixture was gently mixed overnight by rotation with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) buffered with Tris-HCl pH 8.0. The layers were separated by centrifuging for 15 minutes at 5000 g, and the top layer was gently transferred to a new tube with a pipet. A second extraction with phenol:chloroform:isoamyl alcohol (25:24:1) was carried out, followed by an extraction with chloroform:isoamyl alcohol (24:1). One-tenth volume of 3 M NaOAc was added, the tubes were inverted several times, and 0.9 volume of room temperature isopropanol was added and mixed. Precipitated DNA was spooled out with a heat-sealed glass Pasteur pipet, transferred to a tube containing 600 L of 10 mM Tris-HCl, 1 mM EDTA, pH 8, and 200 g/mL RNaseA, and incubated for 1-2 hours at 37C. A phenol:chloroform:isoamyl alcohol and a chloroform extraction were carried out, 93

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followed by precipitation with NaOAc and 2 volumes of room temperature 95% ethanol. After careful mixing, the total DNA was spooled out as before and resuspended in 200 L sterile distilled water. To analyze the DNA, 1L was digested with a restriction enzyme and run on a 0.7% agarose gel. 94

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APPENDIX C PRIMERS USED Number Sequence 5' To 3' Tm Gene Ext. Time For marker interruption AC-5 GGAATTCAGAATAGGAA CCTTCAATTCATGGGCAGGAAGCGCACTG >72 XCC1629 30" AC-6 GGGATCCAGTATAGGAA CTTCTCTGAATCCGTTTGTCCTGTCCAG >72 XCC1629 30" AC-23 GGATCC ATGATGAGAGACTGCATGTAC 68 XCC2099 30" AC-24 GAGCTC TGACCGTTCATTACGAAATTC 68 XCC2099 30" AC-29 GGATCC ACGCTGCATGACATTGTC 66 XCC2100 30" AC-30 GAGCTC ATTTCACGGATATGACTTCC 66 XCC2100 30" AC-27 GGATCC AAGGAACTGCTACAACTATC 66 XCC2109 30" AC-28 GAGCTC GCACTAATGGCATTATCATC 68 XCC2109 30" AC-25 GGCATCCAGTTCTACAGCGGCGG 68 XCC2396 30" AC-26 GAGCTC AGCGGCGTCAACGG 68 XCC2396 30" AC-19 GTTAAC GTGGAGCGGATCCATG 68 XCC3731 30" AC-20 GAGCTC ACATAGAGCACGTCAGAG 70 XCC3731 30" AC-17 CCACCTGGATCCGGGCTTCG 68 XCC4229 30" AC-18 GAGCTC AGGGTCACGCTCCACG 68 XCC4229 30" AC-C1 GGCGACGGCGTGTCCAGCGCC >72 XCC4229 30" AC-C2 GTGTAGTCCCAGTTGACGTTGC 68 XCC4229 30" AC-60 TGGCCGCGAATTCGACCTCAAC 68 XCC2896 30" AC-61 CGACGACGAGCAATGACCAATGAAAGT >72 XCC2896 30" PEC-1 CCCGCAGTGGACCGAACGATG 70 Pectate Lyase 30" PEC-2 CGCTTTGCAAGTAGGTAGCGGC 70 Pectate Lyase 30" MAC-1 TACCAGGCCAGGCTTTGGACG 68 Unknown 30" MAC-2 CCTAGGCGAGTTTTCCGACG 64 Unknown 30" UNK-1 TTCCCTAGGCGAGTTTTCCGAC 68 Unknown 30" 95

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96 Number Sequence 5' To 3' Tm Gene Ext. Time UNK-2 AGGTACCAGCCAAGCTTTGGAC 66 Unknown 30" HYP-1 CATCGCTGCACTTGTAGGCCG 68 Hypot-hetical 30" HYP-2 GCAGTCCATATGCGTAAGCGG 66 Hypothetical 30" For interruption confirmation AC-13 GCAGGCGGCCTACCAGCTTG 68 XCC1629 1' AC-14 TGCGCGGCGAAATGGGCTGC 68 XCC1629 1' AC-15 GGTCATCATCTGCCCGCCATG 68 XCC1629 1' AC-37 ATGTCAATGGAGCGGGAGATGG 68 XCC2099 1' AC-43 TTATGCATTGTGGTCGAGCCATTC 70 XCC2099 1' AC-49 GCGAGCGCGGCAGGACTAC 66 XCC2099 1' AC-50 CCAGCAAGGTGGTGCAATCGG 68 XCC2099 1' AC-34 ATGTCCGACATGAAAGTTAATTTCTG 70 XCC2100 1' AC-41 TTACGCTTCTCCTGCATTTGTAAC 68 XCC2100 1' AC-35 ATGTGGTCTCAGCCCGTATGG 66 XCC2109 1' AC-39 TTAGGATAATCAGCCACAAATTGG 66 XCC2109 1' AC-45 CTGCAGTTTTTGTACGAATCCCTACCGATC 68 XCC2109 1' AC-36 GTGCTGGAGAGTGCCGATGGC 70 XCC2396 1' AC-44 GTGAGACCACAGTGAATCGCC 66 XCC2396 1' AC-32 GTGGTGGCGGCCCAGAATCAC 66 XCC3731 1' AC-48 TTAGCTCCAGTACTCGGCGTC 66 XCC3731 1' AC-33 TGCCCGAGCGCCCTCATGC 66 XCC4229 1' AC-42 AGTTCCAGATCGCCACGCACC 68 XCC4229 1' FW-XopD ATGGAATATATACCAAGATA 50 XCC2896 1 RV-XopD CTAGAACTTTTTCCACCACTT 58 XCC2896 1 For splice overlap PCR 52-A1 CGCTGGCCGCCGAATGGATG 68 XCC052 1' 52-F TCATACGCGTTCAGATCTTACTGTTCTAGCGCAGGCGATG 60 XCC052 1' 52-C TAAGATCTGAACGCGTATGAGCAGACCATTCGCACGATG 60 XCC052 1' 52-D1 CCGATGGATCTATTGTTCTTCG 64 XCC052 1' 2099-A GGAATTCTCGATGACCTGCTCCAACG >72 XCC2099, XCC2100 1'

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97 Number Sequence 5' To 3' Tm Gene Ext. Time 2099-B TCATACGCGTTCAGATCTTACATCTATGGGGCCTGTTCG 60 XCC2099, XCC2100 1' 2099-C TAAGATCTGAACGCGTATGAAGAGAAGAAGTATCCGCCAC 60 2099, 2100 1' 2099-D GGAATTCGGACGAACTCGCCCAGCC >72 2099, 2100 1' 2109-A TTTGTCGAGCGAGCGTCAC 60 2109 1' 2109-B TCATACGCGTTCAGATCTTACCAATTTGTGGCTGATTATCC 60 2109 1' 2109-E TAAGATCTGAACGCGTATGAGTCCGAAATCTGGTGAAGAG 60 2109 1' 2109-F GTGAGTTCGGCCTACAACCA 62 2109 1' 2396-A1 GCTGATCTGGAAGTTGTAGG 60 2396 1' 2396-B TCATACGCGTTCAGATCTTATACCTGCTGATGCACATGTC 60 2396 1' 2396-C TAAGATCTGAACGCGTATGAGGTCGTGCAAGTGGGCAGTGG 60 2396 1' 2396-D GCAGTGCGGATGGCAGCC 62 2396 1' 4229-A GGCGTTTTCCATGCTGATGTAC 66 4229 1' 4229-B TCATACGCGTTCAGATCTTAGCAGGCGGCGGGGCAATGCAGGC 60 4229 1' 4229-C TAAGATCTGAACGCGTATGAGCCGATCAACAGCCTGCGCTC 60 4229 1' 4229-D CGCGTGGTCGACTGACAACG 66 4229 1' For deletion confirmation 52-E GCTGGATCTGATCCGCAGT 60 52 1' 30" 52-H CCTGGGTGGACCACGATGTG 66 52 1' 30" 2099-A1 GTGCTGCGCTGATGTATTCGG 66 2099, 2100 2 2099-D1 AAGACAAGAGCGACCAACACC 64 2099, 2100 2 2109-A1 GCAATCGAGGTATCGTCATG 60 2109 1' 30" 2109-F GTGAGTTCGGCCTACAACCA 62 2109 1' 30" 2396-A1 GCTGATCTGGAAGTTGTAGG 60 2396 1' 30" 2396-D1 CCGTACTACCGCCATGCC 60 2396 1' 30" 4229-A1 AATCGGCGAACTCGTTGTTG 60 4229 1' 30" 4229-D1 CTCCGGGCGCACCATCCAGATC 74 4229 1' 30"

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98 Number Sequence 5' To 3' Tm Gene Ext. Time For complementation assays 9.03 GAATTC CATGGGTCTATGCGCT TCAAAACC 68 2109 1'30" 9.04 GAATTC CAGGAGATCGACATG GGTCTATG 70 2109 1'30" AC-35 ATGTGGTCTCAGCCCGTATGG 66 2109 1'30" AC-45 CTGCAGTTTTTGTACGAATCCCT ACCGATC 68 2109 1'30" SSH adaptor 1 CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGAGGT 3-GGCCCGTCCA-5 SSH NA NA adaptor 2R CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT 3GCCGGCTCCA5 SSH NA NA Primer 1 CTAATACGACTCACTATAGGGC SSH NA NA Nested primer 1 TCGAGCGGCCGCCCGGGCAGGT SSH NA NA Nested primer 2R AGCGTGGTCGCGGCCGAGGT SSH NA NA Vector-based primers M13R (-48) AGCGGATAACAATTTCACACAGGA 64 Vector based V M13 (-47) GCCAGGGTTTTCCCAGTCACG 64 Vector based V UDG CLONING LZP2 ACUGCAUCCAUGG CUGGACGTC 70 Leucine Zipper 30 LZP1 CCAUGGAUGCAGUGAAAAAGGGAATGCCG >72 Leucine Zipper 30 YP04 ACGAGUUCGGUGACUCCCACTC 70 Casein Kinase 30 YP03 AGUCACCGAACUCGUAGCCCG 68 Casein Kinase 30 YP02 AACCACUUGAGCGUGGTCGGC 68 Upstream Casein kinase 30

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99 Number Sequence 5' To 3' Tm Gene Ext. Time YP01 ACGCUCAAGUGGUUCCCGTGC 68 Upstream Casein kinase 30 Underlined sequences indicate restriction site added. Bold sequences indicate homology region for splice overlap PCR. Italic sequences indicate the nucleotide changed by UDG cloning.

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APPENDIX D PLANTS USED Species Variety Common name Company Brassica oleracea var. capitata Early jersey Wakefield cabbage Sawan seed co., Pelham, GA Brassica rapa Seven Top turnip Sieger seed co., Zeeland, MI Brassica rapa Hakurei hybrid turnip Ferry-Morse seed Co., Phoenix, AZ Raphanus sativa Sparkler Radish radish Excel seeds, Camp Point, IL Brassica juncea Florida Broadleaf Indian Mustard Indian mustard Siegers seed Co., Zeeland, MI Brassica oleracea var. botrytis Miracle F1 cauliflower Jeff Jones, PC Brassica carinata PI 199947 Ethiopian mustard Jeff Jones, PC Phaseolus vulgaris California Redloud Kidney Bean common bean Agway Seeds, Syracuse, NY Phaseolus vulgaris California Redlight Kidney Bean common bean Sacramento Valley Milling Ordbend, CA Capsicum annum California Wonder pepper Jeff Jones, PC Capsicum annum 10R pepper Jeff Jones, PC Capsicum annus 20R pepper Jeff Jones, PC Capsicum annum 30R pepper Jeff Jones, PC Lycopersicum esculentum tomato Jeff Jones, PC Nicotiana benthamiana tobacco Plant Pathology greenhouse A rabidopsis thaliana eco. Coimbra-1 Jeff Rollins, PC A rabidopsis thaliana eco. Columbia Jeff Rollins, PC Gossypium hirsutum Acala Bs6 cotton M. Essenberg Oklahoma St.University Gossypium hirsutum Acala 44 cotton M. Essenberg Oklahoma St.University Citrus paradisi Duncan grapefruit grapefruit DPI 100

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APPENDIX E PLASMID EXTRACTION For extraction of plasmid DNA from E. coli is grown 2 mL of LB plus antibiotics overnight at 37C at 200 rpm and then cells are harvested at 8000 rpm by centrifugation. The supernatant is discarded and then the cells were resuspended in 90 L solution I by vortexing. Ten microliters of lysozyme solution (50 /ml of Solution I) are added, then mixed by inversion and the tubes incubated for 5 to 10 min at 37C. The samples are then frozen at C for 10 min and defrosted at 37C and 200l are added and mixed evenly by rolling the tubes. Two hundred l of solution III are added and the tubes are inverted three to four times and placed on ice for 5 to 10 m to complete precipitation. The precipitate is then centrifuged for 15 m at full speed in the cold room and the supernatant transferred to a new tube. Four hundred microliters of phenol/chloroform/isoamylalcohol are added and the tubes are vortexed then centrifuged in the cold room for 10 m at full speed. The top phase is removed with a pipet and mixed with 400 l of chloroform/isoamylalcohol, vortexed for few seconds and spun down for 15 m at full speed. The top phase is transferred to a new 1.5 ml tube and mixed with 1.0 ml of 95% ethanol, spun down at full speed for 10 m and the supernatant discarded. Seventy percent alcohol is added, mixed and spun down for 5 m at high speed then discarded and dried. The pellet are resuspended in 25 l of water. 101

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102 Solution I: 50 m glucose 25 mM Tris HCl pH 8 10 mM EDTA pH 8 Solution II 0.2 N NaOH 1% SDS Solution III 3 M potassium acetate 5 M Glacial acetic acid

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LIST OF REFERENCES Abramovitch, R.B., Kim, Y.J., Chen, S., Dickman, M.B., and Martin, G.B. 2003. Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death. EMBO J. 22:60-69. Akopyants, N.S., Fradkov, A., Diatchenko, L., Hill, J.E., Siebert, P.D., Lukyanov, S.A., Sverdlov, E.D., and Berg, D.E. 1998. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc. Natl. Acad. Sci. U. S. A. 95:13108-13113. Alfano, J.R., and Collmer, A. 2004. Type III secretion system effector proteins: double agents in bacterial disease and plant defense. Annu. Rev. Phytopathol. 42:385-414. Al-Saadi, A. 2005. Phenotypic characterization and sequence analysis of pthA homologs from five pathogenic variant groups of Xanthomonas citri. University of Florida Ph.D. dissertation. Alvarez, A.M., Benedict, A.A., Mizumoto, C.Y., Hunter, J.E., and Gabriel, D.W. 1994. Serological, pathological, and genetic diversity among strains of Xanthomonas campestris infecting crucifers. Phytopathology. 84:1449-1457. Arlat, M., Gough, C.L., Barber, C.E., Boucher, C. and Daniels, M.J. 1991. Xanthomonas campestris contains a cluster of hrp genes related to the larger hrp cluster of Pseudomonas solanacearum. Mol. Plant-Microb. Interact. 6:593-601. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. 1997. Short Protocols in Molecular Biology, 3rd edition. New York: John Wiley and Sons Inc. Badel, J.L., Nomura, K., Bandyopadhay, S., Shimizu, R., Collmer, A. and Yang He, S. 2003. Pseudomonas syringae pv. tomato DC3000 HopPtoM (CEL ORF3) is important for lesion formation but not growth in tomato and is secreted and translocated by the Hrp type III secretion system in a chaperone-dependent manner. Mol. Microbiol. 5:1239-1251. Bai, J., Choi, S. H., Ponciano, G., Leung, H., and Leach, J.E. 2000. Xanthomonas oryzae pv. oryzae avirulence genes contribute differently and specifically to pathogen aggressiveness. Mol. Plant-Microbe Interact. 13:1322-1329. 103

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104 Balague, C., Lin, B., Alcon, C., Flottes, G., Malmstrom, S., Kohler, C., Neuhaus, G., Pelletier, G., Gaymard, F., and Roby, D. 2003. HLM1, an essential signaling component in the hypersensitive response, is a member of the cyclic nucleotide-gated channel ion channel family. Plant Cell 2:365-379. Barber, C.E., Tang, J.L., Feng, J.X., Pan, M.Q., Wilson, T.J., Slater, H., Dow, J.M., Williams, P. and Daniels, M.J. 1997. A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule. Mol. Microbiol. 24:555-566. Basu, P. K. 1974. Glucose inhibition of the characteristic melanoid pigment of Xanthomonas phaseoli var. fuscans. Can. J. Bot. 44:1239-1245. Black, L.L. and Machmud, M. 1983. Xanthomonas leaf spot of crucifers. In: Int. Congr. Plant Pathol., 4th. Melbourne, Aust. P 126 (abs). Boch, J., Joardar, V., Gao, L., Robertson, T.L., Lim, M. and Kunkel, B.N. 2002. Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana. Mol. Microbiol. 1:73-88. Bogdanove, A.J., Kim, J.F., Wei, Z., Kolchinsky, P., Charkowski, A.O., Conlin, A.K., Collmer, A. and Beer, S.V. 1998. Homology and functional similarity of a hrp-linked pathogenicity locus, dspEF, of Erwinia amylovora and the avirulence locus avrE of Pseudomonas syringae pathovar tomato. Proc. Natl. Acad. Sci. U. S. A. 95:1325-1330. Bogush, M.L., Velikodvorskaya, T.V., Lebedev, Y.B., Nikolaev, L.G., Lukyanov, S.A., Fradkov, A.F., Pliyev, B.K., Boichenko, M.N., Usatova, G.N., Vorobiev, A.A., Andersen, G.L. and Sverdlov, E.D. 1999. Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization. Mol. Gen. Genetics 262:721-729. Bonas, U., Schulte, R., Fenselau, S., Minsavage, G.V., Staskawicz, B.J., and Stall, R.E. 1991. Isolation of a gene cluster from Xanthomonas campestris pv. vesicatoria that determines pathogenicity and the hypersensitive response on peper and tomato. Mol. Plant-Microbe Interact. 4:81-88. Bonas, U., Staskawicz, B.J., and Stall R.E. 1989. Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol. Gen. Genet. 218:127-136. Boucher, C.A., Van Gijsegem, F, Barberis, P.A., Arlat, M., and Zischek, C. 1987. Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered. J. Bacteriol. 12:5626-32. Boyce JD, Cullen PA, Adler B. 2004. Genomic-scale analysis of bacterial gene and protein expression in the host. Emerg. Infect. Dis. 10:1357-1362.

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105 Bradbury, J.F. 1984. Xanthomonas Dowson. 1939. In Bergeys manuals of systematic bacteriology. N.R. Krieg and J.G. Holt, eds. Williams & Wilkins, Baltimore. Bretschneider, K.E., Gonella, M.P. and Robeson, D.J. 1989. A comparative light and electron microscopical study of compatible and incompatible interactions between Xanthomonas-campestris pv. campestris and cabbage (Brassica-oleracea) Physiol. Mol. Plant Pathol. 34:285-297. Broughton, W.J., Hernandez, G., Blair, M., Beebe, S., Gepts, P., and Varderleyden, J. 2003. Beans (Phaseolus spp), model food legumes. Plant Soil 252:55-128. Buell, C.R. 2002. Interaction between Xanthomonas species and Arabidopsis thaliana. In The Arabidopsis Book, eds. C.R. Somerville and E.M. Meyerowitz, American Society of Plant Biologists, Rockville, MD. Buell, C.R., Joardar, V, Lindeberg, M, Selengut, J., Paulsen, I.T., Gwinn, M.L., Dodson, R.J., Deboy, R.T., Durkin, A.S., Kolonay, J.F., Madupu, R., Daugherty, S., Brinkac, L., Beanan, M.J., Haft, D.H., Nelson, W.C., Davidsen, T., Zafar, N., Zhou, L., Liu, J., Yuan, Q., Khouri, H., Fedorova, N., Tran, B., Russell, D., Berry, K., Utterback, T., Van Aken, S.E., Feldblyum, T.V., D'Ascenzo, M., Deng, W.L., Ramos, A.R., Alfano, J.R., Cartinhour, S., Chatterjee, A.K., Delaney, T.P., Lazarowitz, S.G., Martin, G.B., Schneider, D.J., Tang, X., Bender, C.L., White, O., Fraser, C.M. and Collmer, A. 2003. The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc. Natl. Acad. Sci. U.S.A. 100:10181-10186. Bttner, D. and Bonas, U. 2002. Getting across-bacterial type III effector proteins on their way to the plant cell. EMBO J. 21:5313-5322. Bttner, D., Gurlebeck, D., Noel, L.D., and Bonas, U. 2004. HpaB from Xanthomonas campestris pv. vesicatoria acts as an exit control protein in type III-dependent protein secretion. Mol. Microbiol. 3:755-768. Canteros, B., Minsavage, G., Bonas, U., Pring, D., and Stall, R. 1991. A gene from Xanthomonas campestris pv. vesicatoria that determines avirulence in tomato is related to avrBs3. Mol. Plant-Microbe Interact. 4:628-632. Cawly, J., Cole, A.B., Kiraly, L., Qiu, W., and Schoelz, J.E. 2005. The plant gene CCD1 selectively blocks cell death during the hypersensitive response to Cauliflower mosaic virus infection. Mol. Plant-Microbe Interact. 18:212-219. Chan, J.W., and Goodwin, P.H. 1999. A physical map of the chromosome of Xanthomonas campestris pv. phaseoli var. fuscans BXPF65. FEMS Microbiol. Lett. 1:85-90. Chang, J.H., Goel, A.K., Grant, S.R., and Dangl J.L. 2004. Wake of the flood: ascribing functions to the wave of type III effector proteins of phytopathogenic bacteria. Curr. Opin. Microbiol. 1:11-18

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106 Chang, K.W., Weng, S.F., and Tseng Y.H. 2001. UDP-glucose dehydrogenase gene of Xanthomonas campestris is required for virulence. Biochem. Biophys. Res. commun. 287:550-555. Chauvatcharin, N., Atichartpongkul, S., Utamapongchai, S., Whangsuk, W., Vattanaviboon, P., and Mongkolsuk, S. 2005. Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli. Microbiology 151:597-605. Chauvatcharin, N., Vattanaviboon, P., Switala, J., Loewen, P.C., and Mongkolsuk, S. 2003. Cloning and characterization of katA, encoding the major monofunctional catalase from Xanthomonas campestris pv. phaseoli and characterization of the encoded catalase KatA. Curr. Microbiol. 2:83-87. Clough, S.J., Fengler, K.A., Yu, I.C., Lippok, B., Smith, Jr. R.K., and Bent, A.F. 2000. The Arabidopsis dnd1 "defense, no death" gene encodes a mutated cyclic nucleotide-gated ion channel. Proc. Natl. Acad. Sci. U.S.A. 97:9323-9328. Cook, A.A., Walker, J.C., and Larson, R.H. 1952. Studies on the disease cycle of black rot of crucifers. Phytopathology 42:162-167. Crossman, L., and Dow J.M. 2004. Biofilm formation and dispersal in Xanthomonas campestris. Microbes Infect. 6:623-629. Cunnac, S., Occhialini, A., Barberis, P., Boucher, C., and Genin, S. 2004. Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identification of novel effector proteins translocated to plant host cells through the type III secretion system. Mol. Microbiol. 1:115-128. Daniels, M.J., Barber, C.E., Turner, P.C., Cleary, W.G., and Sawczyc, M.K. 1984a. Isolation of mutants of Xanthomonas-campestris pv. campestris showing altered pathogenicity. J. Gen. Microbiol. 130:2447-2455. Daniels, M.J., Barber, C.E., Turner, P.C, Sawczyc, M.K., Byrde, R.J.W., and Fielding, A.H. 1984b. Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid-pLAFR1. EMBO J. 13:3323-3328. Daniels, M.J., Collinge, D.B., and Dow, J.M. 1987. Molecular biology of the interaction of Xanthomonas campestris with plants. Plant Physiol. Biochem. 3: 353-359. Datsenko, K.A., and Wanner, B.L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97:6640-6645. Davidson, A.L., and Chen, J. 2004. ATP-binding cassette transporters in bacteria. Annu. Rev. Biochem. 73:241-268.

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107 da Silva, A.C., Ferro, J.A., Reinach, F.C., Farah, C.S., Furlan, L.R., Quaggio, R.B., Monteiro-Vitorello, C.B., Van Sluys, M.A., Almeida, N.F., Alves, L.M., do Amaral, A.M., Bertolini, M.C., Camargo, L.E., Camarotte, G., Cannavan, F., Cardozo, J., Chambergo, F., Ciapina, L.P., Cicarelli, R.M., Coutinho, L.L., Cursino-Santos, J.R., El-Dorry, H., Faria, J.B., Ferreira, A.J., Ferreira, R.C., Ferro, M.I., Formighieri, E.F., Franco, M.C., Greggio, C.C., Gruber, A., Katsuyama, A.M., Kishi L.T., Leite, R.P., Lemos, E.G., Lemos, M.V., Locali, E.C., Machado, M.A., Madeira, A.M., Martinez-Rossi, N.M., Martins, E.C., Meidanis, J., Menck, C.F., Miyaki, C.Y., Moon, D.H., Moreira, L.M., Novo, M.T., Okura, V.K., Oliveira, M.C., Oliveira, V.R., Pereira, H.A., Rossi, A., Sena, J.A., Silva, C., de Souza, R.F., Spinola, L.A., Takita, M.A., Tamura, R.E., Teixeira, E.C., Tezza, R.I., Trindade dos Santos, M., Truffi, D., Tsai, S.M., White, F.F., Setubal J.C., and Kitajima, J.P. 2002. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417:459-463. Debouck, D.G. 1994. In Neglected Crops: 1492 from a Different Perspective. J.E. Hernndo Bermejo and J. Len (eds.). Plant Production and Protection Series No. 26. FAO, Rome, Italy. 47-62. 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., and Gabriel, D.W. 1991. Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum. J. Bacteriol. 173:6421-6427. 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. Diatchenko, L., Lau, Y.F.C., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., and Siebert, P.D. 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U.S.A. 93: 6025-6030. Dong, X., Mindrinos, M., Davis, K.R., and Ausubel, F.M. 1991. Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene. Plant Cell 1:61-72. Dow, J.M., Clarke, B.R., Milligan, D.E., Tang, J.L., and Daniels M.J. 1990. Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen. Appl. Environ. Microbiol. 10:2994-2998.

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108 Dow, J.M., Crossman, L., Findlay, K., He, Y.Q., Feng, J.X., and Tang, J.L. 2003. Biofilm dispersal in Xanthomonas campestris is controlled by cell-cell signaling and is required for full virulence to plants. Proc. Natl. Acad. Sci. U.S.A. 19:10995-11000. Dow, J.M., Davies, H.A., and Daniels, M.J. 1998. A metalloprotease from Xanthomonas campestris that specifically degrades proline/hydroxyproline-rich glycoproteins of the plant extracellular matrix. Mol. Plant-Microbe Interact. 11:1085-1093. Dow, J.M., Feng, J.X., Barber, C.E., Tang, J.L., and Daniels, M.J. 2000. Novel genes involved in the regulation of pathogenicity factor production within the rpf gene cluster of Xanthomonas campestris. Microbiology 146:885-891. Duan, Y.P., Castaeda, A., Zhao, 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. Egler, M., Grosse, C., Grass, G., and Nies, D.H. 2005. Role of the extracytoplasmic function protein family sigma factor RpoE in metal resistance of Escherichia coli. J. Bacteriol. 187:2297-2307. Ellingboe, A.H. 1976. Genetics of host-parasite interactions. In: Encyclopedia of Plant Physiology (Heitefass, R. and Williams, P.H., eds.). Springer-Verlag, Berlin, Germany 4:761-778. El Yacoubi, B. 2005. Bacterial citrus canker: molecular aspects of a compatible plant-microbe interaction. University of Florida, Ph.D. dissertation. Esnault, R., Buffard, D., Breda, C., Sallaud, C., el Turk, J., and Kondorosi, A. 1993. Pathological and molecular characterizations of alfalfa interactions with compatible and incompatible bacteria, Xanthomonas campestris pv.. alfalfae and Pseudomonas syringae pv. pisi. Mol. Plant-Microbe Interact. 5:655-664. FAOSTAT data 2004. http://apps.fao.org/faostat/notes/citation.htm. 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. U.S.A. 76:1648-1652. Flor, H.H. 1956. The complementary genic systems in flax and flax rust. Advances in Genetics Incorporating Molecular Genetic Medicine 8: 29-54 Frank, S. A. 1992. Models of plant-pathogen coevolution. Trends Genet. 6:213-219. Gabriel, D.W. 1997. Targeting of protein signals from Xanthomonas to the plant nucleus. Trends Plant Sci. 2:204-206.

PAGE 123

109 Gabriel, D. W. 1999. Why do pathogens carry avirulence genes? Physiol. Molec. Plant Pathol. 55:205-214. Gabriel, D.W., Burges, A., and Lazo G.R. 1986. Gene-for-gene recognition of five cloned avirulence genes from Xanthomonas campestris pv. malvacearum by specific resistance genes in cotton. Proc. Natl. Acad. Sci. U.S A. 83:6415-6419. Gabriel, D.W., Kingsley, M.T., 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. Galan, J.E., and Collmer A. 1999. Type III secretion machines: Bacterial devices for protein delivery into host cells. Science 284:1322-1328. Garcia-Vallv, S., Guzman, E., Montero, M.A., and Romeu, A. 2003. HGT-DB: a database of putative horizontally transferred genes in prokaryotic complete genomes. Nucl. Acids Res. 1:187-189. Gauthier, A., Thomas, N.A., and Finlay, B.B. 2003. Bacterial injection machines. J. Biol. Chem. 28:25273-25276. Gillings, M.R., Holley, M.P., Stokes, H.W., and Holmes, A.J. 2005. Integrons in Xanthomonas: a source of species genome diversity. Proc. Natl. Acad. Sci. U.S.A. 102:4419-4424. Goodner, B., Hinkle, G., Gattung, S., Miller, N., Blanchard, M., Qurollo, B., Goldman, B.S., Cao, Y., Askenazi, M., Halling, C., Mullin, L., Houmiel, K., Gordon, J., Vaudin, M., Iartchouk, O., Epp, A., Liu, F., Wollam, C., Allinger, M., Doughty, D., Scott, C., Lappas, C., Markelz, B., Flanagan, C., Crowell, C., Gurson, J., Lomo, C., Sear, C., Strub, G., Cielo, C., and Slater, S. 2001. Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 5550:2323-2328. Gopalan, S., Bauer, D.W., Alfano, J.R., Loniello, A.O., He, S.Y., and Collmer A. 1996. Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death. Plant Cell 8:1095-1105. Greenberg, J.T., and Vinatzer B.A. 2003. Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells. Curr. Opin. Microbiol. 6:20-28. Gurlebeck, D., Szurek, B., and Bonas, U. 2005. Dimerization of the bacterial effector protein AvrBs3 in the plant cell cytoplasm prior to nuclear import. Plant J. 42:175-187.

PAGE 124

110 Guttman, D.S., Vinatzer, B.A., Sarkar, S.F., Ranall, M.V., Kettler, G., and Greenberg, J.T. 2002. A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae. Science 295:1722-1726. Hacker, J., and Kaper, J.B. 2000. Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54:641-679. Harakava, R., and Gabriel, D.W. 2003. Genetic differences between two strains of Xylella fastidiosa revealed by suppression subtractive hybridization. Appl. Environ. Microbiol. 69:1315-1319. Hayward, A.C. 1993. The hosts of Xanthomonas. In Xanthomonas. Chapman & Hall. New York. He, S.Y., Nomura, K., and Whittam, T.S. 2004. Type III protein secretion mechanism in mammalian and plant pathogens. Biochim. Biophys. Acta 1694:181-206. Hendrix, R.W., Smith, M.C., Burns, R.N., Ford, M.E. and Hatfull, G.F. 1999. Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage. Proc. Natl. Acad. Sci. U.S.A. 96:2192-2197. Hibberd, A.M., Stall, R.E., and Basset, M.J. 1987. Different phenotypes associated with incompatible races and resistance genes in bacterial spot disease of pepper. Plant Dis. 71:1075-1078. Hieter, P., and Boguse, M. 1997. Functional genomics: Its how you read it. Science 278:601-602. Hildebrand, D.C., Palleroni, N.J., and Schroth, M.N. 1990. Deoxyribonucleic acid relatedness of 24 xanthomonad strains representing 23 Xanthomonas campestris pathovars and Xanthomonas fragariae. J. Appl. Bacteriol. 68: 263-269. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease L.R. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1:51-59. Hoang, T.T., Karkhoff-Schweizer, R.R., Kutchma, A.J., and Schweizer, H.P. 1998. A broad host range FLP-FRT recombination system for site-specific excision of chromosome located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77-86. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61-68. Hotson, A., Chosed, R., Shu, H., Orth, K., and Mudgett, M.B. 2003. Xanthomonas type III effector XopD targets SUMO-conjugated proteins in planta. Mol. Microbiol. 2:377-389.

PAGE 125

111 Hugouvieux, V., Barber, C.E., and Daniels, M.J. 1998. Entry of Xanthomonas campestris pv. campestris into hydathodes of Arabidopsis thaliana leaves: a system for studying early infection events in bacterial pathogenesis. Mol. Plant-Microbe Interact. 6:537-543. Ignatov, A.N., Monakhos, G.F., Dzhalilov, F.S., and Pozmogova G.V. 2003. Avirulence gene from Xanthomonas campestris pv. campestris homologous to the avrBs2 locus is recognized in race-specific reaction by two different resistance genes in Brassicas. Russian Journal of Genetics 12: 1404-1410. Innes, R.W., Bent, A.F., Kunkel, B.N., Bisgrove, S.R., and Staskawicz B.J. 1993. Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes. J. Bacteriol. 15:4859-4869. James, B.D., and Higgins, S.J. 1985. Nucleic Acid Hybridization. IRL Press Ltd., Oxford. Jamir, Y., Guo, M., Oh, H.S., Petnicki-Ocwieja, T., Chen, S., Tang, X., Dickman, M.B., Collmer, A., and Alfano, J.R. 2004. Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast. Plant J. 4:554-565. Jakoby, M., Weisshaar, B., Droge-Laser, W., Vicente-Carbajosa, J., Tiedemann, J., Kroj, T., and Parcy, F. 2002. bZIP transcription factors in Arabidopsis. Trends Plant Sci. 7:106-111. Jensen, A.B., Goday, A., Figueras, M., and Jessop, A.C. 1998. Phosphorylation mediates the nuclear targeting of the maize Rab17 protein. Plant J. 13:691-697. Jin, Q.L., and He, S.Y. 2001. Role of the Hrp pilus in type III protein secretion in Pseudomonas syringae. Science 294: 2556-2558. Jin, Q.L., Thilmony, R., Zwiesler-Vollick, J., and He, S.Y. 2003. Type III protein secretion in Pseudomonas syringae. Microbes and Infection 5:301-310. Jurkowski, G.I., Smith, Jr. R.K., Yu, I.C., Ham, J.H., Sharma, S.B., Klessig, D.F., Fengler, K.A. and Bent, A.F. 2004. Arabidopsis dnd2, a second cyclic nucleotide-gated ion channel gene for which mutation causes the "defense, no death" phenotype. Mol. Plant-Microbe Interact. 17:511-520. Kamoun, S., Hamada, W., and Huitema, E. 2003. Agrosuppression: a bioassay for the hypersensitive response suited to high-throughput screening. Mol. Plant-Microbe Interact. 1:7-13. Kamoun, S., and Kado, C.I. 1990. A plant-inducible gene of Xanthomonas campestris pv. campestris encodes an exocellular component required for growth in the host and hypersensitivity on nonhosts. J. Bacteriol. 9:5165-5172.

PAGE 126

112 Kamoun, S., Kamdar, H.V., Tola, E., and Kado, C.I. 1992. Incompatible interactions between crucifers and Xanthomonas campestris involve a vascular hypersensitive response: Role of the hrpX locus. Mol. Plant-Microbe Interact. 5:22-33. Kapila, J., DeRycke, R., Van Montagu, M., and Angenon, G. 1997. An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci. 122:101-108. Kearny, B., and Staskawicz, B.J. 1990. Widespread distribution and fitness contribution of Xanthomonas campestris avirulence gene avrBs2. Nature 332:541-543. Keck, A.S., and Finley, J.W. 2004. Cruciferous vegetables: cancer protective mechanisms of glucosinolate hydrolysis products and selenium. Integr. Cancer Ther. 3:5-12. Kucharek, T., and Strandberg, J. 1981. Black rot of crucifers. Plant Pathology Fact Sheet. IFAS/University of Florida, Gainesville. Lawrence, J.G., and Ochman, H. 1997. Amelioration of bacterial genomes: rates of change and exchange. J. Mol. Evol. 4:383-397. 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. Lazo, G. R., Roffey, R., and Gabriel, D. W. 1987. Pathovars of Xanthomonas campestris are distinguishable by restriction fragment length polymorphisms. Int. J. Syst. Bacteriol. 37:214-221. Leach, J.E., and White, F.F. 1996. Bacterial avirulence genes. Annu. Rev. Phytopathol. 34:153-179. Lee, B.M., Park, Y.J., Park, D.S., Kang, H.W., Kim, J.G., Song, E.S., Park, I.C., Yoon, U.H., Hahn, J.H., Koo, B.S., Lee, G.B., Kim, H., Park, H.S., Yoon, K.O., Kim, J.H., Jung, C.H., Koh, N.H., Seo J.S., Go, S.J. 2005. The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice. Nucleic Acids Res. 33:577-586. Liang, B., Yu, T.G., Guo, B., Yang, C., Dai, L., and Shen, D.L. 2004. Cloning and characterization of a novel avirulence gene (arp3) from Xanthomonas oryzae pv. oryzae. DNA Seq. 15:110-117. Lim, M.T., and Kunkel, B.N. 2004. The Pseudomonas syringae type III effector AvrRpt2 promotes virulence independently of RIN4, a predicted virulence target in Arabidopsis thaliana. Plant J. 40:790-798.

PAGE 127

113 Lin, N.C., and Martin, G.B. 2005. An avrPto/avrPtoB Mutant of Pseudomonas syringae pv. tomato DC3000 does not elicit Pto-mediated resistance and is less virulent on tomato. Mol. Plant-Microbe Interact. 1:43-51. Lloyd, S.A., Sjostrom, M., Andersson, S., and Wolf-Watz, H. 2002. Molecular characterization of type III secretion signals via analysis of synthetic N-terminal amino acid sequences. Mol. Microbiol. 1:51-59. Lorang, J.M., Shen, H., Kobayashi, D., Cooksey, D., and Keen N.T. 1994. avrA and avrE in Pseudomonas syringae pv. tomato PT23 play a role in virulence on tomato plants. Mol. Plant-Microbe Interact. 7:508-515. Losada, L., Sussan, T., Pak, K., Zeyad, S., Rozenbaum, I. and Hutcheson, S.W. 2004. Identification of a novel Pseudomonas syringae Psy61 effector with virulence and avirulence functions by a HrpL-dependent promoter-trap assay. Mol. Plant-Microbe Interact. 3:254-262. Mackey, D., Holt, B.F., Wiig, A. and Dangl, J.L. 2002. RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108:743-754. Maniatis, T., Fritsch, E.T., and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. McKusik, V.A, Kucherlapati, R.S, and Ruddle, F.H. 1993. Genomics: stock-taking after 5 years. Genomics 1:1-2. Mezencev, R., Mojzis, J., Pilatova, M., and Kutschy, P. 2003. Antiproliferative and cancer chemopreventive activity of phytoalexins: focus on indole phytoalexins from crucifers. Neoplasma 4:239-245. Miller, S.A., Sahin, F., and Rowe, R.C. 2002. Black rot of crucifers. Plant Pathology fact sheet. Plant Pathology Department, the Ohio State University, Columbus. Mills, D. 1985. Transposon mutagenesis and its potential for studying virulence genes in plant pathogens. Annu. Rev. Phytopathol. 23:297-320. Minsavage, G.V., Dahlbeck, D., and Whalen, M.C. 1990. Gene-for-gene relationships specifying disease resistance in Xanthomonas campestris pv. vesicatoria-pepper Interactions. Mol. Plant-Microbe Interact. 1:41-47. Moffett, M.L., and. Irwin, J.A.G. 1975. Bacterial leaf and stem spot (Xanthomonas alfalfae)of lucerne in Queensland. Aust. J. Exp. Agric. 15: 223-226. Newman, K.L, Almeida, R.P., Purcell, A.H., and Lindow, S.E. 2003. Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera. Appl. Environ. Microbiol. 69:7319-7327.

PAGE 128

114 Noel, L., Thieme, F., Nennstiel, D., and Bonas, U. 2001. cDNA-AFLP analysis unravels a genome-wide hrpG-regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria. Mol. Microbiol. 6:1271-1281. Nomura, K., and He, S.Y. 2005. Powerful screens for bacterial virulence proteins. Proc. Natl. Acad. Sci. U.S.A. 102:3527-3528. O'Garro, L.W., Gibbs, H., and Newton A. 1997. Mutation in the avrBs1 avirulence gene of Xanthomonas campestris pv. vesicatoria influences survival of the bacterium in soil and detached leaf tissue. Phytopathology 87:960-966. O'Reilly, M., de Azavedo, J.C., Kennedy, S., and Foster T.J. 1986. Inactivation of the alpha-haemolysin gene of Staphylococcus aureus 8325-4 by site-directed mutagenesis and studies on the expression of its haemolysins. Microb. Pathog. 2:125-138. Osbourn, A.E., Clarke, B.R., and Daniels, M.J. 1990. Identification and DNA-sequence of a pathogenicity gene of Xanthomonas campestris pv. campestris. Mol. Plant-Microbe Interact. 5:280-285. Parker, J.E., Barber, C.E., Fan, M.J., and Daniels, M.J. 1993. Interaction of Xanthomonas campestris with Arabidopsis thaliana: characterization of a gene from X. c. pv. raphani that confers avirulence to most A. thaliana accessions. Mol. Plant-Microbe Interact. 2:216-224. Pernezny, K., and Jones, J.B. 2002. Common bacterial blight of snap bean in Florida. Institute of Food and Agricultural Sciences. Fact sheet. University of Florida Extension. Petnicki-Ocwieja, T., Schneider, D.J., Tam, V.C., Chancey, S.T., Shan, L., Jamir, Y., Schechter, L.M, Buell, C.R., Tang, X., Collmer, A., and. Alfano, J.R. 2002. Genomewide identification of proteins secreted by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000. Proc. Natl. Acad. Sci. U. S. A. 99:7652-7657. Pomati, F., and Neilan, B.A. 2004. PCR-based positive hybridization to detect genomic diversity associated with bacterial secondary metabolism. Nucleic Acids Res. 32:e7. Qian, W., Jia, Y., Ren, S.X., He, Y.Q., Feng, J.X., Lu, L.F., Sun, Q., Ying, G., Tang, D.J., Tang, H., Wu, W., Hao, P., Wang, L., Jiang, B.L., Zeng, S., Gu, W.Y., Lu, G., Rong, L., Tian, Y., Yao, Z., Fu, G., Chen, B., Fang, R., Qiang, B., Chen, Z., Zhao, G.P., Tang, J.L. and He, C. 2005. Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris. Genome Res. 15:757-767.

PAGE 129

115 Rashtchian, A., Buchman, G.W., Schuster, D.M. and Berninger, M.S. 1992. Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning. Ann. Biochem. 206:91-97. Remaut, H. and Waksman, G. 2004. Structural biology of bacterial pathogenesis. Curr. Opin. Struct. Biol. 14:161-170. Roden, J.A., Belt, B., Ross, J.B., Tachibana, T., Vargas, J., and Mudgett, M.B. 2004. A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection. Proc. Natl. Acad. Sci. U.S.A. 47:16624-16629. Roden, J., Eardley, L., Hotson, A., Cao, Y., and Mudgett, M.B. 2004. Characterization of the Xanthomonas AvrXv4 effector, a SUMO protease translocated into plant cells. Mol. Plant-Microbe Interact. 6:633-643. Rohmer, L., Guttman, D.S., and Dangl J.L. 2004. Diverse evolutionary mechanisms shape the type III effector virulence factor repertoire in the plant pathogen Pseudomonas syringae. Genetics 3:1341-1360. Ronald, P.C., and Staskawicz, B.J. 1988. The avirulence gene avrBs1 from Xanthomonas campestris pv. vesicatoria encodes a 50-kD protein. Mol. Plant-Microbe Interact. 5:191-198. Ronald, P.C., Salmeron, J.M., Carland, F.M., and Staskawicz, B.J. 1992. The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene. J. Bacteriol. 5:1604-1611. Rossier, O., Wengelnik, K., Hahn, K., and Bonas U. 1999. The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens. Proc. Natl. Acad. Sci. U.S.A. 16:9368-9673. RPD 924. 1999. Black rot of cabbage and other crucifers. Reports on Plant Diseases. University of Illinois Extension. Rubatzky, V.E., and Yamaguchi, M. 1997. World Vegetables. Principles, Production and Nutritive Values. Second Edition. Chapman & Hall, New York. Ruzin, S.E. 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York. Sagulenko, E., Sagulenko, V., Chen, J., and Christie, P.J. 2001. Role of Agrobacterium VirB11 ATPase in T-pilus assembly and substrate selection. J Bacteriol. 183:5813-5825.

PAGE 130

116 Salanoubat, M., Lemcke, K., Rieger, M., Ansorge, W., Unseld, M., Fartmann, B., Valle, G., Blocker, H., Perez-Alonso, M., Obermaier, B., Delseny, M., Boutry, M., Grivell, L.A., Mache, R., Puigdomenech, P., De Simone, V., Choisne, N., Artiguenave, F., Robert, C., Brottier, P., Wincker, P., Cattolico, L., Weissenbach, J., Saurin, W., Quetier, F., Schafer, M., Muller-Auer, S., Gabel, C., Fuchs, M., Benes, V., Wurmbach, E., Drzonek, H., Erfle, H., Jordan, N., Bangert, S., Wiedelmann, R., Kranz, H., Voss, H., Holland, R., Brandt, P., Nyakatura, G., Vezzi, A., D'Angelo, M., Pallavicini, A., Toppo, S., Simionati, B., Conrad, A., Hornischer, K., Kauer, G., Lohnert, T.H., Nordsiek, G., Reichelt, J., Scharfe, M., Schon, O., Bargues, M., Terol, J., Climent, J., Navarro, P., Collado, C., Perez-Perez, A., Ottenwalder, B., Duchemin, D., Cooke, R., Laudie, M., Berger-Llauro, C., Purnelle, B., Masuy, D., de Haan, M., Maarse, A.C., Alcaraz, J.P., Cottet, A., Casacuberta, E., Monfort, A., Argiriou, A., Flores, M,, Liguori, R., Vitale, D., Mannhaupt, G., Haase, D., Schoof, H., Rudd, S., Zaccaria, P., Mewes, H.W., Mayer, K.F., Kaul, S., Town, C.D., Koo, H.L., Tallon, L.J., Jenkins, J., Rooney, T., Rizzo, M., Walts, A., Utterback, T., Fujii, C.Y., Shea, T.P., Creasy, T.H., Haas, B., Maiti, R., Wu, D., Peterson, J., Van Aken, S., Pai, G., Militscher, J., Sellers, P., Gill, J.E., Feldblyum, T.V., Preuss, D., Lin, X., Nierman, W.C., Salzberg, S.L., White, O., Venter, J.C., Fraser, C.M., Kaneko, T., Nakamura, Y., Sato, S., Kato, T., Asamizu, E., Sasamoto, S., Kimura, T., Idesawa, K., Kawashima, K., Kishida, Y., Kiyokawa, C., Kohara, M., Matsumoto, M., Matsuno, A., Muraki, A., Nakayama, S., Nakazaki, N., Shinpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda M., and Tabata, S.; European Union Chromosome 3 Arabidopsis Sequencing Consortium; Institute for Genomic Research; Kazusa DNA Research Institute. 2002. Genome sequence of the plant pathogen Ralstonia solanacearum. Science 415:497-502. Schechter, L.M., Roberts, K.A., Jamir, Y., Alfano, J.R., and Collmer, A. 2004. Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter. J. Bacteriol. 2:543-555. Schwartz, H. F., and Pastor-Corrales, M.A. 1989. Bean Production in the Tropics (second edition). Centro Internacional de Agricultura Tropical, Cali, Colombia. Schaad, N.W. 1988. Laboratory guide for identification of plant pathogenic bacteria. Second edition. APS press. Schaad, N.W., and Alvarez, A.M. 1993. Xanthomonas campestris pv. campestris in Xanthomonas. Chapman & Hall. New York. Schmidt, H., and Hensel, M. 2004. Pathogenicity islands in bacterial pathogenesis. Clin. Microbiol. 17:14-56. Shaw, J.J., Kado, C.I. 1998. Whole plant wound inoculation for consistent reproduction of black rot of crucifers. Phytopathology 78: 981-986. Silhavy, T.J. 1991. Death by lethal injection. Science 278:1085-1086.

PAGE 131

117 Simpson, A.J., Reinach, F.C., Arruda, P., Abreu, F.A., Acencio, M., Alvarenga, R., Alves, L.M., Araya, J.E., Baia, G.S., Baptista, C.S., Barros, M.H., Bonaccorsi, E.D., Bordin, S., Bove, J.M., Briones, M.R., Bueno, M.R., Camargo, A.A., Camargo, L.E., Carraro, D.M., Carrer, H., Colauto, N.B., Colombo, C., Costa, F.F., Costa, M.C., Costa-Neto, C.M., Coutinho, L.L., Cristofani, M., Dias-Neto, E., Docena, C., El-Dorry, H., Facincani, A.P., Ferreira, A.J., Ferreira, V.C., Ferro, J.A., Fraga, J.S., Franca, S.C., Franco, M.C., Frohme, M., Furlan, L.R., Garnier, M., Goldman, G.H., Goldman, M.H., Gomes, S.L., Gruber, A., Ho, P.L., Hoheisel, J.D., Junqueira, M.L., Kemper, E.L., Kitajima, J.P., Krieger, J,E,, Kuramae, E.E., Laigret, F., Lambais, M.R., Leite, L.C., Lemos, E.G., Lemos, M.V., Lopes, S.A., Lopes, C.R., Machado, J.A., Machado, M.A., Madeira, A.M., Madeira, H.M., Marino, C.L., Marques, M.V., Martins, E.A., Martins, E.M., Matsukuma, A.Y., Menck, C.F., Miracca, E.C., Miyaki, C.Y., Monteriro-Vitorello, C.B., Moon, D.H., Nagai, M.A., Nascimento, A.L., Netto, L.E., Nhani, A. Jr., Nobrega, F.G., Nunes, L.R., Oliveira, M.A., de Oliveira, M.C., de Oliveira, R.C., Palmieri, D.A., Paris, A., Peixoto, B.R., Pereira, G.A., Pereira, H.A. Jr., Pesquero, J.B., Quaggio, R.B., Roberto, P.G., Rodrigues, V., de M Rosa, A.J., de Rosa, V.E. Jr., de Sa, R.G., Santelli, R.V., Sawasaki, H.E., da Silva, A.C., da Silva, A.M., da Silva, F.R., da Silva, W.A. Jr., da Silveira, J.F., Silvestri, M.L., Siqueira, W.J., de Souza, A.A., de Souza, A.P., Terenzi, M.F., Truffi, D., Tsai, S.M., Tsuhako, M.H., Vallada, H., Van Sluys, M.A., Verjovski-Almeida, S., Vettore, A.L., Zago, M.A., Zatz, M., Meidanis, J., and Setubal, J.C. 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406:151-157. Snyder, M., and Gerstein, M. 2003. Defining genes in the genomics era. Science 300:258-260. Steller, S., Angenendt, P., Cahill, D.J., Heuberger, S., Lehrach, H., and Kreutzberger, J. 2005. Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections. Proteomics 5:2048-2055. 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 cankerlike lesions on citrus. Phytopathology 81:802-809. Swarup, S., Yang, Y., Kingsley, M.K., and Gabriel, D.W. 1992. A Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhost. Mol. Plant-Microbe Interact. 5:204-213. Swings, J.G., and Civerolo, E.L. 1993. Xanthomonas. Chapman & Hall. New York. Szurek, B., Marois, E., Bonas, U., and Van den Ackerveken, G. 2001. Eukaryotic features of the Xanthomonas type III effector AvrBs3: protein domains involved in transcriptional activation and the interaction with nuclear import receptors from pepper. Plant J. 5:523-534.

PAGE 132

118 Talalay, P., and Fahey, J.W. 2001. Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. J. Nutr. 131:3027S-3033S. Tang, J.L., Liu, Y.N., Barber, C.E., Dow, J.M., Wootton, J.C., and Daniels, M.J. 1991. Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris. Mol. Gen. Genet. 3:409-417. Taylor, J.D., Conway, S.W., Roberts, S.J., Astley, D., and Vicente J.G. 2002. Sources and origin of resistance to Xanthomonas campestris pv. campestris in Brassica Genomes. Phytopathology 92:105-111. USDA. 2004. National Agricultural Statistics Service NASS. http://www.usda.gov/nass/pubs/estindx.htm Van den Ackerveken, G., Marois, E., and Bonas, U. 1996. Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell 87:1307-1316. Vattanaviboon, P., Whangsuk, W., Panmanee, W., Klomsiri, C., Dharmsthiti, S., and Mongkolsuk, S. 2002. Evaluation of the roles that alkyl hydroperoxide reductase and Ohr play in organic peroxide-induced gene expression and protection against organic peroxides in Xanthomonas campestris. Biochem. Biophys. Res. Commun. 2:177-182. Vattanaviboon, P., Whangsuk, W., and Mongkolsuk, S. 2003. A suppressor of the menadione-hypersensitive phenotype of a Xanthomonas campestris pv. phaseoli oxyR mutant reveals a novel mechanism of toxicity and the protective role of alkyl hydroperoxide reductase. J. Bacteriol. 5:1734-1738. Vauterin, L., Hoste, B., Yang, P., Alvarez, A., Kersters K., and Swings, J. 1993. Taxonomy of the genus Xanthomonas. In Xanthomonas. Chapman and Hall, New York. Vauterin, L., Hoste, B., Kersters K., and Swings, J. 1995. Reclassification of Xanthomonas. Int. J. Syst. Bacteriol. 3: 472-489. Vera Cruz, C.M., Bai, J., Ona, I., Leung, H., Nelson, R.J., Mew, T.W., and Leach, J.E. 2000. Predicting durability of a disease resistance gene based on an assessment of the fitness loss and epidemiological consequences of avirulence gene mutation. Proc. Natl. Acad. Sci. U.S.A. 25:13500-13505. Vicente, J.G., Conway, J., Roberts, S.J., and Taylor J.D. 2001. Identification and origin of Xanthomonas campestris pv campestris races and related pathovars. Phytopathology 91:492-499. Vidaver, A.K. 1993. Xanthomonas campestris pv. phaseoli. In Xanthomonas Chapman & Hall. New York.

PAGE 133

119 Vojnov, A.A., Slater, H., Newman, M.A., Daniels, M.J., and Dow, J.M. 2001. Regulation of the synthesis of cyclic glucan in Xanthomonas campestris by a diffusible signal molecule. Arch. Microbiol. 6:415-420. Vorhlter, F.J., Thias, T., Meyer, F., Bekel, T., Kaiser, O., Phler, A., and Niehaus, K. 2003. Comparison of two Xanthomonas campestris pathovar campestris genomes revealed differences in their gene composition. J. Biotechnol. 106:193-202. Wallen, V. R., and Galway, D.S. 1979. Effective management of bacterial blight of field beans in Ontario: a 10-year program. Can. J. Plant Pathol. 1:42-46. Whalen, M.C., Innes, R.W., Bent, A.F., and Staskawicz, B.J. 1991. Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell 1:49-59. Whalen, M.C., Wang, J.F., Carland, F.M., Heiskell, M.E., Dahlbeck, D., Minsavage, G.V., Jones, J.B., Scott, J.W., Stall, R.E., and Staskawicz, B.J. 1993. Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998. Mol. Plant-Microbe Interact. 5:616-627. White, H.E. 1930. Bacterial spot of radish and turnip. Phytopathology 20:653-662. Wichmann, G., and Bergelson, J. 2004. Effector genes of Xanthomonas axonopodis pv. vesicatoria promote transmission and enhance other fitness traits in the field. Genetics 166: 693-706. Williams, P.H. 1980. Black rot: a continuing threat to world crucifers. Plant Dis. 64:736-742. Wilson, T.J., Bertrand, N., Tang, J.L., Feng, J.X., Pan, M.Q., Barber, C.E., Dow, J.M., and Daniels, M.J. 1998. The rpfA gene of Xanthomonas campestris pathovar campestris, which is involved in the regulation of pathogenicity factor production, encodes an aconitase. Mol. Microbiol. 5:961-970. Winstanley, C. 2002. Spot the difference: applications of subtractive hybridisation to the study of bacterial pathogens. J. Med. Microbiol. 51:459-467. Wood, D.W., Setubal, J.C., Kaul, R., Monks, D.E., Kitajima, J.P., Okura, V.K., Zhou, Y., Chen, L., Wood, G.E., Almeida, N.F. Jr., Woo, L., Chen, Y., Paulsen, I.T., Eisen, J.A., Karp, P.D., Bovee, D. Sr., Chapman, P., Clendenning, J., Deatherage, G., Gillet, W., Grant, C., Kutyavin, T., Levy, R., Li, M.J., McClelland, E., Palmieri, A., Raymond, C., Rouse, G., Saenphimmachak, C., Wu, Z., Romero, P., Gordon, D., Zhang, S., Yoo, H., Tao, Y., Biddle, P., Jung, M., Krespan, W., Perry, M., Gordon-Kamm, B., Liao, L., Kim, S., Hendrick, C., Zhao, Z.Y., Dolan, M., Chumley, F., Tingey, S.V., Tomb, J.F., Gordon, M.P., Olson, M.V., and Nester, E.W. 2001. The Genome of the Natural Genetic Engineer Agrobacterium tumefaciens C58. Science 5550:2317-2323.

PAGE 134

120 Xanthomonas ONSA FAPESP network. 2001/2002. http://cancer.lbi.ic.unicamp.br/xanthomonas/ Yang, B., and White, F.F. 2004. Diverse members of the AvrBs3/PthA family of type III effectors are major virulence determinants in bacterial blight disease of rice. Mol. Plant-Microbe Interact. 11:1192-1200. Yang, B., Sugio, A., and White, F.F. 2005. Avoidance of host recognition by alterations in the repetitive and C-terminal regions of AvrXa7, a type III effector of Xanthomonas oryzae pv. oryzae. Mol. Plant-Microbe Interact. 18:142-149. Yang, B., Zhu, W., Johnson, L.B., and White, F.F. 2000. The virulence factor AvrXa7 of Xanthomonas oryzae pv. oryzae is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein. Proc. Natl. Acad. Sci. U.S.A. 97:9807-9812. Yang, Y., De Feyter, R., and Gabriel, D.W. 1994. Host-specific symptoms and increased release of Xanthomonas citri and X. campestris pv. malvacearum from leaves are determined by the 102 bp tandem repeats of pthA and avrb6, respectively. Mol. Plant-Microbe Interact. 5:204-213. Yang, Y., and Gabriel, D.W. 1995a. Xanthomonas avirulence/pathogenicity gene family encodes functional plant nuclear targeting signals. Mol. Plant-Microbe Interact. 8:627-631. Yang, Y., and Gabriel, D.W. 1995b. Intragenic recombination of a single plant pathogen gene provides a mechanism for the evolution of new host specificities. J. Bacteriol. 177:4963-4968. Yang, Y., Yuan, Q., and Gabriel, D.W. 1996. Water soaking functions(s) of Xcm H1005 are redundantly encoded by members of the Xanthomonas avr/pth gene family. Mol. Plant-Microbe Interact. 5:204-213. Yanisch-Perron, C., Vieira, J., and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119. Yoshii, K., Galvez, G.E., and Alvarez-Ayala, G. 1976. Estimation of yield losses in beans caused by common blight. Proc. Am. Phytopathol. Soc. 3:298-299. Yu, I.C., Parker, J., and Bent AF. 1998. Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant. Proc. Natl. Acad. Sci. U.S.A. 95:7819-7824. Zhu, W., Yang, B., Chittoor, J.M., Johnson, L.B., and White, F.F. 1998. AvrXa10 contains an acidic transcriptional activation domain in the functionally conserved C terminus. Mol. Plant-Microbe Interact. 8:824-832.

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BIOGRAPHICAL SKETCH Adriana is native of Colombia, South America. She graduated with a B.S. degree in microbiology from Andes University in Bogot and went on to work for the Colombia Agriculture and Livestock Institute in the Plant Quarantine Facility, and she became its director on 1994. In 1996 she was awarded a scholarship from the same institute and came to the University of Florida to get an M.S. in the Plant Pathology Department, that she obtained in 1999. She went back to Colombia to work for the same Institute in the Seed Division and in 2000 she became the director of the Seed National Laboratory. In 2002, with ICAs permission she came back to UF to pursue a Ph.D. 121