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Phenotypic Characterization and Sequence Analysis of pthA Homologs from Five Pathogenic Variant Groups of Xanthomonas citri


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PHENOTYPIC CHARACTERIZATI ON AND SEQUENCE ANALYSIS OF pthA HOMOLOGS FROM FIVE PATHOGENIC VARIANT GROUPS OF Xanthomonas citri By ABDULWAHID AL-SAADI 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 Abdulwahid Al-Saadi

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This dissertation is dedicated to H.E Ma hmood A. Makki for his continuous support, encouragement and belief in me. He passed aw ay last year without seeing me make it to the end and successfully complete my PhD. I am sure that he would have been very happy and appreciative for my accomplishment.

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ACKNOWLEDGMENTS I would thank God for giving patience and blessing me with a lot of colleagues and friends who stood by my side when I needed them most. The completion of this dissertation would not have been possible without the help and support of many people both in Gainesville and back home in Oman. First I would like to express my gratitude to my advisor Dr. Dean W. Gabriel for his support, guidance and patience with me during my time in his lab. I would also like to thank him for providing me financial support towards the end my PhD. I would like to thank Dr. Jeff Jones for his support, encouragement, guidance, friendship and listening to me when I was feeling down and ready to give up. I would also like to thank my other committee members Dr. G. Moore and Dr. R. Lee for being on my committee, support and correcting this manuscript I would like to thank Dr. G. Wisler for her support and encouragement. I would like to give special thanks to Mr. Gary Marlow for his continued help, support, scientific discussions and just being a good friend that was there for me when I needed him both in and outside the lab. I want to also thank Dr. Joseph Ready and Dr. Young Duan for their help, discussions and advice in the lab. I would like to thanks other students in my lab especially Basma, Adriana and Asha for their help and support. I also thank all former members of the lab. I thank faculty, staff and students in the PMCB program and the Department of Plant Pathology who helped and encouraged me in many ways. I would like to thank everyone in the Department of Plant Industry (DPI) for providing help in iv

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maintenance of citrus plants in the quarantine facility. Special thanks go to my friend Eduardo Carlos and his family for all their support and encouragement. I also want to thank my two study partners Mohamed Al-Khairy and Yousef Al-Dligan. I also thank Mohamed Al-Matar and Fahad Al-Saqqaf. I would like to recognize two people in the Diwan of Royal Court who have recently passed away. H.E Said Seif bin Hamed and H.E Mahmood Maki who supported me coming to the US to get my PhD. Without their support and encouragement I would have not been able to complete my degree. I would like to thank my advisor and mentor in Oman, Dr. Ahmed Hamooda, for continuously supporting and encouraging me throughout my time here. I thank him for being my advocate and believing in me even when many were ready to give up on me. I cannot say thank you enough to this man who took me under his wings and guided me through difficult times. I would like to thank Mr. Yahya Al-Zidjali for his support. Special thanks go to Dr. Yahya Al-Hinai for his support and friendship. I want to thank Mrs. Nihaya, Dr. Magdy, Dr. Deitz, Abdulrahman Al-Siyabi, Abduljalil Attiya, Ammer Al-Manthiri and everyone in the Diwan of Royal Court who helped and supported me. I thank my parents, Abubaker and Khadija, for unconditional support, love and encouragement. I also thank my brothers and sisters for their support and encouragement during my study here. I thank them for making me the person I am. I also like to thank Yasser Al-Ajmi, Mohamed Al-Balushi, Fida Al-Raissy, Aqeel Abdawani and all my friends in Oman. I thank and remember my twin brother Abdulrahman who passed away a few days after our birth. He has always been with me and provided me with strength, v

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patience and hope. Finally I have to say special thanks to my wife May and son Muadh for being there for me as they waited patiently for me during my time here. vi

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 Citrus.............................................................................................................................1 Florida Citrus................................................................................................................2 Citrus Canker................................................................................................................3 Resistance to Citrus Canker..........................................................................................5 Controlling Citrus Canker.............................................................................................5 Objectives.....................................................................................................................6 2 USE OF TWO DIFFERENT CITRUS HOSTS TO DISTINGUISH ALL FORMS OF CITRUS CANKER DISEASE.............................................................................10 Introduction.................................................................................................................10 Material and Methods.................................................................................................11 Strains, Plasmids and Culture Media...................................................................11 Recombinant DNA Techniques...........................................................................11 Plant Inoculations................................................................................................12 In vitro Growth Kinetics......................................................................................12 In planta Growth Kinetics...................................................................................13 Results.........................................................................................................................13 Pathogenicity Phenotypes of X. citri Strains.......................................................13 In vitro Growth....................................................................................................14 Growth Kinetics in planta...................................................................................14 Discussion...................................................................................................................15 vii

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3 IDENTIFICATION AND CHARACTERIZATION OF HOST RANGE FACTOR(S) IN CITRUS CANKER STRAINS........................................................24 Introduction.................................................................................................................24 Material and Methods.................................................................................................26 Bacterial Strains, Plasmids and Culture Media...................................................26 Recombinant DNA Techniques...........................................................................26 Vector Preparation...............................................................................................27 Packaging and Transfection................................................................................27 Plant Inoculations................................................................................................28 Triparental Matings.............................................................................................28 Results.........................................................................................................................29 Xanthomonas citri pv citri A 3213 Strain Genomic Library...............................29 Screening of 3213 Library in Xc270...................................................................29 Discussion...................................................................................................................29 4 SEQUENCE COMPARISON AND CHARACTERIZATION OF FIVE NEW pthA HOMOLOGS FROM FOUR DIFFERENT Xanthomonas citri STRAINS......35 Introduction.................................................................................................................35 Material and Methods.................................................................................................36 Bacterial Strains, Plasmids and Culture Media...................................................36 Recombinant DNA Techniques...........................................................................37 DNA Library Construction..................................................................................37 Plant Inoculations................................................................................................38 Southern Hybridization Analysis........................................................................38 Colony Hybridization..........................................................................................38 Triparental Matings.............................................................................................39 Sequence Analysis of pth Genes.........................................................................40 Results.........................................................................................................................41 Southern Blot Analysis........................................................................................41 Cloning, Characterization and Sequencing of pthA Homologs from X. citri A*, A w B and C Strains...................................................................................41 Inactivation and Complementation of Genes pthB and pthC in X. citri pv aurantifolii........................................................................................................43 None of the pthA Homologs from Group A Strain 3213 Increased the Host Range of Group A* Strain 270 to Include Grapefruit......................................44 Sequence Analysis of pthA Homologs from All Known X. citri Groups............44 Discussion...................................................................................................................45 5 SUMMARY AND CONCLUSION...........................................................................60 APPENDIX A SEQUENCE OF pthC.................................................................................................63 B SEQUENCE OF pthAW.............................................................................................66 viii

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C SEQUENCE OF pthA*...............................................................................................70 D SEQUENCE OF pthA*-2...........................................................................................74 E ALIGNMENT OF PATHOGENECITY GENES FROM X. citri STRAINS............77 LIST OF REFERENCES...................................................................................................87 BIOGRAPHICAL SKETCH.............................................................................................95 ix

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LIST OF TABLES Table page 2-1. Strains and plasmids used in this study....................................................................18 2-2. Phenotypic differences among X. citri strains..........................................................19 3-1. Strains and plasmids used in this study....................................................................31 4-1. Strains and plasmids used in this study....................................................................49 4-2. Phenotypic responses of X. citri strains in 2 citrus hosts.........................................51 4-3. Amino acid sequence identity between pathogenicity genes from X. citri strains...52 x

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LIST OF FIGURES Figure page 1-1. World citrus production.............................................................................................7 1-2. Status of citrus canker disease in the state of Florida................................................8 1-3. Citrus canker symptoms in citrus...............................................................................9 2-1. Inoculation of strains of different citrus canker groups in grapefruit and Key lime...........................................................................................................................20 2-2. Inoculation of several different A* strains in Duncan grapefruit.............................21 2-3. Growth of X. citri strains in PYGM medium...........................................................22 2-4. in planta growth of X. citri strains in Mexican/Key lime and Duncan grapefruit...23 3-1. Scheme for cosmid vector preparation and DNA cloning.......................................32 3-2. DNA fractionation of X. citri 3213 genomic DNA..................................................33 3-3. Restriction profiles of random clones from X. citi 3213 genomic library................34 4-1. Southern Hybridization analysis of X. citri strains hybridized with the BamHI internal fragment of pthA.........................................................................................53 4-2. Colony Hybridization of E. coli with cloned X0053 A w plasmid DNA fragments using 32 P-labeled pthA..............................................................................................54 4-3. Complementation of A strain knockout B21.2 (pthA::Tn5) with pthA homologs from A w strain X0053 on citrus................................................................................55 4-4. Complementation of A strain knockout B21.2 (pthA::Tn5) with pthA homologs in citrus.....................................................................................................................56 4-5. Analysis of pthA and its three homologs in A* strain Xc270..................................57 xi

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4-6. Neighbor-joining dengogram of members of avrBs3/pthA genes from different species and pathovars of Xanthomonas....................................................................58 4-7. Sequence alignment of the predicted amino acids encoded in the main variable portion of the repeat region of all 13 pthA homologs..............................................59 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 PHENOTYPIC CHARACTERIZATION AND SEQUENCE ANALYSIS OF pthA HOMOLOGS FROM FIVE PATHOGENIC VARIANT GROUPS OF Xanthomonas citri By Abdulwahid Al-Saadi August 2005 Chair: Dean W. Gabriel Major Department: Plant Molecular and Cellular Biology Citrus canker is an economically important disease that is caused by five different groups of Xanthomonas citri strains: three from Asia (A, A* and A w ) and two from South America (B and C). In artificial inoculations of grapefruit, only strains of the A and B groups appear to be virulent; strains of the C and A w group elicit an hypersensitive response (HR) and the A* strains show various levels of reduced virulence. The tested A* and A w strains also grew to much lower concentrations in grapefruit compared to the A strain. The strains from all five citrus canker groups were virulent in Mexican lime, but the B and C strains elicited a distinctive canker lesion that was almost white in appearance. Strains from the Asiatic groups grew faster than South American B and C group strains in artificial media. Mexican lime and grapefruit can be used in artificial inoculations to readily distinguish all known strains causing citrus canker disease within 10 days without the need for other laboratory tests. Attempts to identify positive host xiii

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range determinants from X. citri were unsuccessful suggesting the possibility of negative factors (avirulence) being involved in determining host range of X. citri. Every X. citri strain carries multiple DNA fragments that hybridize with pthA, a member of the avrBs3/pthA gene family from X. citri group A that is required for pathogenicity and growth of X. citri in citrus. Three new pthA homologs were cloned and sequenced from canker groups A w (pthAW) and A* (pthA* and pthA*-2), and compared with pthA, pthB and pthC. Homologs pthA, pthB, pthC pthAW and pthA* all have 17.5, nearly identical, direct tandem repeats of 34 amino acids and all complemented a pthA::Tn5 knockout mutation in an X. citri group A strain B21.2. Although grapefruit is a differential host and groups A* and A w are avirulent in grapefruit, none of the pthA homologs appeared responsible for the avirulence phenotype by cross complementation tests. Furthermore, none of the four pthA homologs from the wide host range group A strain 3213, including pthA, conferred an increase in host range of group A* or A w strains to include grapefruit. pthA*-2 carried only 15.5 repeats and did not confer either pathogenicity or avirulence to B21.2 in any citrus species tested. Phylogenetic studies separate pthA homologues into two groups, Asiatic and South American groups. Analysis of the predicted amino acid sequences of all sequenced pthA homologs from X. citri indicated that a specific set of amino acid residues in two variable regions of the 17 th direct tandem repeat may be required for pathogenicity in citrus. xiv

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CHAPTER 1 INTRODUCTION Citrus Citrus is one of the major fruit crops in the world. It is thought to have originated in Southeast Asia and India. Citrus was introduced into the new world in the 16 th century by Spanish and Portuguese explorers (Allen, 2000). World production of citrus is estimated to be about one hundred million metric tons (FAOSTAT data, 2004, http://apps.fao.org ). World citrus production has seen a substantial increase over the last 40 years (Figure 1-1). Major citrus producing countries include the United States, Brazil, China, Argentina, Spain and Mexico (Figure 1-1b). Currently, citrus produced in North and South America account for the majority of citrus production worldwide. Although citrus is mainly grown for the fresh fruit market, large citrus-based juice industries have developed in many countries such as Brazil and the United States. Generally citrus is grown between 40 North and 40 South latitudes where minimum temperatures stay above 20 24 F (Timmer and Duncan, 1999). Citrus is a perennial evergreen with an expected economical production expectancy of about 50 years (Timmer and Duncan, 1999). Originally citrus was grown on its own root system, but now most citrus production plants are grafted onto various rootstocks. Rootstocks are selected for their inherent characteristics that affect production, cold hardiness, salinity tolerance, disease resistance and most importantly compatibility with scion tissue. 1

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2 The majority of citrus varieties grown for commercial purposes are in the genus Citrus including grapefruit (Citrus paradisi Macfad), sweet orange (C. sinensis (L.) Osbeck), tangerine/mandarin (C. reticulata Blanco), lemon (C. limon Burm), lime (C. aurantifolia (Christm.) Swingle), pummelo (C. grandis Osbeck) and citron (C. medica L.). Other citrus relatives that are not in this genus are kumquats (Fortunella spp.) and trifoliate orange (Poncirus trifoliata). The latter is used only as a rootstock. Florida Citrus Total citrus production in the U.S. in 2004 is estimated at 16.2 million tons with an estimated value of $ 2.4 billion (USDA, 2004). States that produce citrus include Florida, California, Texas, Arizona, Alabama, Mississippi and Louisiana. Florida produces about 80% of the U.S. citrus, of which 20% 25% is sold for fresh fruit consumption. In 2004, Florida produced 242 million boxes of oranges and 40.9 million boxes of grapefruit (USDA, 2004). Citrus is an important economic crop for the state of Florida, as the worth of the commercial citrus industry in Florida is estimated to be more than $8.5 billion. Diseases play a critical role in limiting citrus production as citrus is mainly grown in the same tropical and subtropical areas that also favor the growth of microorganisms. This provides great challenges to citrus growers since they must balance cost of controlling diseases against lower projected profit margins. An example of a citrus disease that is a serious problem for citrus growers is citrus canker disease. Citrus canker has destroyed many citrus growing areas around the world. Florida authorities are putting major resources towards completely eradicating this disease. It is important to gain a better understanding of this disease because of the quarantine of citrus canker as a

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3 pest. Citrus canker it is still spreading, despite $50 million spent between 1996 and 1999 in eradication efforts (Schubert et al., 2001). Citrus Canker Citrus canker is one of the major disease problems facing citrus producers in Florida and many areas of the world (Danos et al., 1981; Elgoorani, 1989; Gottwald et al., 2001). Citrus canker, also known as bacterial canker, has destroyed large areas of citrus production (Fegan et al., 2004; Schubert et al., 2001). The pathogen is thought to have originated in Southeast Asia, from where it has spread to other citrus producing areas. Asiatic citrus canker was introduced into the United States for the first time in 1912 from infected nursery material. It took approximately 20 years to eliminate this outbreak of citrus canker (Loucks and Florida. Division of Plant Industry., 1934). In 1986, citrus canker reappeared for the second time in both residential and commercial areas around Tampa, Florida. As a result of this outbreak a new citrus canker eradication program was initiated (Brown, 2001). Eight years later, Florida declared it had eradicated citrus canker at a cost of $27 million (Agrios, 1997). Citrus canker reappeared for the third time in Florida in 1995 in Dade County, and has resulted in the destruction of more than four million trees in both residential and commercial areas (FDACS data, 2005). Figure 1-2 shows the status of citrus canker disease in the state of Florida in 2004. Currently, citrus canker has been detected in 20 Florida counties. The total area under quarantine is estimated at 1,397.82 sq. miles (FDACS data, 2005, www.doacs.state.fl.us). Strong regulatory and quarantine measures were implemented in the latest effort to eradicate the disease. Healthy citrus trees anywhere within a radius of 1900 ft from infected trees are deemed exposed and are destroyed (Gottwald et al., 2002).

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4 Citrus canker is caused by several pathogenic variants of Xanthomonas citri. In general, five different groups of pathogenic variants are recognized: A, B, C, A* and A w (Gabriel et al., 1989; Stall et al., 1982; Verniere et al., 1998). In the literature two other groups of citrus canker are described: D and E strains. Although there is a single extant D strain that was reported in Mexico, it is thought that the fungal pathogen Alternaria limicola was responsible for that disease outbreak (Schubert et al., 2001). The E strain group was found in grapefruit only in nurseries in Florida and was described as a new form of citrus canker. However, strains in this group do not cause hyperplasia and do not infect fruit or mature citrus in groves. The disease is now recognized as distinct from citrus canker and was named citrus bacterial leaf spot caused by Xanthomonas axonopodis pv. citrumelo Citrus canker symptoms appear after the pathogen enters the leaves through the stomata or wounds and multiplies in the intercellular spaces of the spongy mesophyll (Gottwald et al., 1988; 1989; Graham et al., 1992; Pruvost et al., 2002). The initial symptom is the formation of water-soaked tissue followed by growth of yellow halos on the infection margins. As the disease progresses, erumpent necrotic lesions are formed on leaves, stems and fruits (Figure 1-3). At advanced disease stages, plants defoliate and fruit can drop prematurely. At the microscopic level, infected cells divide (hyperplasia) and enlarge (hypertrophy); and the pustules rupture the surface of the leaf tissue and release bacteria that become a source of inoculum for further infections (Swarup et al., 1991). The citrus canker bacterium is transmitted by wind-blown rain, although machinery, animals and humans can also transmit it (Bock et al., 2005; Danos et al.,

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5 1984). An important factor that contributed to the spread of citrus canker in this last infection in Florida was the Asian citrus leaf miner Phyllocnistis citrella (Cook, 1988). The leaf miner is probably not a vector for canker, but instead it provides wounds that allow entry of bacteria into citrus leaves (Belasque et al., 2005). Although citrus canker does not cause systemic damage, it results in reduced marketability of citrus fruit especially those produced for the fresh market. Resistance to Citrus Canker Citrus genotypes show differences in susceptibility to this disease. Grapefruit, sweet orange and Mexican lime are highly susceptible. Sour orange, lemon and tangelo are moderately susceptible, whereas mandarin, citron and kumquat are less susceptible (Schubert et al., 2001). It is not clear if resistance in citrus is a result of active defense responses or if it is due to physical characteristics of different citrus genotypes, e.g. number of stomata or thickness of the leaf tissue that may influence the number of bacterial particles entering citrus leaves (Goto, 1969; McLean and Lee, 1922). Controlling Citrus Canker The most effective control of citrus canker is application of strict regulatory and quarantine measures that will protect against the introduction of new infections (Graham et al., 2004). Most citrus producing areas put many resources into monitoring and regulating citrus canker. That is because it is so difficult to eliminate the bacteria once it has become established. Once the disease is established in an area, eradication of both infected and exposed trees and burning plant material are used to help eliminate and prevent spread of disease. Multiple applications of copper based compounds were found to help control the disease to some extent (Hwang, 1949). In some cases pruning infected branches is used to control and eliminate the source of infection. Since citrus canker

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6 spreads by wind driven rain, wind brakes were found to be useful in controlling this disease (Gottwald and Timmer, 1995). Objectives The aim of this study was to identify host range determinants of canker causing strains. I was interested in identifying genes that are necessary for increasing the host range of canker causing strains of X. citri. These new strains that are limited in host range were used to screen for genes involved in host range determination. Further understanding of how host range is determined may provide important tools in developing control measures. The specific objectives of this work include the following: Objective 1. Characterizing canker causing Xanthomonas citri A* and A w group strains. Objective 2. Attempting to identify and characterize positive host range factor(s) in canker causing Xanthomonas citri. Objective 3. Isolating pathogenicity gene (pthA) homologs from A* and A w groups and characterize their role in host range determination.

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7 19%13%12%6%6%4%4%3%3%2%2%2%2%2%1%1%1%1%14% Brazil USA China Mexico Spain India Iran Nigeria Italy Egypt Argentina Turkey Pakistan South Africa Japan Greece Morocco Thailand others 020406080100120Millions of tons 196119651970197519801985199019952000year A B Figure 1-1. World citrus production. A. world citrus production between 1961 2000 expressed in metric tons. B. Percent production by countries (FAOSTAT data, 2004, http://apps.fao.org ).

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8 Figure 1-2. Status of citrus canker disease in the state of Florida. (FDACS data, 2005, www.doacs.state.fl.us)

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9 Figure 1-3. Citrus canker symptoms in citrus. Citrus canker symptoms on leaves, fruit and stem. At advanced disease stages, plants defoliate and fruit can drop prematurely.

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CHAPTER 2. USE OF TWO DIFFERENT CITRUS HOSTS TO DISTINGUISH ALL FORMS OF CITRUS CANKER DISEASE Introduction Citrus canker disease is caused by several different pathogenic variants of Xanthomonas citri (ex Hasse) (Brunings and Gabriel, 2003; Gabriel et al., 1989). Although the taxonomy of these strains is controversial (Gabriel et al., 1989; Vauterin et al., 1995), five groups of pathogenic variants are recognized, based primarily on field symptoms: A, B, C, A* and A W (Gabriel et al., 1989; Stall et al., 1982; Sun et al., 2004; Verniere et al., 1998). The Asiatic (A) group (X. citri pv citri A) is the most severe and widely spread throughout the world. The B and C groups (X. citri pv aurantifolii B and C), which are also known as cancrosis B and cancrosis C, respectively, have been found only in South America. These South American groups are phylogenetically distinct and grow more slowly on artificial media than strains from all other groups (Brunings and Gabriel, 2003; Goto, 1969; Stall et al., 1982). The B and C strains also have a reduced host range compared to the A group. In addition, the C strains elicit an hypersensitive response (HR) in grapefruit (Citrus paradisi) (Stall et al., 1982). Recently two new variants of the A group of citrus canker strains were identified and designated A* and A w (Sun et al., 2004; Verniere et al., 1998). Both new groups are limited in host range to Key/Mexican lime (C. aurantifolia); the A w strain causes an HR when inoculated in grapefruit at high concentrations (Sun et al., 2004). The A* and A w strains of X. citri are phylogenetically most closely related to the A group (Cubero and Graham, 2002) 10

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11 Various diagnostic aids have been used to confirm citrus canker disease, including PCR (Cubero and Graham, 2002; Mavrodieva et al., 2004), antibodies (Alvarez et al., 1991) and microscopy. These tests can be critical if the disease appears in regions where it has not previously been seen or has not been recently observed. Indeed, a fungal disease was misdiagnosed as citrus canker disease in Mexico (Stapleton, 1986; Stapleton and Garza-lopez, 1988), and a bacterial leaf spot disease was misdiagnosed as citrus canker in Florida in 1984 (Schubert et al., 1996). Once confirmation of citrus canker disease has been made, only host range tests can be used to reliably determine the strain group or pathovar. Historically, these studies relied on sweet orange, mandarin orange, lemon, lime and grapefruit (Stall and Civerolo, 1991). In this study, we report the use of Duncan grapefruit and Mexican lime as differential hosts to differentiate strains from all variant groups of citrus canker disease. Material and Methods Strains, Plasmids and Culture Media Strains of Escherichia coli, Xanthomonas spp. and plasmids used in this study are listed in Table 2-1 along with their relevant characteristics and source or reference. E. coli strains were grown in Luria-Broth (LB) medium at 37 C (Sambrook et al. 1989). Xanthomonas spp. were grown in PYGM (peptone yeast extract-glycerol-MOPS) medium at 30 C as described by Gabriel et al. (1989). Antibiotics were used at the following final concentrations (g/ml): rifampin (Rif), 75; spectinomycin (Sp), 35. Recombinant DNA Techniques Xanthomonas total DNA was prepared as described by Gabriel and De Feyter (1992) and also using Amersham Biosciences DNA Isolation Kit as described by the

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12 manufacturer. Plasmids were isolated by alkaline lysis from E. coli (Sambrook et al., 1989) and Xanthomonas (Defeyter and Gabriel, 1991). QIAGENs QIAprep and plasmid midi kits were also used to isolate plasmid DNA from E. coli and Xanthomonas as described by the manufacturer. Southern hybridizations were performed using nylon membranes as described (Lazo and Gabriel, 1987). Plant Inoculations Duncan grapefruit and Mexican lime plants were grown and maintained under natural light in the quarantine greenhouse facility at the Division of Plant Industry, Florida Department of Agriculture, in Gainesville. Temperatures in this greenhouse ranged from 25 C to 35 C, with 50 % to 100 % relative humidity. All inoculations were carried out in this facility. Liquid cultures of the tested strains were grown in PYGM medium at 30 C for approximately 24 hr. Cultures were centrifuged @ 1000g for 3 min and cells resuspended in equal volumes of sterile tap water (saturated with CaCO 3 ) and infiltrated into the abaxial surface of young, freshly flushed partially expanded citrus leaves at two concentrations (10 5 cfu/ml for low and 10 8 cfu/ml for high levels) using the blunt end of tuberculin syringe as described (Gabriel et al., 1989). Observations were taken 5-10 days after inoculation. In vitro Growth Kinetics Liquid cultures of X. citri 3213, B69, Xc270 and X0053 were prepared in PYGM medium and grown at 37 C overnight with slow shaking. The following day, 100 ml of fresh PYGM was inoculated with 50 l of the starter culture. Optical density (OD 600 ) readings were taken at 0, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38 hr. This experiment was repeated three times.

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13 In planta Growth Kinetics For bacterial cell counts, whole leaves were infiltrated as described. For each strain, three leaves of each host were infiltrated. Bacterial cell counts from the inoculated leaves were taken at days 0, 1, 6, 10 and 14. A number 7 cork borer (about 1 cm 2 ) was used to cut one leaf disk from each inoculated leaf per time point. Each treatment, a leaf disk from each of the three inoculated leaves was placed in a mortar and pestle and macerated together (in 1 ml sterile tap water saturated with CaCO 3 ). Once homogeneity was obtained, ten-fold serial dilution were made ranging from 10 -1 to 10 -9 Ten microliters droplets of each dilution were spread on PYGM plates without antibiotics and allowed to grow for 48 hr at 28 C. Colonies were counted from the most readily scored dilution, and the number of cfu per cm 2 of leaf tissue was calculated. The experiments were repeated three times. Populations were expressed as log cfu/cm 2 of leaf tissue. Results Pathogenicity Phenotypes of X. citri Strains In addition to the previously described A (3213), B (B69) and C (Xc340) strains, newly described A* (Xc205, Xc270, Xc280, Xc290, Xc322 and Xc406) and A w (X0053) strains (Sun et al., 2004; Verniere et al., 1998) were inoculated on citrus. All strains tested caused hyperplastic lesions in Mexican lime that developed 59 days after inoculation (Figure 2-1). In Duncan grapefruit (Citrus paradisi) differential reactions were observed that distinguished each group. Strains from group A elicited typical canker symptoms in grapefruit within 6 days, but B strains elicited a whitish canker phenotype within 10 days. C and A w strains elicited a hypersensitive response (HR) in grapefruit. The A* strains, which were originally isolated from Southwest Asia, also gave distinct phenotypes in Duncan grapefruit (Figure 2-2). Strains Xc205 and Xc322

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14 did not elicit canker symptoms at low concentration (10 4 cfu/ml). Strain Xc406 elicited a weak canker at low concentration, but when inoculated at high concentration, elicited typical canker lesions. Strains Xc270, Xc280 and Xc290 did not elicit canker in Duncan grapefruit at either low or high inoculum concentrations. A* strains could be subdivided into three groups; A*-1, A*-2 and A*-3 (Figure 2-2). Table 2-2 summarizes symptoms of different X. citri groups on grapefruit and lime. In vitro Growth In vitro growth of X. citri strains in liquid medium was measured by optical density (OD 600 ) changes recorded over time (Figure 2-3). Strains 3213 (A), X0053 (A w ) and Xc270 (A*) were very similar in their growth in PYGM medium. B69 (B) strain was significantly slower in its growth compared to A, A w and A* strains. On agar plates, the South American B and C strains grew similarly on a variety of media, and always slower than the A, A w and A* strains. Growth Kinetics in planta Growth kinetics of X. citri strains 3213 (A), 270 (A*) and X0053 (A w ) were studied in Duncan grapefruit and Mexican lime leaves. In Mexican lime, growth of all three strains was similar (Figure 2-4). However the growth kinetics of these strains was different in Duncan grapefruit (Figure 2-4). Growth of the A w strain X0053 was reduced by one order of magnitude as compared to A strain 3213. This reduction in growth was noticeable 6 days post-inoculation and continued through day 14 when the comparison ended. Growth of A* strain 270 was reduced by at least two orders of magnitude after 6 days post-inoculation as compared to strain 3213. Strain 270 did not continue to increase after 6 days growth in planta.

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15 Discussion Inoculation of different X. citri strains on just two citrus host species allowed the differentiation of all known X. citri groups based on their symptoms. All tested strains of X. citri caused canker in Key/Mexican lime. Lime is a very susceptible host compared to other citrus species or types. The B and C strains elicited a characteristic whitish canker in key/Mexican lime that is readily distinguished from canker symptoms caused by other strains. This is probably due to the fact that both strains are phylogeneticlly very similar and likely share common pathogenicity factors. Indeed, the pathogenicity elicitors pthB and pthC from the B and C strains were found to be closely related to each other (98% similarity) at the amino acid level and different from pthA homologues from A, A* and A w strains. The latter were closely related to each other (Chapter 4). Strains from different groups of X. citri exhibited quite different phenotypic responses when inoculated in Duncan grapefruit leaves. Except for the A and B groups, which elicited typical canker symptoms, strains from all other groups appeared much less virulent. X0053 from the A w group elicited necrotic symptoms in grapefruit that took 5 to 10 days to appear. Unlike the A W strain, the C group strain C340 elicited a relatively fast HR reaction in grapefruit that took only 3 days to appear. In both cases, the necrosis and HR, potential avr gene function is indicated. The A* group showed the most within-group variation among strains in grapefruit; all A* strains were characterized by reduced virulence as compared to A and B strains and lacked any evidence of eliciting necrosis or an HR. Strains Xc205 and Xc322 were only capable of causing canker in Duncan grapefruit when inoculated at high concentrations (10 8 -10 9 cfu/ml). At (lower) concentrations that more closely resemble field conditions, no canker symptoms were observed with these strains. Strain Xc406 elicited a very weak canker phenotype when

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16 inoculated at low concentration, but elicited normal canker symptoms when artificially inoculated at high concentrations. Strains Xc270, Xc280 and Xc290 were not able to elicit canker in grapefruit leaves at either concentration. All tested A* strains were unable to cause canker at low inoculum concentrations or an HR at high concentrations. In planta growth kinetics of strains representing the fast growing A, A* and A w groups showed interesting differences (Figure 2-4). All three strains appeared to grow similarly to each other in Mexican/Key lime. Asiatic strain 3213 inoculated in Duncan grapefruit grew to levels similar to those seen in lime. A w strain X0053, which elicits necrotic symptoms in grapefruit, grew to a final level that was only one log lower than strain 3213. A* strain Xc270, which does not cause canker or HR in grapefruit, was unable to grow well in grapefruit, increasing only 2 logs after inoculation and grew to a final level that was more than two logs lower compared to strain 3213. It is possible that A* strains carry an avr gene that specifically triggers grapefruit defenses, but without an HR. An HR is not always observed with gene-for-gene resistance (Bendahmane et al., 1999; Goulden and Baulcombe, 1993; Jurkowski et al., 2004; Lehnackers and Knogge, 1990; Ori et al., 1997; Schiffer et al., 1997; Yu et al., 2000). Indeed, a Xanthomonas avr gene that elicits host defense without an HR was recently reported (Castaneda, 2005). An alternative explanation is that this group is missing a factor or perhaps factors that are specifically required for growth in grapefruit, such as the extracellular polysaccharides (EPS) and lipopolysaccharides (LPS) that are needed by X. axonopodis pv. citrumelo for virulence on citrus (Kingsley et al., 1993). Only further experimental testing can distinguish between these explanations.

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17 Although five different citrus host species have traditionally been used to distinguish pathovars of X. citri, all known groups can be readily distinguished by inoculation of only two host differentials, lime and grapefruit. Even if positive control cultures are not available, if low inoculations are used, then: 1) only the A strains elicit green cankers in both lime and grapefruit; 2) only the B strains elicit whitish cankers in lime and grapefruit; 3) only the C stains elicit whitish cankers in lime and an HR in grapefruit; 4) only the A* strains elicit green canker in lime and at best very weak cankers in grapefruit, and 5) only the A w strains elicit green cankers in lime and an HR in grapefruit.

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18 Table 2-1. Bacterial strains and plasmids used in this study Strain or plasmid Relevant Characteristics Reference or source Escherichia coli DH5 F endA1, hsdR17(r k m k ), supE44, thi-1, recA1 Gibco-BRL Xanthomonas citri 3213 Group A, wild type Gabriel et al. 1989 3213Sp Spontaneous Sp r derivative of 3213, Sp r Gabriel et al. 1989 B21.2 pthA::Tn5-gusA, marker exchanged mutant of 3213Sp, Sp r Kn r Swarup et al. 1991 B69 Group B, wild type Stall et al. 1982 B69Sp Spontaneous Sp r derivative of B69, Sp r El Yacoubi, 2005 C340 Group C, wild type Stall et al. 1982 Xc205 Group A*, wild type Verniere et al. 1989 Xc205Rif Spontaneous Rif r derivative of Xc205, Rif r This study Xc270 Group A*, wild type Verniere et al. 1989 Xc270Rif Spontaneous Rif r derivative of Xc270, Rif r This study Xc280 Group A*, wild type Verniere et al. 1989 Xc290 Group A*, wild type Verniere et al. 1989 Xc322 Group A*, wild type Verniere et al. 1989 Xc406 Group A*, wild type Verniere et al. 1989 X0053 Group A w wild type Sun et al. 2004 X0053Rif Spontaneous Rif r derivative of X0053, Rif r This study

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19 Table 2-2. Phenotypic differences among X. citri strains. Mexican Lime Grapefruit Strain Low (10 4 -10 5 ) cfu/ml High (10 8 -10 9 ) cfu/ml Low (10 4 -10 5 ) cfu/ml High (10 8 -10 9 ) cfu/ml A C a C C C B Wt C b Wt C Wt C Wt C C C C HR c HR A* -1 C C 0 d C A* -2 C C WC e C A* -3 C C 0 0 A w C C 0 HR a=canker, b= white canker, c=Hypersensitive response, d=no canker and e=weak canker

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20 Figure 2-1. Inoculation of strains of different citrus canker groups in grapefruit and key lime. A and B strains are able to cause canker in both hosts. A* and A w strains can only cause canker in Key lime. Note the HR in grapefruit caused by A w

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21 322 322 406 222290 fu/ml 10 8 -10 9 c 80 70 05 205 270 280 290 406 406 322 290 280 270 22223445fu/ml 10 -10 c 06 22 90 80 70 05 205 Figure 2-2. Inoculation of several different A* strains in Duncan grapefruit. High (left) and low (right) concentrations of bacteria were used for inoculation.

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00.20.40.60.811.2024681012141618202224262830323436384042Time (hr) 3213 B69 Xc270 X0053 22 Figure 2-3. Growth of X. citri strains in PYGM medium. A strain 3213, A* strain Xc270, A strain X0053 and B strain B69 were used in this comparison. w

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23 012345678910061014Days after inoculation 3213-KL Xc270-KL X0053-KL 0123456789061014Days post inoculati o 3213-GF Xc270-G F X0053-G F B A Figure 2-4. in planta growth of X. citri strains in A) Mexican/Key lime and B) Duncan grapefruit. A strain 3213, A* strain Xc270 and A w strain X0053 were used.

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CHAPTER 3 IDENTIFICATION AND CHARACTERIZATION OF HOST RANGE FACTOR(S) IN CITRUS CANKER STRAINS. Introduction Both positive and negative genetic factors have been found to affect the host range of phytopathogenic bacteria. For example, a given Rhizobium species can only nodulate a restricted number of hosts, and this specificity is determined by specific signal molecules that are exchanged between the bacteria and host plants (Fisher and Long, 1993; Kondorosi et al., 1991). Some of the host specific nodulation genes needed to actively condition the host range include NodD, NodZ, NodW, NolA and NodC (Kamst et al., 1997). Some negatively acting factors have also been found in some rhizobia and these have avirulence (avr) function in some hosts. For example, nodFE of R. leguminosarum bv trifolii, which is virulent in white and red clover, condition avirulence in pea (Djordjevic et al., 1987), and nodQ and nodH were found to confer avirulence to R. l. bv. trifolii and R. l. bv. viceae in their respective hosts, white clover and common vetch (Debelle et al., 1988; Faucher et al., 1989). Agrobacterium tumefaciens and A. rhizogenes generally have a wide host range that includes most dicotyledonous plants. Host range in A. tumefaciens is thought to be generally determined by positive factors; however some negative factors of host range determination have also been found (Keen, 1990). For example, certain virulence (vir) genes on the Ti plasmid condition host range. Progressive deletions of the 3 end of virE were found to progressively reduce the number of plant species on which crown galls are 24

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25 formed. virG from supervirulent A. tumefaciens strain extends the host range of certain Agrobacterium strains (Chen et al., 1991; Hood et al., 1986). On the other hand virA and virC appear to act as negative regulators of host range in grapevine. virA is involved in detection of host specific phenolics compounds. virC was shown to act as an avr gene to trigger host defenses in incompatible interactions and thus limit the number of plants A. tumefaciens can infect (Yanofsky et al., 1985; Yanofsky and Nester, 1986). Strains of the genus Xanthomonas are always found associated with plants. Different xanthomonads attack a very wide range of plant species. However, individual species show limited host range (Gabriel, 1999b). Members of this genus are divided into species and pathovars, based on phylogeny, host range and disease symptom variation. The molecular basis of host range determination at the pathovar level is not well understood. Azad and Kado (1984) showed that elimination of the HR in tobacco to Erwinia rubrifaciens did not increase the host range of this pathogen to include tobacco. Similarly, Swarup et al (1992) showed that elimination of the nonhost HR did not extend the host range of X. citri. Factors that positively enhance the host range of Xanthomonas include the extracellular polysaccharide (EPS) and lipopolysaccharide (LPS); the opsX locus is involved in the biosynthesis of EPS and LPS, and is also needed by X. axonopodis pv. citrumelo for virulence in citrus (Kingsley et al., 1993). Other positive factors that could influence the host range of pathogens are suppressors of host defenses (Ponciano et al., 2003). For example, HopPtoD2, from Pseudomonas syringae, was found to suppress programmed cell death in plants resulting in infection (Bretz et al., 2003; Espinosa et al., 2003; Hauck et al., 2003). Similarly, Abramovitch et al. (2003) found that the P. syringae effector, AvrPtoB, induced plant

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26 disease susceptibility by preventing a programmed cell death response from occurring in tobacco plants. These results suggest that bacterial host range is determined by positive and negative acting factors. This chapter describes attempts to identify host range determinants of X. citri. A virulence enhancement approach (Swarup et al., 1991) was used in an attempt to identify positive factor(s) required to increase host range of a narrow host range, A* strain Xc270, to include grapefruit. Material and Methods Bacterial Strains, Plasmids and Culture Media Strains of Escherichia coli, Xanthomonas spp. and plasmids used in this study are listed in Table 3-1 along with their relevant characteristics, source and/or reference. E. coli strains were grown in Luria-Broth (LB) medium at 37 C (Sambrook et al., 1989). Xanthomonas spp. were grown in PYGM (peptone yeast extract-glycerol-MOPS) medium at 30 C as described (Gabriel et al.1989). Antibiotics were used at the following final concentrations (g/ml): rifampin (Rif), 75; spectinomycin (Sp), 35; ampicillin (Ap), 100; gentamycin (Gm), 5. Recombinant DNA Techniques Xanthomonas total DNA was prepared as described by Gabriel and De Feyter (1992). Plasmids were isolated by alkaline lysis from E. coli (Sambrook et al. 1989) and Xanthomonas (De Feyter and Gabriel 1991). Restriction enzyme digestion was performed as recommended by the manufacturers. Southern hybridization was performed by using nylon membranes as previously described (Lazo et al., 1987).

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27 Vector Preparation To identify genes involved in determining the host range of X. citri pv citri, genomic DNA from the wide host range X. citri pv citri A strain 3213 was partially digested with MboI and size fractioned on a sucrose gradient. Cosmid vector pUFR43 was used to make a DNA library of 3213 DNA fragments. This cosmid vector (Defeyter et al., 1990) was split into two pools and cut with either EcoRI or SalI restriction enzyme to produce the two arms and then treated with shrimp alkaline phosphatase. To create common cloning ends the arms were then cut with BamHI and used for ligations to the 20 25kb 3213 DNA fraction (Figure 3-1 and 3-2). Packaging and Transfection The recombinant DNA was packaged using stratagene packaging mix (Gigapack III Gold Packaging Extract), and introduced into E. coli strain DH5(mcr) via transfection as described by the manufacturer protocol. Positive white plaques were then picked and placed onto LB plates containing the antibiotic Kanamycin (20 g/l). Using a 48 pin replicating fork, these colonies were transferred into 96 well microtiter plates containing liquid LB (with 14% glycerol) and stored at C. At the same time a replicate of each plate was made and maintained by replicating each plate once every month. DNA from eighteen randomly selected library clones was extracted and digested with BamHI and electrophoresed on agarose gels in order to estimate insert size and evaluate the quality of the library. The total number of cosmid clones required to cover the entire 3213 genome (N) was determined using the following formula (Clarke and Carbon, 1976):

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28 N I n(1 0.99)In1insert sizeTotal genome size Plant Inoculations Duncan grapefruit and Mexican lime plants were grown and maintained under natural light in the quarantine greenhouse facility at the Division of Plant Industry, Florida Department of Agriculture, Gainesville, Fl. Temperatures in this greenhouse ranged from 25C to 35 C with 50% to 100% relative humidity. All inoculations were carried out in this facility. Liquid cultures of all tested strains were grown in PYGM medium at 30 C for approximately 24 hr. Cultures were centrifuged and resuspended in equal volumes of sterile tap water (saturated with CaCO 3 ) and pressure infiltrated at appropriate concentrations (10 5 for low and 10 8 cfu/ml for high) into the abaxial surface of citrus leaf using the blunt end of tuberculin syringes. Observations were taken 5-10 days after inoculation. For screening of large numbers of clones, colonies were streaked onto PYGM agar plates incubated at 30 C for approximately 24 hr, resuspended in sterile tap water (saturated with CaCO 3 ) and pressure infiltrated into citrus as described. Triparental Matings To transfer the 3213 library to the limited host range Xc270 (A*) strain, triparental matings were performed as described by Defeyter et al (1990). Strain pRK2073 was used as a helper strain. The recipient was concentrated 50 100 fold. Transconjugants were screened on PYGM plates containing Rif 75l g/ml and Gm 3l g/ml at 28 C and 2-3 days later colonies were transferred onto new selection plates.

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29 Results Xanthomonas citri pv citri A 3213 Strain Genomic Library A genomic library of X. citri pv citri A, strain 3213, was made and 18 randomly picked clones were evaluated for insert size and pattern (Figure 3-3). All 18 clones gave different restriction patterns indicating random insertions in the vector. The average size of the inserts was 39 kb. Based on the Clark and Carbon formula (Clarke and Carbon, 1976), 610 clones were required to cover the whole X. citri 3213 genome with 99% probability. Seven hundred and fifty clones were maintained in E. coli strain DH5 and stored in 15% glycerol at -80 C. Screening of 3213 Library in Xc270 Five hundred and fifty clones were transferred from the 3213 library into X. citri pv. citri A*-3 strain Xc270 by triparental mating and transconjugants were individually screened for symptoms in Duncan grapefruit. No clones were identified that consistently increased the pathogenicity of Xc270. Discussion Attempts to identify positive host range determinants from X. citri were unsuccessful when a library from the wide host range group A strain 3213 was moved into the narrow host range group A* strain Xc270. Initially six clones (pAW377, pAW378, pAW380, pAW400, pAW413 and pAW419) seemed to elicit canker-like symptoms in Duncan grapefruit, but when those clones were re-conjugated into Xc270, the initial results were not confirmed. The in planta growth of Xc270 described in chapter 2 showed that the Xc270 grew poorly in Duncan grapefruit, suggesting that the only clones that would complement Xc270 and cause canker in grapefruit would be those that would increase growth. It is likely that in planta growth requires multiple effectors,

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30 and that no individual cosmid would carry enough factors to reveal a strong difference. Another possibility is that Xc270 may carry avr genes that function in grapefruit and prevent Xc270 from growing. Avirulence is usually epistatic over virulence and therefore a screen for positive factors would fail if this were the case. Perhaps a better approach would be to construct a library of Xc270 DNA and screen in 3213 in order to identify any avirulence gene function.

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31 Table 3-1. Strains and plasmids used in this study Strain or plasmid Relevant Characteristics Reference or source Escherichia coli DH5 F endA1, hsdR17(r k m k ), supE44, thi-1, recA1 Gibco-BRL Xanthomonas citri 3213 Group A, wild type Gabriel et al. 1989 3213Sp Spontaneous Sp r derivative 3213, Sp r Gabriel et al. 1989 B21.2 pthA::Tn5-gusA, marker exchanged mutant of 3213Sp, Sp r Kn r Swarup et al. 1991 Xc270 Group A*, wild type Verniere et al. 1989 Xc270Rif Spontaneous Rif r derivative of Xc270, Rif r This study Plasmid pRK2013 ColE1, Km r ,Tra + helper plasmid Figurski and Helinski, 1979 pRK2073 pRK2013 derivative,npt::Tn7, Km s Sp r ,Tra + helper plasmid Leong et al. 1982 pURF043 IncW, Mob + lacZ + Gm r Nm r cos, shuttle vector De Feyter and Gabriel, 1991 pAW377 Fragment from X. citri 3213 library cloned in pUFR43 This study pAW378 15 kb fragment from X. citri 3213 library cloned in pUFR43 This study pAW380 Fragment from X. citri 3213 library cloned in pUFR43 This study pAW400 Fragment from X. citri 3213 library cloned in pUFR43 This study pAW413 24 kb fragment from X. citri 3213 library cloned in pUFR43 This study pAW419 Fragment from X. citri 3213 library cloned in pUFR43 This study

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32 E=EcoRIEEEEEB=BamHIEEBBBBBBEES=SalISBSSSSSSSScoscoscosSalIEcoRIPhosphotaseBamHImix******pUFR43Phosphotase E=EcoRIEEEEEB=BamHIEEBBBBBBEES=SalISBSSSSSSSScoscoscosSalIEcoRIPhosphotaseBamHImix******pUFR43Phosphotase headsTransfect E. coliEESSSS*EMboIEBBBB MboI***Ligase headsTransfect E. coliEESSSS*EMboIEBBBB MboI***Ligase + inserts Figure 3-1. Scheme for cosmid vector preparation and DNA cloning. 32

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33 A 4.3kb 23kb 6.5kb 9.4kb 23kb B Figure 3-2. DNA fractionation. A). Partial digestion of X. citri 3213 genomic DNA (0.7% agarose gel). B) Size fractionation of MboI partial digest of 3213 DNA (0.7% agarose gel).

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34 Figure 3-3. Restriction profiles of random clones from X. citi 3213 genomic library. DNA was digested with EcoRI. As a merker, DNA digested with HindIII (M). 2.0 kb 2.3 kb 4.3 kb 6.5 kb 9.4 kb 23 kb 1 3 2 5 M M 6 7 8 11 12 13 17 18 10 16 9 14 4 15

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CHAPTER 4. SEQUENCE COMPARISON AND CHARACTERIZATION OF FIVE NEW pthA HOMOLOGS FROM FOUR DIFFERENT Xanthomonas citri STRAINS. Introduction All strains of Xanthomonas citri cause hyperplastic pustules in citrus that are dignostic of citrus canker disease (Gabriel, 2001). The Asiatic (A) group (X. citri pv citri A) has the widest host range and is widespread throughout the world. The B and C groups (X. citri pv aurantifolii B and C) have only been found in South America and have a reduced host range compared to the A groups (Stall and Seymour, 1983). New groups of X. citri pv citri (A w from Florida and A* from Southwest Asia) were more recently identified that are primarily restricted in host range to Mexican lime (Citrus aurantifolia) (Stall et al., 1982b; Sun et al., 2004). Grapefruit (C. paradisi) serves as a differential host that is resistant to the A*, A w and C strains; the A w and C strains elicit a strong hypersensitive response (HR) in grapefruit, while some A* strains show reduced growth in grapefruit (Chapter 2). The molecular basis for avirulence of the A*, A w and C strains in grapefruit and the wide host range of the A strains is unknown. Pathogenicity gene pthA encodes the primary causal effector of the citrus canker disease phenotype (Duan et al., 1999; Swarup et al., 1991; Swarup et al., 1992). All strains of X. citri tested carry pthA homologs (Cubero and Graham, 2002; Mavrodieva et al., 2004). pthA is capable of conferring ability to cause canker-like symptoms to strains that cannot otherwise cause canker, such as X. campestris pv citrumelo (Swarup et al., 1991) or even E. coli carrying a functional hrp system (Kanamori and Tsuyumu, 1998). 35

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36 When pthA is transiently expressed in citrus using either Agrobacterium tumefaciens or particle bombardment, small canker-like lesions are elicited (Duan et al., 1999). pthA is the first member of the avrBs3/pthA gene family demonstrated to function for pathogenicity. The vast majority of cloned or described Xanthomonas avirulence genes belong to this family; many have demonstrated pathogenicity functions (Leach and White, 1996). Members of this gene family show very high levels of homology at the DNA sequence level (De Feyter et al., 1993; Hopkins et al., 1992; Leach and White, 1996; Yang et al., 2000). All members encode more than 11 nearly perfect, 34 amino acid, leucine rich, tandemly arranged, direct repeats. Swapping repeat regions between members of the gene family results in chimeric genes that confer the pathogenicity and/or avirulence phenotypes expected from the source genes (Herbers et al., 1992; Yang et al., 1994). Although pthA can confer avirulence to other xanthamonads (Swarup et al., 1992), no pthA homolog from X. citri is known to function for avirulence in citrus. Conversely, although the pthA homolog aplI from X. citri pv citri group A has been suggested as a suppressor of the tobaco defense response (Ponciano et al., 2003), no pthA homolog from X. citri is known to suppress citrus host defenses. The purpose of this study was to clone, isolate, sequence and characterize pthA homologs that function to determine pathogenicity from all known X. citri groups. A secondary purpose was to determine if any of these pthA homologs also determined avirulence in grapefruit or could increase the pathogenicity of an A* strain in grapefruit. Material and Methods Bacterial Strains, Plasmids and Culture Media Strains of Escherichia coli, Xanthomonas spp. and plasmids used in this study are listed in Table 4-1 along with their relevant characteristics and source or reference. E.

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37 coli strains were grown in Luria-Broth (LB) medium at 37 C (Sambrook et al., 1989). Xanthomonas spp. were grown in PYGM (peptone yeast extract-glycerol-MOPS) medium at 30 C as described (Gabriel et al. 1989). Antibiotics were used at the following final concentrations (g/ml): rifampin (Rif), 75; spectinomycin (Sp), 35; chloramphenicol (Cm), 35; ampicillin (Ap), 100; gentamycin (Gm), 5; kanamycin (Kn), 25. Recombinant DNA Techniques Xanthomonas total DNA was prepared as described (Gabriel and De Feyter, 1992). Plasmids were isolated by alkaline lysis from E. coli (Sambrook et al., 1989) and Xanthomonas (De Feyter and Gabriel, 1991). Southern hybridization was performed by using nylon membranes as described by Lazo and Gabriel (1987). DNA Library Construction Genomic DNA from the wide host range Xanthomonas citri pv citri group A strain 3213 was partially digested with MboI and size fractioned on a sucrose gradient. The cosmid vector pUFR43 was used to make a DNA library of 3213 DNA fragments. This cosmid vector was split into two pools and cut with either EcoRI or SalI restriction enzyme to produce the two arms and treated with shrimp alkaline phosphatase. To create common cloning ends, the arms were then cut with BamHI and used for ligations to the 20 25kb 3213 DNA fraction. Recombinant DNA was packaged using Stratagene packaging mix (Gigapack III Gold Packaging Extract), and introduced into E. coli strain DH5 via transfection as described by the manufacturer protocol. Positive white plaques were then picked and placed onto LB plates containing the antibiotic Kn (20 g/l). Colonies were transferred into 96 well micro titer plates containing liquid LB

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38 (with 14% glycerol) and stored at C. DNA from eighteen randomly selected library clones was extracted, digested with BamHI and run on agarose gels in order to estimate insert size and evaluate the quality of the library. Plant Inoculations Duncan grapefruit and Mexican lime plants were grown, maintained and inoculated under natural light in the quarantine greenhouse facility at the Division of Plant Industry, Florida Department of Agriculture in Gainesville, Fl. Temperatures in this greenhouse ranged from 25C to 35 C with 50% to 100% relative humidity. Liquid cultures of tested Xanthomonas strains were grown in PYGM at 30 C for approximately 24 hr. Cultures were centrifuged @ 1000g for 3 min at room teperature, and resuspended in equal volumes of sterile tap water (saturated with CaCO 3 ) and pressure infiltrated at appropriate concentrations (10 5 for low and 10 8 cfu/ml for high) into the abaxial citrus leaf surface using the blunt end of a tuberculin syringe. Observations were taken 510 days after inoculation. Southern Hybridization Analysis Genomic DNA from all canker causing strains were isolated as described, digested with either EcoRI or BamHI restriction enzyme and the digested DNA was analysed by electrophoresis on 0.6% agarose gels. DNA was then transferred onto GeneScreen Plus (DuPont, Wilmington, Delaware) nylon membranes as described by the manufacturer. Membranes were hybridized with a 32 P-labeled BamHI internal fragment of pthA. Colony Hybridization Plasmid DNA from A W strain X0053 was digested with EcoRI and KpnI and ligated into shuttle vector pUFR047. Recombinant DNA was transformed into DH5 competent cells, and transformed clones were selected on Ap100 and X-Gal/IPTG in LB

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39 agar. White colonies were transferred onto a registry plate and pZit45 was included at specific positions as a control. Plasmid DNA from A* strain Xc270 was digested with EcoRI and HindIII and ligated into shuttle vector pUFR71. Recombinant DNA was transformed into DH5 and selected on Cm35 LB and X-Gal/IPTG plates. White colonies were transferred from registry plates onto Colony/PlaqueScreen hybridization transfer nylon membranes and placed colony side up on plain LB plates and incubated for 24 hr at 37 C. DNA was fixed on membranes as described by the manufacturer and hybridized with a 32 P-labeled BamHI fragment of pthA. Group B strain B69 plasmid DNA was digested with EcoRI, and a 23 kb and a 4.3 kb fragment that hybridized with pthA were cloned in pUFR53 resulting in pQY93.3 and pQY22.1, respectively. A 14kb HindIII fragment within the pQY93.3 EcoRI fragment was subcloned in pUFR53, resulting in pQY96. Group C strain C340 plasmid DNA was digested with SalI, and a 20 kb and a 6 kb fragment that hybridized with pthA were cloned into pUFR53, resulting in pQYC2.1 and pQYC1.1, respectively. The 6 kb insert from pQYC1.1 fragment was cloned into the high copy vector pUC119 resulting in pQY103.5. Triparental Matings Clones that hybridized to pthA were conjugated into Xanthomonas strain B21.2 (pthA::Tn5) by triparental mating as described by Defeyter et al (De Feyter et al., 1990). Strain pRK2073 was used as a helper strain. The recipient strain was concentrated 50 100 fold for higher conjugation rate. 10 l of each recipient, donor and helper were mixed together on PYGM plate and allowed to grow for 6 hr to overnight at 28 C. Transconjugants were screened on PYGM plates containing Sp 35 l g/ml and Gm 5l g/ml at 28 C. Two to three days later colonies were transferred onto new selection

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40 plates. Successful transconjugants were infiltrated into Duncan grapefruit and Mexican/Key lime. Southern blot analysis was used to further analyze clones. Marker Integration Mutagenesis The mutants BIM2 (pthB::pUFR004) of B69Sp and CIM1 (pthC::pUFR004) of C340 were created by the integration of pYY40.10 (2.0 kb internal StuI-HincII fragment of pthA in pUFR004), and Cm resistant colonies were selected. Sequence Analysis of pth Genes pthA homologs from A*, A w B and C strains were sequenced using primers based on the sequence of pthA (Swarup et al., 1992) and designed to cover the entire gene. Seven primers were used for sequencing reactions; DG8: gaggtggtcgttggtcaacgc, DG35: agttatctcgccctgatc, DP35: caggtcactgaagctgcccgc, DP36: gcgggcagcttcagtgacctg, DP37: ccgaaggttcgttcgaca, DP38:ctgtcgaacgaaccttcg, DP45: gcatggcgcaatgcactgac, and YP03: tagctccatcaaccatgc. Sequencing was done at the UF ICBR DNA Sequencing Core, Gainesville, FL. When necessary, fragments were cloned into high copy vectors such as pUC119 or pUC19 to obtain larger amounts of DNA. Sequence information of these genes was used to construct the full DNA sequence using Vector NTi software (Invitrogen, Carlsbad, California). Nucleotide and predicted amino acid sequence alignments were carried out with the program CLUSTAL W. Percent amino acid identity was calculated using the needle program in EMBOSS package which uses the Needleman-Wunsch algorithm to do global alignment of sequences. The DNA sequences of pthA1, pthA2, pthA3 and pthA4 (da Silva et al., 2002) were taken from GenBank Accessions # NC_003921, NC_003921, NC_003922 and NC_003922, respectively. The DNA sequences of apl1,apl2 and apl3 (Kanamori and

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41 Tsuyumu, 1998) were taken from GenBank Accessions # AB021363, AB021364 and AB021365, respectively. Dendograms showing phylogenetic relationships of these genes were generated with TREECON (version 1.3b) (Van de Peer and De Wachter, 1994) using neighbor-joining algorithm with Poisson correction. RSc1815, an avrBs3/pthA gene from Ralstonia solanacearum was used as an outgroup for phylogenetic tree construction. The percentage of trees from 100 bootstrap resamples supporting the topology is indicated when the percentage is above 70. Results Southern Blot Analysis Southern blot analyses revealed that all tested X. citri strains have at least two BamHI DNA fragments that strongly hybridized to an internal BamHI fragment from pthA (Figure 4-1a; some data not shown). With the exception of group A strains, which had four BamHI fragments that hybridized with pthA, all other strains from all other groups, including the A*, A w B and C groups, had only two such BamH1 fragments. All strains tested appeared to share a 3.4 kb BamHI hybridizing fragment of a size similar or identical to that of pthA. Otherwise, each of the different phenotypic groups exhibited distinct and characteristic banding patterns. Based on the hybridization intensity of both BamHI and EcoRI digested DNA fragments and other results (not shown), the C and A w strains appeared to carry their two hybridizing DNA fragments on a single plasmid (Figure 4-1). Cloning, Characterization and Sequencing of pthA Homologs from X. citri A*, A w B and C Strains Using an internal fragment of pthA as a probe, colony hybridization of E. coli carrying cloned group A w strain X0053 plasmid DNA revealed eight colonies with

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42 hybridizing inserts (Figure 4-2). The plasmids from these colonies were designated as pAW5.1 5.8, and all carried hybridizing inserts of identical size (Table 4-1). Four of these inserts were separately introduced into strain B21.2 (pthA::Tn5) and screened for pathogenicity. All four clones complemented the knockout phenotype of B21.2 and restored ability to cause canker in both Duncan grapefruit and Mexican lime (Figure 4-3; Table 4-2). The pthA homolog encoded on pAW5.2 was sequenced and designated pthAW. Similarly, colony hybridization of cloned group A* strain Xc270 plasmid DNA revealed three hybridizing clones, designated as pAW12.1 12.3. The inserts carried on pAW12.1 and 12.2 were identical in size; pAW12.3 was smaller. When transferred to B21.2, pAW12.1 complemented B21.2 and resulted in canker symptoms in both grapefruit and lime (Figure 4-4; Table 4-2). The pthA homolog encoded on pAW12.1 was sequenced and designated pthA*. pAW12.3 did not complement B21.2 in either host (Table 4-2). The pthA homolog from pAW12.3 was sequenced and designated pthA*-2. pthA*-2 carried only 15.5 internal repeats. To verify that the lack of evident activity of pthA*-2 was not due to a cloning artifact, the promoter region and Shine-Dalgarno (SD) sequence were verified to be present on pAW12.3. In addition, no premature stop codons or frame shifts were found in pthA*-2. Colony hybridization of cloned group B strain B69 plasmid DNA revealed several hybridizing clones of two different sizes. Representative clones of both sizes were selected for complementation tests. pQY93.3 (23 kb insert) and pQY22.1 (4.3 kb insert) were mobilized by conjugation into B21.2; only pQY93.3 was found to complement B21.2, resulting in canker symptoms in both grapefruit and lime (Table 4-2). The pthA

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43 homolog was subcloned from pQY93.3 on pQY96, verified as functional in B21.2 designated as pthB and sequenced. Finally, colony hybridization of cloned group C strain C340 plasmid DNA revealed several hybridizing clones of two different sizes, and again representative clones of both sizes were selected for complementation tests. pQYC2.1 (20 kb insert) and pQYC1.1 (6 kb) were mobilized by conjugation into B21.2; only pQYC1.1 was found to complement B21.2, resulting in canker symptoms in both grapefruit and lime (Table 4-2). The pthA homolog encoded on pQYC1.1 was designated as pthC and sequenced. Even when inoculated at high concentrations, none of the pthA homologs (pthAW, pthA*, pthA*-2, pthB or pthC) in B21.2 elicited an HR in grapefruit. Inactivation and Complementation of Genes pthB and pthC in X. citri pv aurantifolii In order to determine the role of pthB in the pathogenicity of X. citri pv aurantifolii group B strain B69Sp in citrus, marker integration mutagenesis was carried out. Southern blot analysis showed that pthB had been interrupted in BIM2 (pthB::pUFR004). BIM2 was unable to cause canker (data not shown). BIM2 was fully complemented by pAB2.1, pZit45, pAB18.1 (all carrying pthA), pQY96 (carrying pthB) and pQYC1.1 (carrying pthC) to elicit wild type response in grapefruit and lime (data not shown). In order to determine the role of gene pthC in the pathogenicity of group C strain C340 in citrus, marker integration mutagenesis was carried out. Southern blot analysis showed that pthC had been interrupted in CIM1 (pthC::pUFR004). CIM1 was unable to cause typical canker symptom in lime, but elicited an HR in grapefruit that was as strong as the HR elicited by the wild type strain C340. CIM1 was fully complemented by pZit45 (pthA), pQY96 (pthB) and pQYC1.1 (pthC) to elicit a wild type response in lime (data not shown).

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44 None of the pthA Homologs from Group A Strain 3213 Increased the Host Range of Group A* Strain 270 to Include Grapefruit All four pthA homologs from group A strain 3213 were isolated and cloned from the 3213 library by colony hybridization with an internal fragment of pthA: pAW20.2, pAW20.4, pAW20.7 and pAW20.11 carry pthA, pthA1, pthA2 and pthA3, respectively (Figure 4-5). None of these clones complemented B21.2. When these clones were conjugated into the A* strain Xc270, none extended the host range of the strain to include Duncan grapefruit. As with Xc270, all three transconjugants elicited cankers in Mexican lime. When pZit45, which carries pthA from 3213 and complements B21.2 (Swarup et al., 1992), and pAW20.2 were introduced into Xc270, they similarly did not extend the host range of Xc270 to include grapefruit (Figure 4-5). Sequence Analysis of pthA Homologs from All Known X. citri Groups The DNA sequences of all 13 available pthA homologs were analyzed and the predicted amino acid sequences were found to be >75% identical (Table 4-3). With the notable exception of Apl3, all seven other PthA homologs within X. citri pv citri group A (PthA, PthA1, PthA2, PthA3, PthA4, Apl1 and Apl2) were more closely related to each other (> 92% identical), than the active PthA homologs from all X. citri groups (PthA, PthB, PthC, PthAW and PthA*) which were >97% identical (Figure 4-6). Comparative analysis of the 34 aa direct repeat regions of all thirteen genes revealed three primary regions of variation within each repeat, at positions 3 and 4 (region 1), positions 11-13 (region 2) and positions 30-32 (region 3) (Figure 4-7). In region 1, no particular set of amino acids was universally conserved among any of the repeats of active pthA homologs. However, in regions 2 and 3, and only in repeat number 17 in each gene,

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45 N(12)G(13) in region 2 and Q(31)A(32) in region 3 were correlated with active pathogenicity gene function. Discussion Southern hybridization analyses of a limited number of X. citri strains revealed a common 3.4 kb BamHI band shared by all strains examined in all five described groups of strains; all group A strains tested carried four hybridizing fragments, while all other strains examined carried only two. Among the 13 sequenced and functionally tested pthA homologs, including three tested by others [Apl1, Apl2 and Apl3; (Kanamori and Tsuyumu, 1998)] and the ten tested in this study, only the 3.4 kb fragment appeared to encode the active pathogenicity gene that is required for elicitation of citrus canker. This includes genes pthA*, pthAW, pthB and pthC from the A*, A w B and C strains, respectively, as well as pthA. All five of these genes were found to be fully isofunctional, and capable of eliciting the typical canker phenotype in grapefruit in B21.2, even though the source A*, A w and C strains were unable to elicit the canker phenotype in grapefruit. Furthermore, pthA*, pthAW and pthC did not elicit an avirulence phenotype of any type in B21.2, despite being members of an avr gene family, and despite the avirulence of the respective source strains in grapefruit. Indeed, the pthC knockout mutation in CIM1 eliminated pathogenicity in lime, but did not affect the HR in grapefruit, which remained as strong as that elicited by the wild type. The HR elicited by the wild type C group strain C340 is therefore independent of pthC. These results suggest that the A*, A w and C strains likely carry yet to be identified avr genes that prevent compatible phenotypes from developing in grapefruit. The C strain C340 and A* strain Xc270 fragments that hybridized with pthA did not complement B21.2 to pathogenicity in lime or grapefruit. The sequenced Xc270

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46 homolog that failed to complement, pthA*-2, carried 15.5 repeats and appeared to have intact promoter, a SD region and an open reading frame. This gene was 97% identical to PthA2 and Apl2 (Table 4-3) and carried the same number of repeats. All three of these genes appear intact and yet also appear non-functional in terms of pathogenicity or avirulence. Although the C340 homolog (on pQYC2.1) that did not complement B21.2 was not sequenced, restriction enzyme analysis (not shown) of the 20 kb insert indicated that the promoter region was intact, making this homolog unlikely to be responsible for avirulence in grapefruit. The other three group A 3213 pthA homologs did not complement B21.2 and also appeared to be non-functional, confirming and extending the work of Kanamori and Tsuyumu (Kanamori and Tsuyumu, 1998) on group A strain L-9. However, the fact that all wide host range group A strains examined carry two additional pthA homologs that are not present in the more narrow host range B, C, A* and A w strains suggests a potential role in determining host range. Indeed, Ponciano et al (2003) reported that apl1, a pthA homolog that is functionally equivalent to pthA but found in a different group A strain, suppressed tobacco defense response and HR. However, when pthA or any of its 3213 homologs (pthA1, pthA2 or pthA3) were transferred into Xc270, no increase in host range of A* strain Xc270 to include grapefruit was observed (Figure 4-5). Although, additional pthA homologs in a given X. citri strain may contribute marginally to pathogenicity (Kanamori and Tsuyumu, 1998), the primary value of multiple copies of the gene family in a given strain may be to facilitate recombination and the potential for rapid adaptation to new hosts (Gabriel, 1999a; Yang and Gabriel, 1995).

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47 All pthA homologs that are required for citrus canker disease from all five known X. citri groups carried exactly 17.5 repeats. All other homologs, even those nearly identical to pthA (e.g., from X. citri pv citri group A) were not required for canker and had a different number of repeats. Interestingly, deletion mutants of various repeats and numbers of repeats in pthA can result in a gene that confers a weak canker phenotype in citrus to B21.2 (Yang and Gabriel, 1995). In that study, however, repeat numbers 1-5 and 16,17 were not affected in deletion derivatives capable of conferring canker. This indicates that while the total number of repeats may be important, the number of repeats may be less important than the relative location of the specific repeats within the gene. Surprisingly, sequence variation among these active pthA genes (PthA, PthAW, PthA*, PthB and PthC) was greater than variation among the pthA homologs within the A group. Even the nonfunctional homologs were closer to the active genes within the A group than to active homologs from B and C groups. The relatively high level of variation within the active homologs from different phylogenetic groups allowed the possibility of identifying amino acids within the 34 aa direct repeat that might be critical for pathogenic specificity in citrus. Three somewhat variable regions were found in each of the repeats, at amino acid positions 3 and 4, 11-13, 30-32. The aligned repeat regions of all active genes revealed that only amino acids N(12)G(13) in the second and Q(31)A(32) in the third variable regions of the 17 th repeat were conserved. No such conservation of identical amino acids was found in any other repeat (Figure 4-6). Interestingly, only the 17 th repeat of the South American group B and C strains show a sequence identity to Asiatic strains in the third variable region. Q(31),A(32) is not seen in any other PthB repeat and in only two other PthC repeats,

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48 which favor E(31)Q(32) at that position. In addition, the deletion mutants evaluated by Yang and Gabriel (Yang and Gabriel, 1995) never affected the 17 th repeat. These results suggest that the 17th repeat may be critical for pathogenicity of X. citri.

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49 Table 4-1. Strains and plasmids used in this study Strain or plasmid Relevant Characteristics Reference or source Escherichia coli DH5 F endA1, hsdR17(r k m k ), supE44, thi-1, recA1 Gibco-BRL Xanthomonas citri 3213 Group A, wild type Gabriel et al. 1989 3213Sp Spontaneous Sp r derivative 3213, Sp r Gabriel et al. 1989 B21.2 pthA::Tn5-gusA, marker exchanged mutant of 3213Sp, Sp r Kn r Swarup et al. 1991 B69 Group B, wild type Stall et al. 1982 B69Sp Spontaneous Sp r derivative of B69, Sp r C340 Group C, wild type Stall et al. 1982 Xc205 Group A*, wild type Verniere et al. 1989 Xc205 Spontaneous Rif r derivative of Xc205, Rif r This study Xc270 Group A*, wild type Verniere et al. 1989 Xc270Rif Spontaneous Rif r derivative of Xc270, Rif r This study Xc280 Group A*, wild type Verniere et al. 1989 Xc290 Group A*, wild type Verniere et al. 1989 Xc322 Group A*, wild type Verniere et al. 1989 Xc406 Group A*, wild type Verniere et al. 1989 X0053 Group A w wild type Sun et al. 2004 X0053Rif Spontaneous Rif r derivative of X0053, Rif r This study BIM2 pthB::pUFR004, marker integrated mutant of B69Sp El-Yacoobi, 2005 CIM1 pthC::pUFR004, marker integrated mutant of C340 This study Plasmids pRK2013 ColE1, Km r ,Tra + helper plasmid Figurski and Helinski 1979 pRK2073 pRK2013 derivative, npt::Tn7, Km s Sp r Tra + helper plasmid Leong et al. 1982 pUC119 ColE1, M13 lg, Ap r lacZ + Vieira and Messing, 1987 pUFR004 ColE1, Mob + Cm r lacZ + De Feyter et al. 1990 pUFR043 IncW, Mob + lacZ + Gm r Nm r cos, shuttle vector De Feyter and Gabriel, 1991 pUFR047 IncW, Mob + lacZ + Par + Gm r Ap r De Feyter et al. 1993

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50 Table 4-1. Continued. Strain or plasmid Relevant Characteristics Reference or source pUFR053 IncW, Gm r ,Cm r Mob + mob(P), lacZ + Par + El-Yacoobi, 2005 pUFR071 IncW, Mob + Cm r Gm r lacZ + Par + Castaneda, 2005 pYD9.3 pthA in pUC118, Ap r Duan et al. 1999 pZit45 4.5Kb fragment containing pthA from 3213 cloned in pUFR47, Ap r Swarup et al. 1992 pAB2.1 EcoRI/HindIII fragment of pZit45, containing pthA, in pLAFR3 This study pAB18.1 EcoRI/HindIII fragment of pYD9.3, containing pthA, in pUFR47 This study pQY93.3 23 kb EcoRI fragment containing pthB in pUFR53 This study pQY22.1 4.3 kb EcoRI fragment containing pthB 0 (non-functional) in pUFR53 This study pQY99.3 8.8 Kb SalI fragment containing pthB from B69 was cloned in pUC119 This study pQY96 14 kb HindIII fragment containing pthB cloned in pUFR53 This study pQY103.5 5 Kb SalI fragment containing pthC from C340 cloned in pUC119 This study pQYC1.1 6 kb SalI fragment containing pthC cloned in pUFR47 This study pQYC2.1 20 kb SalI fragment containing pthC 0 (non-functional) cloned in pUFR47 This study pAW5.15.8 5Kb EcoRI-KpnI fragment containing pthAW from X0053 cloned in pUFR47 This study pAW12.1-12.2 22 kb EcoRI/HindIII fragment containing pthA* from A* group strain Xc270 cloned in pUFR71 This study pAW12.3 6 kb EcoRI/HindIII fragment containing pthA*-2 from A* group strain Xc270 cloned in pUFR71 This study pAW20.2 36 kb MboI fragment containing pthA from 3213 cloned in pUFR43 This study pAW20.4 17 kb MboI fragment containing pthA1 homolog from 3213 cloned in pUFR43 This study pAW20.7 32 kb MboI fragment containing pthA2 homolog from 3213 cloned in pUFR43 This study pAW20.11 40 kb MboI fragment containing pthA3 homolog from 3213 cloned in pUFR43 This study

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51 Table 4-2. Phenotypic responses of X. citri strains in 2 citrus hosts Mexican Lime Grapefruit Strains/Plasmid Low a High b Low High 3213 + c + + + B21.2 0 d 0 0 0 B21.2/pZit45( pthA) + + + + B21.2/pQY96( pthB ) + + + + B21.2/pQYC1.1( pthC ) + + + + B21.2/pAW5.2( pthAW ) + + + + B21.2/ pAW12.1( pthA*) + + + + B21.2/ pAW12.3( pthA*2) 0 0 0 0 a=10 4 -10 5 cfu/ml, b=10 8 -10 9 cfu/ml, c= canker, d= no canker,

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52 Table 4-3. Amino acid sequence identity between pathogenicity genes from X. citri strains PthA PthA4 Apl1 PthAW PthA* PthA*-2 PthA1 PthA2 PthA3 Apl2 Apl3 PthB PthC PthA 100 100 100 99 98 92 95 93 92 94 84 87 87 PthA4 100 100 99 98 92 95 93 92 94 84 87 87 Apl1 100 99 98 92 95 93 92 94 84 87 87 PthAW 100 97 92 95 93 92 93 84 87 87 PthA* 100 92 95 93 92 93 84 87 87 PthA*-2 100 95 97 97 97 79 82 83 PthA1 100 95 95 95 81 84 85 PthA2 100 98 99 79 82 82 PthA3 100 98 79 82 82 Apl2 100 80 82 82 Apl3 100 75 75 PthB 100 98 PthC 100

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53 Figure 4-1. Southern Hybridization analysis of X. citri strains hybridized with the BamHI internal fragment of pthA. A). BamHI restriction digested genomic DNA from X. citri strains. B). EcoRI restriction digested genomic DNA.

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54 + control Hybridizing colonies Figure 4-2. Colony Hybridization of E. coli with cloned X0053 A w plasmid DNA fragments using 32 P-labeled pthA. pZit45 (pthA) was used as a positive control.

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55 3213 B21.2/ pAW5.2 B21.2/ pAW5.4 B21.2/ pAW5.5B21.2/ pAW5.8B21.2B21.2B21.2/ pAW5.5B21.2/ pAW5.83213B21.2/ pAW5.2B21.2/ pAW5.4GF KL Figure 4-3. Complementation of A strain knockout B21.2 (pthA::Tn5) with pthA homologs from A w strain X0053 in citrus. pAW5.2, pAW5.4, pAW5.5 and pAW5.8 carry fragments that hybridized with pthA in grapefruit (left) and Key lime (right).

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56 Figure 4-4. Complementation of A strain knockout B21.2 (pthA::Tn5) with pthA homologs in citrus. pthAW (pAW5.2), pthA* (pAW12.1) and pthA*-2 (pAW12.3) in B21.2 and pthA (3213) in grapefruit (left) and Key lime (right).

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57 Figure 4-5. Analysis of pthA and its three homologs in A* strain Xc270. Key lime (left) and grapefruit (right)

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58 Figure 4-6. Neighbor-joining dengogram depicting phylogenetic relastionship based on pairwise comparison of neucleotide sequences of members of avrBs3/pthA genes from different species and pathovars of Xanthomonas. Numbers at the nodes represent bootstrap values (based on 100 replicates). GenBank Accesions numbers are presented to the right of the gene for genes not mentioned in material and methods.

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59 PthA3 CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA Apl2 CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA Apl3 CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA Figure 4-7. Sequence alignment of the predicted amino acids encoded in the main variable portion of the repeat region of all 13 pthA homologs. Boxed areas indicate the regions in the 17 th repeat that are conserved among all pthA homologs with experimental evidence of active pathogenic function. PthA*-2 PE PG PE PA PA PE PE PE PE PA PE PE PE PE PA PthA1 PE PD PA PA PA PE PE PE PA PE PE PE PE PA PE PE PthA2 PE PE PE PE PE PD PD PQ PE PE PE PE LD PE PE PthA3 PE PE PE PE PE PE PE PD PD PD PD PE PE PE PE Apl2 PE PE PE PE PE PE PD PQ PE PE PE PE LD PE PE Apl3 PE PE PE PE PE PE PD PQ PE PE PE PE PE PE PE PE PD PE PE PE PE PE PE PthA SNI SNG SNI SNI SNI SNG SHD SNG SHD SNG SNG SNG SNG SNS SHD SHD CNG PthA4 SNI SNG SNI SNI SNI SHD SHD SNG SHD SNG SNG SNG SNG SNS SHD SHD CNG Apl1 SNI SNG SNI SNI SNI SHD SHD SNG SHD SNG SNG SNG SNG SNS SHD SHD CNG PthA* SNI SNG SNI SHD SNI SHD SHD SNG SHD SNG SNG SNG SNS SHD SNS CNG SNG PthAW SNI SNG SNG SNG SNS SHD SHD SNS SHD SNG SNC SNG SNG SNS SHD SHD CNG PthB SHD SNG SHD SNG SNI SNG SHD SNG SHD SNI SNI SHD SHD SHD SHD SNG SNG Veriable region II PthC SHD SNG SHD SHD SNI SNG SNI SNG SNI SNI SHD SNG SHD SHD SHD SNG SNG PthA*-2 SNI SNI SNSNI SNSNI SHD SHD SNG SNI SHD SNI SHD SHD SHD PthA1 SNI SNG SNI SNSNI SHD SHD SNSNI SHD SNI SHD SNSNI SHD SHD PthA2 SNI SHD SNI SHD SNI SHD SHD SNG SHD SNG SNG SNG SNG SNI SNI PthA3 SNI SHD SNI SHD SNI SNG SHD SNG SNG SNG SNI SNI SNI SNI SHD Apl2 SNI SNG SNI SHD SNI SHD SHD SNG SHD SNG SNG SNG SNG SNI SHD Apl3 SNI SHD SNI SHD SNI SHD SHD SNG SHD SHD SNG SNI SHD SHD SNI SHD SHD SNG SHD SNG SNI SHD CNG PthA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA PthA4 CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA Apl1 CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA PthA* CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA PthAW CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA PthB CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ RQA Veriable region III PthC CEQ CEQ CEQ CEQ CEQ CEQ CEQ CEQ CQA CEQ CEQ CEQ CEQ RQA CEQ CEQ RQA PthA*-2 CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA PthA1 CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA PthA2 CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA CQA Repeat 1 2 3 45678910111213141516 17 181920212223 Gene PthA PE PE PE PE PA PE PD PQ PE PE PE PE LD PE PD PE PE PthA4 PE PE PE PE PA PE PD PE PE PE PE PE LD PE PD PE PE Apl1 PE PE PE PE PA PE PE PE PE PE PE PE LD PE PD PE PE PthA* PE PQ PE PE PA PE PD PQ PE PE PE LD PE PD PE PE PE PthAW PE PE PE LD PE PE PE PE PE PE PE PE LD PE PD PE PE Veriable region I PthB PD PA PD PA PD PA PD PA PD PD PD PD PD PD PD PD PD PthC PD PD PD PD PD PD PD PA PD PD PD PD PD PD PA PD PD

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CHAPTER 5 SUMMARY AND CONCLUSION The main objective of this dissertation was to study host range determination factors among all described Xanthomonas citri groups that are known world-wide. Five variant groups of X. citri have been described in the literature, and all are known from field observations to differ in host range and/or pathogenicity. In this study, all known groups were studied together in lime, grapefruit and sweet orange. All groups were readily distinguished by inoculation of only two host differentials, lime and grapefruit. The in planta growth of strains from two different groups that did not elicit an obvious defense response in grapefruit was found to be poor. This indicated that either these strains carry negative acting (avirulence) factors that limited growth in grapefruit, or that they are missing positive acting (pathogenicity) factors that are present in strains from groups that can attack grapefruit. The lack of a grapefruit defense response that is typical of bacterial infections limited by avirulence factors led to an attempt to identify positive pathogenicity factors. A DNA library of an X. citri strain able to attack grapefruit was moved into one of the strains unable to attack grapefruit in an attempt to identify one or more positive acting host range factors. Despite using a DNA library that theoretically covered the wide host range X. citri genome with 99% probability, no pathogenicity factors were found, despite multiple screens of all library clones. It is possible that in planta growth requires multiple effectors, and that no individual cosmid would carry enough factors to reveal a strong difference. Another possibility is that Xc270 may carry avr genes that function in 60

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61 grapefruit and prevent Xc270 from growing. Avirulence is usually epistatic over virulence and therefore a screen for positive factors would fail if this were the case. In addition to the DNA library screen, particular attention was paid to the pthA homologs from all five X. citri strain groups, since pthA is known to be required by at least three strain groups for citrus canker disease. In this study, pthA was demonstrated to be required by the remaining strain groups. The fact that all wide host range group A strains examined carried two additional pthA homologs that were not present in the narrow host range B, C, A* and A w strain groups suggested a potential role for these additional homologs in determining host range. However, when pthA or any of its group A homologs (pthA1, pthA2 or pthA3) were transferred into a narrow host range group (A*) strain, no increase in host range to include grapefruit was observed. Three new pthA homologs were cloned, isolated and sequenced from strains of group A* (pthA* and pthA*-2) and A w (pthAW) and functionally compared with pthA homologs previously isolated from strains of the three remaining groups: A (pthA), B (pthB) and C (pthC). pthA*, pthAW, pthB and pthC were found to be fully isofunctional with pthA, and capable of eliciting the typical canker phenotype in grapefruit in complementation tests using an X. citri group A pthAmutant strain (B21.2), even though the source A*, A w and C strains were unable to elicit the canker phenotype in grapefruit. Furthermore, pthA*, pthAW pthA* and pthC did not elicit an avirulence phenotype of any type in B21.2, despite the fact that pthA homologs are all members of an avirulence gene family, and despite the avirulence of the respective source strains in grapefruit. DNA sequence comparisons of the three new pthA homologs cloned, sequenced and characterized in this study with ten previously sequenced pthA homologs revealed

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62 that all functional pthA homologs (i.e., those that are required for citrus canker disease in their respective strains) from all five known X. citri groups carried exactly 17.5, 102bp direct tandem repeats. All other homologs that are not functional for citrus canker pathogenicity carried a different number of repeats. Phylogenetic comparisons of the DNA and predicted protein sequences of the thirteen available pthA homologs revealed the same phylogenetic distinctions that are found by more general phylogenetic studies. In addition, comparisons of the five functional pthA homologs from each group against those that were nonfunctional revealed that amino acids N(12)G(13) in the second and Q(31)A(32) in the third variable regions of the 17 th direct tandem repeat were only conserved in functional genes. These results suggest that the 17th repeat plays a critical role in citrus canker pathogenicity and may help explain the origination of new citrus canker strains.

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APPENDIX A SEQUENCE OF pthC DNA sequence of pthC: atggatcccattcgtccgcgcacgtcaagtcctgcccacgaacttttggccggaccccagccggatagggttcagccgcagccgactgcagatcgtgggggggctccgcctgctggcagccccctggatggcttgcccgctcgacggacgatgtcccgaacccgtctcccgtctccccctgcccccttgcctgcgttctcagcgggcagtttcagcgatctgctctgtcagttcgatccgttgcttcttgacacattgctttttgattcgatgtctgccttcggcgctcctcatacagaggctgccccaggagaggcggatgaagtgcaatcgggtctgcgtgcagtcgatgacccgcaccccaccgtgcacgtcgctgtgacggccgcgcgaccgccgcgcgccaagccggcgccgcgacggcgtgctgcgcacacctctgacgcttcgccggccgggcaggttgatctatgcacgctcggctacagccagcagcagcaagacgagatcaaaccgaaggcgcgtgcgacagtggcgcagcaccaccaggcactgatgggccatgggtttacacgtgcgcacatcgttgcgctcagccaacacccggcagccttggggaccgtcgctgtcaagtaccaggccatgatcgcggcgttgccggaggcgacacacgaagacatcgttggcgtcggcaaacagtggtccggcgcacgcgccctggaagcattgctcacggtgtcgggagagttgagaggtccaccgttacagttggacacaggtcaacttctcaagattgcaaaacgtggcggcgtgaccgcggtggaggcagtgcatgcatggcgcaatgcactgacgggcgctcccctgaacctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagcaatggcggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagcaatattggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagcaatggcggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagcaatattggcggcaagcaggcgctggagacggtgcagcgg 63

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64 ctgttgccggtgctgtgcgagcaacatggcctgaccccggcgcaggtggtggccatcgccagcaatggcggcggcaagcaggcgctggaaacggtgcagcagctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagcaatattggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcacatggcctgaccccggaccaggtggtggccatcgccagcaatattggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggttgccatcgccagcaatggcggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgcgccaggcacatggcctgaccccggcgcaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagcaatggcggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagcaatggcggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgcgccaggcacatggcctgaccccggcgcaggtggtggccatcgccagcaatggcggcggcaggccggcactggagagcatttttgcccagttatctcgccctgatcaggcgttggccgcgttgaccaacgaccacctcgtcgccttggcctgcctcggcgggcgtcctgcgctggaggcagtgaaaaagggattgccgcacgcgccgaccttgatcaaaagaaccaatcgccgtcttcccgaacgcacgtcccatcgcgttgccgaccacgcgcaagtggctcgcgtgctgggttttttccagtgccactcccacccagcgcaagcatttgatgaagccatgacgcagttcgggatgagcaggcacgggttgttacagctatttcgcagagtgggcgtcaccgaactcgaggcccgcggtggaacgctccccccagccccgcagcgttggcaccgtatcctccaggcatcagggatgaaaagggccgaaccgtccggtgcttcggctcaaacgccggaccaggcgtctttgcatgcattcgccgatgcgctggagcgtgagctggatgcgcccagcccaatagaccaagcaggccaggcgctggcaagcagcagccgtaaacggtcccgatcggagagttctgtcaccggctccttcgcacagcaagctgtcgaggtgcgcgttcccgaacagcgcgatgcgctgcatttaccccccctcagctggggtgtaaaacgcccgcgtaccaggatcgggggcgg

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65 cctcccggatcctggtacccccatggacgccgacctggcagcgtccagcaccgtgatgtgggaacaagatgcggaccccttcgcaggggcagcggatgatttcccggcattcaacgaagaggagatggcatggttgatggagctatttcctcagtga Predicted amino acid sequence of pthC: MDPIRPRTSSPAHELLAGPQPDRVQPQPTADRGGAPPAGSPLDGLPARRTMSRTRLPSPPAPLPAFSAGSFSDLLCQFDPLLLDTLLFDSMSAFGAPHTEAAPGEADEVQSGLRAVDDPHPTVHVAVTAARPPRAKPAPRRRAAHTSDASPAGQVDLCTLGYSQQQQDEIKPKARATVAQHHQALMGHGFTRAHIVALSQHPAALGTVAVKYQAMIAALPEATHEDIVGVGKQWSGARALEALLTVSGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNIGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNIGGKQALETVQRLLPVLCEQHGLTPAQVVAIASNGGGKQALETVQQLLPVLCEQHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLRQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNGGGKQALETVQRLLPVLRQAHGLTPAQVVAIASNGGGRPALESIFAQLSRPDQALAALTNDHLVALACLGGRPALEAVKKGLPHAPTLIKRTNRRLPERTSHRVADHAQVARVLGFFQCHSHPAQAFDEAMTQFGMSRHGLLQLFRRVGVTELEARGGTLPPAPQRWHRILQASGMKRAEPSGASAQTPDQASLHAFADALERELDAPSPIDQAGQALASSSRKRSRSESSVTGSFAQQAVEVRVPEQRDALHLPPLSWGVKRPRTRIGGGLPDPGTPMDADLAASSTVMWEQDADPFAGAADDFPAFNEEEMAWLMELFPQ

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APPENDIX B SEQUENCE OF pthAW DNA sequence of pthAW: atggatcccattcgttcgcgcacaccaagtcctgcccgcgagcttctgcccggcccccaaccggatagggttcagccgactgcagatcgtggggtgtctccgcctgccggcggccccctggatggcttgcccgctcggcggacgatgtcccggacccggctgccatctccccctgccccctcacctgcgttctcggcgggcagcttcagtgacctgttacgtcagttcgatccgtcactttttaatacatcgctttttgattcattgcctcccttcggcgctcaccatacagaggctgccacaggcgagtgggatgaggtgcaatcgggtctgcgggcagccgacgcccccccacccaccatgcgcgtggctgtcactgccgcgcggccgccgcgcgccaagccggcgccgcgacgacgtgctgcgcaaccctccgacgcttcgccggccgcgcaggtggatctacgcacgctcggctacagccagcagcaacaggagaagatcaaaccgaaggttcgttcgacagtggcgcagcaccacgaggcactggtcggccatgggtttacacacgcgcacatcgttgcgctcagccaacacccggcagcgttagggaccgtcgctgtcaagtatcaggacatgatcgcagcgttgccagaggcgacacacgaagcgatcgttggcgtcggcaaacagtggtccggcgcacgcgctctggaggccttgctcacggtggcgggagagttgagaggtccaccgttacagttggacacaggccaacttctcaagattgcaaaacgtggcggcgtgaccgcagtggaggcagtgcatgcatggcgcaatgcactgacgggtgcccccctgaacctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcaggcgctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccctggaccaggtcgtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatagcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgtt 66

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67 gccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatagcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagcaatgcgggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaattgcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccctggaccaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatagcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgcctgcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggcggcaggccggcgctggagagcattgttgcccagttatctcgccctgatccggcgttggccgcgttgaccaacgaccacctcgtcgccttggcctgcctcggcggacgtcctgcgctggatgcagtgaaaaagggattgccgcacgcgccggccttgatcaaaagaaccaatcgccgtattcccgaacgcacatcccatcgcgttgccgaccacgcgcaagtggttcgcgtgctgggttttttccagtgccactcccacccagcgcaagcatttgatgacgccatgacgcagttcgggatgagcaggcacgggttgttacagctctttcgcagagtgggcgtcaccgaactcgaagcccgcagtggaacgctccccccagcctcgcagcgttgggaccgtatcctccaggcatcagggatgaaaagggccaaaccgtcccctacttcaactcaaacgccggaccaggcgtctttgcatgcattcgccgattcgctggagcgtgaccttgatgcgcccagcccaacgcacgagggagatcagaggcgggcaagcagccgtaaacggtcccgatcggatcgtgctgtcaccggtccctccgcacagcaatcgttcgaggtgcgcgttcccgaacagcgcgatgcgctgcatttgcccctcagttggagggtaaaacgcccgcgtaccagtatcgggggcggcctcccggatcctgg

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68 tacgcccacggctgccgacctggcagcgtccagcaccgtgatgcgggaacaagatgaggaccccttcgcaggggcagcggatgatttcccggcattcaacgaagaggagctcgcatggttgatggagctattgcctcagtga Predicted amino acid sequence of PthAW: MDPIRSRTPSPARELLPGPQPDRVQPTADRGVSPPAGGPLDGLPARRTMSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNCGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIACNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGP

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69 SAQQSFEVRVPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQ

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APPENDIX C SEQUENCE OF pthA* DNA sequence of pthA* atgcggcctcggaagctatgtaggaaccacagaccgctagtctggaggcgaccatgtaaagaggtatgcctgatggatcccattcgttcgcgcacaccaagtcctgcccgcgagcttctgcccggaccccaacccgatggggttcagccgactgcagatcgtggggtgtctccgcctgccggcggccccctggatggcttgcccgctcggcggacgatgtcccggacccggctgccatctccccctgccccctcacctgcgttctcggcgggcagcttcagtgacctgttacgtcagttcgatccgtcactttttaatacatcgctttttgattcattgcctcccttcggcgctcaccatacagaggctgccacaggcgagtgggatgaggtgcaatcgggtctgcgggcagccgacgcccccccacccaccatgcgcgtggctgtcactgccgcgcggccgccgcgcgccaagccggcgccgcgacgacgtgctgcgcaaccctccgacgcttcgccggccgcgcaggtggatctacgcacgctcggctacagccagcagcaacaggagaagatcaaaccgaaggttcgttcgacagtggcgcagcaccacgaggcactggtcggccatgggtttacacacgcgcacatcgttgcgctcagccaacacccggcagcgttagggaccgtcgctgtcaagtatcaggacatgatcgcagcgttgccagaggcgacacacgaagcgatcgttggcgtcggcaaacagtggtccggcgcacgcgccctggaggccttgctcacggtggcgggagagttgagaggtccaccgttacagttggacacaggccaacttctcaagattgcaaaacgtggcggcgtgaccgcagtggaggcagtgcatgcatggcgcaatgcactgacgggtgcccccctgaacctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccgcagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggcacaggtggtggccatcgccagcaatattggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccg 70

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71 gaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccgcagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccctggaccaggtcgtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatagcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatagcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgcctgcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggcggcaggccggcgctggagagcattgttgcccagttatctcgccctgatccggcgttggccgcgttgaccaacgaccacctcgtcgccttggcctgcctcggcggacgtcctgcgctggatgcagtgaaaaagggattgccgcacgcgccggccttgatcaaaagaaccaatcgccgtattcccgaacgcacatcccatcgcgttgccgaccacgcgcaagtggttcgcgtgctgggttttttccagtgccactcccacccagcgcaagcatttgatgacgccatgatgcagttcgggatgagcaggcacgggttgttacagctctttcgcagagtgggcgtcaccgaactcgaagcccgcagtggaacgctccccccagcctcgcagcgttgggaccgtatcctccaggcatcagggatgaaaagggccaaaccgtcccctacttcaactcaaacgccggaccaggcgtctttgcatgcattcgccgattcgctggagcgtgaccttgatgcgcccagcccaacgcacgagggagatcagaggcgggcaagcagccgtaaacggtcccgatcggatcgtgctgtcaccggtccctccgcacagcaatcgttcgaggtgcgcgttcccgaacagcgcgatgcgctgcatttgcccctcagttggagggtaaaacgcccgcgtaccagtatcgggggcggcctcccggatcctggtacgcccacgg

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72 ctgccgacctggcagcgtccagcaccgtgatgcgggaacaagatgaggaccccttcgcaggggcagcggatgatttcccggcattcaacgaagaggagctcgcatggttgatggagctattgcctcagtga Predicted amino acid sequence of PthA* MDPIRSRTPSPARELLPGPQPDGVQPTADRGVSPPAGGPLDGLPARRTMSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPEQVVAIACNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMMQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVT

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73 GPSAQQSFEVRVPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQ

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APPENDIX D SEQUENCE OF pthA*-2 DNA sequence of pthA*-2 atgcggcctcggaagctatgtaggaaccacagaccgctagtctggaggcgaccatgtaaagaggtatgcctgatggatcccattcgttcgcgcacaccaagtcctgcccgcgagcttctgcccggcccccaaccggatagggttcagccgactgcagatcgtggggtgtctccgcctgccggcggccccctggatggcttgcccgctcggcggacgatgtcccggacccggctgccatctccccctgcacccttgcctgcgttctcggcgggcagcttcagtgacctgttacgtcagttcgatccgtcactttttaatacatcgctttttgattcattgcctcccttcggcgctcaccatacagaggctgccacaggcgagtgggatgaggtgcaatcgggtctgcgggcagccgacgcccccccacccaccatgcgcgtggctgtcactgccgcgcggccgccgcgcgccaagccggcgccgcgacgacgtgctgcgcaaccctccgacgcttcgccggccgcgcaggtggatctacgcacgctcggctacagccagcagcaacaggagaagatcaaaccgaaggttcgttcgacagtggcgcagcaccacgaggcactggtcggccatgggtttacacacgcgcacatcgttgcgctcagccaacacccggcagcgttagggaccgtcgctgtcaagtatcaggacatgatcgcagcgttgccagaggcgacacacgaagcgatcgttggcgtcggcaaacagtggtccggcgcacgcgccctggaggccttgctcacggtggcgggagagttgagaggtccaccgttacagttggacacaggccaacttctcaagattgcaaaacgtggcggcgtgaccgcagtggaggcagtgcatgcatggcgcaatgcactgacgggtgcccccctgaacctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgcacccgggacaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagcaatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggcacaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggcacaggtggtggccatcgccagcaatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggt 74

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75 cgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggcacaggtggtggccatcgccagcaatattggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggcacaggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggcggcaggccggcgctggagagcattgttgcccagttatctcgccctgatccggcgttggccgcgttgaccaacgaccacctcgtcgccttggcctgcctcggcggacgtcctgcgctggatgcagtgaaaaagggattgccgcacgcgccggccttgatcaaaagaaccaatcgccgtattcccgaacgcacatcccatcgcgttgccgaccacgcgcaagtggttcgcgtgctgggttttttccagtgccactcccacccagcgcaagcatttgatgacgccatgacgcagttcgggatgagcaggcacgggttgttacagctctttcgcagagtgggcgtcaccgaactcgaagcccgcagtggaacgctccccccagcctcgcagcgttgggaccgtatcctccaggcatcagggatgaaaagggccaaaccgtcccctacttcaactcaaacgccggaccaggcgtctttgcatgcattcgccgattcgctggagcgtgaccttgatgcgcccagcccaacgcacgagggagatcagaggcgggcaagcagccgtaaacggtcccgatcggatcgtgctgtcaccggtccctccgcacagcaatcgttcgaggtgcgcgttcccgaacagcgcgatgcgctgcatttgcccctcagttggagggtaaaacgcccgcgtaccagtatcgggggcggcctcccggatcctggtacgcccacggctgccgacctggcagcgtccagcaccgtgatgcgggaacaagatgaggaccccttcgcaggggcagcggatgatttcccggcattcaacgaagaggagctcgcatggttgatggagctattgcctcagtga

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76 Predicted amino acid sequence of PthA*-2 MDPIRSRTPSPARELLPGPQPDRVQPTADRGVSPPAGGPLDGLPARRTMSRTRLPSPPAPLPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLHPGQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRVPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGAADDFPAFNEEELAWLMELLPQ

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APPENDIX E ALIGNMENT OF PATHOGENECITY GENES FROM X. citri STRAINS 1 50 PthA (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT PthA4 (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT Apl1 (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT PthAW (1) MDPIRSRTPSPARELLPGPQPDRVQP--TADRGVSPPAGGPLDGLPARRT PthA* (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT PthA*-2 (1) MDPIRSRTPSPARELLPGPQPDRVQP--TADRGVSPPAGGPLDGLPARRT PthA1 (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT PthA2 (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT PthA3 (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT Apl3 (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT Apl2 (1) MDPIRSRTPSPARELLPGPQPDGVQP--TADRGVSPPAGGPLDGLPARRT PthB (1) MDPIRPRTSSPAHELLAGPQPDRVQPQPTADRGGAPPAGSPLDGLPARRT PthC (1) MDPIRPRTSSPAHELLAGPQPDRVQPQPTADRGGAPPAGSPLDGLPARRT Consensus (1) MDPIRSRTPSPARELLPGPQPDGVQP TADRGVSPPAGGPLDGLPARRT 51 100 PthA (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE PthA4 (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE Apl1 (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE PthAW (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE PthA* (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE PthA*-2 (49) MSRTRLPSPPAPLPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE PthA1 (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE PthA2 (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE PthA3 (49) ISRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE Apl3 (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE Apl2 (49) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE PthB (51) MSRTRLPSPPAPLPAFSAGSFSDLLCQFDPLLLDTLLFDSMSAFGAPHTE PthC (51) MSRTRLPSPPAPLPAFSAGSFSDLLCQFDPLLLDTLLFDSMSAFGAPHTE Consensus (51) MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE 101 150 PthA (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS PthA4 (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS Apl1 (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS PthAW (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS PthA* (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS PthA*-2 (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS PthA1 (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS PthA2 (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS PthA3 (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS Apl3 (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS Apl2 (99) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS PthB (101) AAPGEADEVQSGLRAVDDPHPTVHVAVTAARPPRAKPAPRRRAAHTSDAS PthC (101) AAPGEADEVQSGLRAVDDPHPTVHVAVTAARPPRAKPAPRRRAAHTSDAS Consensus (101) AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS 77

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78 151 200 PthA (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ PthA4 (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ Apl1 (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ PthAW (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ PthA* (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ PthA*-2 (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ PthA1 (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ PthA2 (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ PthA3 (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ Apl3 (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ Apl2 (149) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ PthB (151) PAGQVDLCTLGYSQQQQDEIKPKARATVAQHHQALMGHGFTRAHIVALSQ PthC (151) PAGQVDLCTLGYSQQQQDEIKPKARATVAQHHQALMGHGFTRAHIVALSQ Consensus (151) PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ 201 250 PthA (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL PthA4 (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL Apl1 (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL PthAW (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL PthA* (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL PthA*-2 (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL PthA1 (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL PthA2 (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL PthA3 (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL Apl3 (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL Apl2 (199) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL PthB (201) HPAALGTVAVKYQAMIAALPEATHEDIVGVGKQWSGARALEALLTVSGEL PthC (201) HPAALGTVAVKYQAMIAALPEATHEDIVGVGKQWSGARALEALLTVSGEL Consensus (201) HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL 251 300 PthA (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA PthA4 (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA Apl1 (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA PthAW (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA PthA* (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA PthA*-2 (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA PthA1 (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVYAWRNALTGAPLNLTPEQVVAIA PthA2 (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA PthA3 (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA Apl3 (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA Apl2 (249) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA PthB (251) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIA PthC (251) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIA Consensus (251) RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIA

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79 301 350 PthA (299) SNIGGK-------------------------------------------PthA4 (299) SNIGGK-------------------------------------------Apl1 (299) SNIGGK-------------------------------------------PthAW (299) SNIGGK-------------------------------------------PthA* (299) SNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLL PthA*-2 (299) SNIGGK-------------------------------------------PthA1 (299) SNIGGK-------------------------------------------PthA2 (299) SNIGGK-------------------------------------------PthA3 (299) SNIGGK-------------------------------------------Apl3 (299) SNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLL Apl2 (299) SNIGGK-------------------------------------------PthB (301) SHDGGK-------------------------------------------PthC (301) SHDGGK-------------------------------------------Consensus (301) SNIGGK 351 400 PthA (305) ------------------------QALETVQRLLPVLCQAHGLTPEQVVA PthA4 (305) ------------------------QALETVQRLLPVLCQAHGLTPEQVVA Apl1 (305) ------------------------QALETVQRLLPVLCQAHGLTPEQVVA PthAW (305) ------------------------QALETVQALLPVLCQAHGLTPEQVVA PthA* (349) PVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVA PthA*-2 (305) ------------------------QALETVQRLLPVLCQAHGLHPGQVVA PthA1 (305) ------------------------QALETVQRLLPVLCQAHGLTPDQVVA PthA2 (305) ------------------------QALETVQALLPVLCQAHGLTPEQVVA PthA3 (305) ------------------------QALETVQALLPVLCQAHGLTPEQVVA Apl3 (349) PVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVA Apl2 (305) ------------------------QALETVQRLLPVLCQAHGLTPEQVVA PthB (307) ------------------------QALETVQRLLPVLCEQHGLTPAQVVA PthC (307) ------------------------QALETVQRLLPVLCEQHGLTPDQVVA Consensus (351) QALETVQRLLPVLCQAHGLTPEQVVA 401 450 PthA (331) IASNGG-KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR PthA4 (331) IASNGG-KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR Apl1 (331) IASNGG-KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR PthAW (331) IASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQR PthA* (399) IASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQR PthA*-2 (331) IASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN-GGKQALETVQR PthA1 (331) IASNGG-KQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQR PthA2 (331) IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR PthA3 (331) IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR Apl3 (399) IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR Apl2 (331) IASNGG-KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR PthB (333) IASNGGGKQALETVQQLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQR PthC (333) IASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQR Consensus (401) IASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR

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80 451 500 PthA (380) LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQV PthA4 (380) LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQV Apl1 (380) LLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQV PthAW (381) LLPVLCQAHGLTLDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQV PthA* (449) LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQV PthA*-2 (380) LLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQV PthA1 (380) LLPVLCQAHGLTPAQVVAIASN-GGKQALETVQRLLPVLCQAHGLTPAQV PthA2 (381) LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQV PthA3 (381) LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQV Apl3 (449) LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQV Apl2 (380) LLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQV PthB (383) LLPVLCEQHGLTPAQVVAIASNGGGKQALETVQQLLPVLCEQHGLTPDQV PthC (383) LLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQV Consensus (451) LLPVLCQAHGLTPEQVVAIASN GGKQALETVQRLLPVLCQAHGLTPDQV 501 550 PthA (430) VAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETV PthA4 (430) VAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV Apl1 (430) VAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV PthAW (431) VAIASNSGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV PthA* (499) VAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETV PthA*-2 (430) VAIASN-GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETV PthA1 (429) VAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV PthA2 (431) VAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETV PthA3 (431) VAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETV Apl3 (499) VAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETV Apl2 (430) VAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV PthB (433) VAIASNIGGKQALETVQRLLPVLCEQHGLTPAQVVAIASNGGGKQALETV PthC (433) VAIASNIGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNGGGKQALETV Consensus (501) VAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN GGKQALETV 551 600 PthA (480) QRLLPVLCQAHGLTPDQVVAIASHDGGK---------------------PthA4 (480) QRLLPVLCQAHGLTPDQVVAIASHDGGK---------------------Apl1 (480) QRLLPVLCQAHGLTPEQVVAIASHDGGK---------------------PthAW (481) QRLLPVLCQAHGLTPEQVVAIASHDGGK---------------------PthA* (549) QRLLPVLCQAHGLTPEQVVAIASHDGGK---------------------PthA*-2 (479) QRLLPVLCQAHGLTPEQVVAIASHDGGK---------------------PthA1 (479) QRLLPVLCQAHGLTPEQVVAIASHDGGK---------------------PthA2 (481) QRLLPVLCQAHGLTPDQVVAIASHDGGK---------------------PthA3 (481) QRLLPVLCQAHGLTPEQVVAIASHDGGK---------------------Apl3 (549) QRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE Apl2 (480) QRLLPVLCQAHGLTPDQVVAIASHDGGK---------------------PthB (483) QQLLPVLCEQHGLTPDQVVAIASHDGGK---------------------PthC (483) QRLLPVLCEQHGLTPDQVVAIASNIGGK---------------------Consensus (551) QRLLPVLCQAHGLTPEQVVAIASHDGGK

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81 601 650 PthA (508) ---------------------------------------------QALET PthA4 (508) ---------------------------------------------QALET Apl1 (508) ---------------------------------------------QALET PthAW (509) ---------------------------------------------QALET PthA* (577) ---------------------------------------------QALET PthA*-2 (507) ---------------------------------------------QALET PthA1 (507) ---------------------------------------------QALET PthA2 (509) ---------------------------------------------QALET PthA3 (509) ---------------------------------------------QALET Apl3 (599) QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALET Apl2 (508) ---------------------------------------------QALET PthB (511) ---------------------------------------------QALET PthC (511) ---------------------------------------------QALET Consensus (601) QALET 651 700 PthA (513) VQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP PthA4 (513) VQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP Apl1 (513) VQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP PthAW (514) VQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTP PthA* (582) VQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP PthA*-2 (512) VQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTP PthA1 (512) VQRLLPVLCQAHGLTPEQVVAIASN-GGKQALETVQRLLPVLCQAHGLTP PthA2 (514) VQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP PthA3 (514) VQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP Apl3 (649) VQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTP Apl2 (513) VQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP PthB (516) VQRLLPVLCEQHGLTPAQVVAIASNGGGKQALKTVQQLLPVLCEQHGLTP PthC (516) VQRLLPVLCEQHGLTPAQVVAIASNGGGKQALETVQQLLPVLCEQHGLTP Consensus (651) VQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP 701 750 PthA (563) EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL PthA4 (563) EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL Apl1 (563) EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL PthAW (564) EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL PthA* (632) EQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQAL PthA*-2 (562) EQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQAL PthA1 (561) AQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQAL PthA2 (564) EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL PthA3 (564) DQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQAL Apl3 (699) EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQAL Apl2 (563) EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL PthB (566) DQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNIGGKQAL PthC (566) DQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQAL Consensus (701) EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL

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82 751 800 PthA (613) ETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGL PthA4 (613) ETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGL Apl1 (613) ETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGL PthAW (614) ETVQRLLPVLCQAHGLTPEQVVAIASNCGGKQALETVQRLLPVLCQAHGL PthA* (682) ETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGL PthA*-2 (612) ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGL PthA1 (611) ETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGL PthA2 (614) ETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGL PthA3 (614) ETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGL Apl3 (749) ETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGL Apl2 (613) ETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGL PthB (616) ETVQRLLPVLCEQHGLTPDQVVAIASNIGGKQALETVQRLLPVLCEQHGL PthC (616) ETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGL Consensus (751) ETVQRLLPVLCQAHGLTPEQVVAIASN GGKQALETVQRLLPVLCQAHGL 801 850 PthA (663) TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQ PthA4 (663) TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQ Apl1 (663) TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQ PthAW (664) TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQ PthA* (732) TPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQ PthA*-2 (662) TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQ PthA1 (661) TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN-GGKQ PthA2 (664) TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQ PthA3 (664) TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQ Apl3 (799) TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQ Apl2 (663) TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQ PthB (666) TPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQ PthC (666) TPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQ Consensus (801) TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASN GGKQ 851 900 PthA (713) ALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAH PthA4 (713) ALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAH Apl1 (713) ALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAH PthAW (714) ALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAH PthA* (782) ALETVQRLLPVLCQAHGLTPEQVVAIACNGGGKQALETVQRLLPVLCQAH PthA*-2 (712) ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAH PthA1 (710) ALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAH PthA2 (714) ALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH PthA3 (714) ALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH Apl3 (849) ALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAH Apl2 (713) ALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH PthB (716) ALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLCEQH PthC (716) ALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLRQAH Consensus (851) ALETVQRLLPVLCQAHGLTPEQVVAIASN GGKQALETVQRLLPVLCQAH

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83 901 950 PthA (763) GLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS---PthA4 (763) GLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS---Apl1 (763) GLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS---PthAW (764) GLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS---PthA* (832) G------------------------------------------------PthA*-2 (762) G------------------------------------------------PthA1 (760) GLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG--------------PthA2 (764) G------------------------------------------------PthA3 (764) G------------------------------------------------Apl3 (899) GLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGG Apl2 (763) G------------------------------------------------PthB (766) GLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIAS---PthC (766) GLTPAQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIAS---Consensus (901) GLTPDQVVAIASHDGGKQALETVQRLLPVLC HGLTPEQVVAIAS 951 1000 PthA (809) -------------------------------------------------PthA4 (809) -------------------------------------------------Apl1 (809) -------------------------------------------------PthAW (810) -------------------------------------------------PthA* (833) -------------------------------------------------PthA*-2 (763) -------------------------------------------------PthA1 (795) -------------------------------------------------PthA2 (765) -------------------------------------------------PthA3 (765) -------------------------------------------------Apl3 (949) KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQ Apl2 (764) -------------------------------------------------PthB (812) -------------------------------------------------PthC (812) -------------------------------------------------Consensus (951) 1001 1050 PthA (809) --------------HDGGKQALETVQRLLPVLCQAHGLTPEQVVAIACNG PthA4 (809) --------------HDGGKQALETVQRLLPVLCQAHGLTPEQVVAIACNG Apl1 (809) --------------HDGGKQALETVQRLLPVLCQAHGLTPEQVVAIACNG PthAW (810) --------------HDGGKQALETVQRLLPVLCQAHGLTPEQVVAIACNG PthA* (833) -------------------------------------LTPEQVVAIASNG PthA*-2 (763) -------------------------------------LTPAQVVAIASHD PthA1 (795) -------------------------------------LTPEQVVAIASHD PthA2 (765) -------------------------------------LTPEQVVAIASNI PthA3 (765) -------------------------------------LTPEQVVAIASHD Apl3 (999) AHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIACNG Apl2 (764) -------------------------------------LTPEQVVAIASHD PthB (812) --------------NGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNG PthC (812) --------------NGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNG Consensus (1001) GGKQALETVQRLLPVLC HGLTPEQVVAIASNG

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84 1051 1100 PthA (845) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP PthA4 (845) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP Apl1 (845) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP PthAW (846) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP PthA* (846) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP PthA*-2 (776) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP PthA1 (808) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP PthA2 (778) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP PthA3 (778) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP Apl3 (1049) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP Apl2 (777) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP PthB (848) GGKQALETVQRLLPVLRQAHGLTPAQVVAIASNGGGRPALESIFAQLSRP PthC (848) GGKQALETVQRLLPVLRQAHGLTPAQVVAIASNGGGRPALESIFAQLSRP Consensus (1051) GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP 1101 1150 PthA (895) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS PthA4 (895) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS Apl1 (895) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS PthAW (896) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS PthA* (896) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS PthA*-2 (826) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS PthA1 (858) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS PthA2 (828) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS PthA3 (828) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS Apl3 (1099) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS Apl2 (827) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS PthB (898) DQALAALTNDHLVALACLGGRPALEAVKKGLPHAPTLIKRTNRRLPERTS PthC (898) DQALAALTNDHLVALACLGGRPALEAVKKGLPHAPTLIKRTNRRLPERTS Consensus (1101) DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS 1151 1200 PthA (945) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT PthA4 (945) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT Apl1 (945) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT PthAW (946) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT PthA* (946) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMMQFGMSRHGLLQLFRRVGVT PthA*-2 (876) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT PthA1 (908) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT PthA2 (878) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT PthA3 (878) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT Apl3 (1149) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT Apl2 (877) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT PthB (948) HRVADHAQVARVLGFFQCHSHPAQAFDEAMTQFGMSRHGLLQLFRRVGVT PthC (948) HRVADHAQVARVLGFFQCHSHPAQAFDEAMTQFGMSRHGLLQLFRRVGVT Consensus (1151) HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT

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85 1201 1250 PthA (995) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL PthA4 (995) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL Apl1 (995) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL PthAW (996) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL PthA* (996) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL PthA*-2 (926) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL PthA1 (958) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL PthA2 (928) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL PthA3 (928) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL Apl3 (1199) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL Apl2 (927) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL PthB (998) ELEARGGTLPPAPQRWHRILQASGMKRAEPSGASAQTPDQASLHAFADAL PthC (998) ELEARGGTLPPAPQRWHRILQASGMKRAEPSGASAQTPDQASLHAFADAL Consensus (1201) ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL 1251 1300 PthA (1045) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRAPEQRDA PthA4 (1045) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRVPEQRDA Apl1 (1045) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRAPEQRDA PthAW (1046) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRVPEQRDA PthA* (1046) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRVPEQRDA PthA*-2 (976) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRVPEQRDA PthA1 (1008) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRVPEQRDA PthA2 (978) ERDLDAPSPMHEGDQTRASS-RKRSRSDRAVTGPSAQQSFEVRVPEQRDA PthA3 (978) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRVPEQRDA Apl3 (1249) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRAPEQRDA Apl2 (977) ERDLDAPSPTHEGDQRRASS-RKRSRSDRAVTGPSAQQSFEVRAPEQRDA PthB (1048) ERELDAPSPIDQAGQALASSSRKRSRSESSVTGSFAQQAVEVRVPEQRDA PthC (1048) ERELDAPSPIDQAGQALASSSRKRSRSESSVTGSFAQQAVEVRVPEQRDA Consensus (1251) ERDLDAPSPTHEGDQRRASS RKRSRSDRAVTGPSAQQSFEVRVPEQRDA 1301 1350 PthA (1094) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA PthA4 (1094) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA Apl1 (1094) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA PthAW (1095) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA PthA* (1095) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA PthA*-2 (1025) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA PthA1 (1057) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA PthA2 (1027) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA PthA3 (1027) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA Apl3 (1298) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA Apl2 (1026) LHLP-LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA PthB (1098) LHLPPLSWGVKRPRTRIGGGLPDPGTPMDADLAASSTVMWEQDADPFAGA PthC (1098) LHLPPLSWGVKRPRTRIGGGLPDPGTPMDADLAASSTVMWEQDADPFAGA Consensus (1301) LHLP LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA

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86 1351 1371 PthA (1143) ADDFPAFNEEELAWLMELLPQ PthA4 (1143) ADDFPAFNEEELAWLMELLPQ Apl1 (1143) ADDFPAFNEEELAWLMELLPQ PthAW (1144) ADDFPAFNEEELAWLMELLPQ PthA* (1144) ADDFPAFNEEELAWLMELLPQ PthA*-2 (1074) ADDFPAFNEEELAWLMELLPQ PthA1 (1106) ADDFPAFNEEELAWLMELLPQ PthA2 (1076) ADDFPAFNEEELAWLMELLPQ PthA3 (1076) ADDFPAFNEEELAWLMELLPQ Apl3 (1347) ADDFPAFNEEELAWLMELLPQ Apl2 (1075) ADDFPAFNEEELAWLMELLPQ PthB (1148) ADDFPAFNEEEMAWLMELFPQ PthC (1148) ADDFPAFNEEEMAWLMELFPQ Consensus (1351) ADDFPAFNEEELAWLMELLPQ

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BIOGRAPHICAL SKETCH Abdulwahid Al-Saadi was born January 25th, 1971, in Mombasa, Kenya. His family moved back to Oman in 1978. He obtained his primary and secondary education in Oman. He earned his Bachelor of Science degree in agriculture at Sultan Qaboos University (S.Q.U) in Oman in October of 1995. After graduating S.Q.U. he worked for the Diwan of Royal court. In 1997 he got a scholarship by the Diwan of Royal Court to pursue the Doctor of Philosophy degree in the field of plant molecular and cellular biology at the University of Florida. After graduating from University of Florida, Abdulwahid will go back to Oman and continue working for the Diwan of Royal Court. 95


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Title: Phenotypic Characterization and Sequence Analysis of pthA Homologs from Five Pathogenic Variant Groups of Xanthomonas citri
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Copyright Date: 2008

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Title: Phenotypic Characterization and Sequence Analysis of pthA Homologs from Five Pathogenic Variant Groups of Xanthomonas citri
Physical Description: Mixed Material
Copyright Date: 2008

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PHENOTYPIC CHARACTERIZATION AND SEQUENCE ANALYSIS OF pthA
HOMOLOGS FROM FIVE PATHOGENIC VARIANT GROUPS OF Xanthomonas
citri















By

ABDULWAHID AL-SAADI


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

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Abdulwahid Al-Saadi

































This dissertation is dedicated to H.E Mahmood A. Makki for his continuous support,
encouragement and belief in me. He passed away last year without seeing me make it to
the end and successfully complete my PhD. I am sure that he would have been very
happy and appreciative for my accomplishment.
















ACKNOWLEDGMENTS

I would thank God for giving patience and blessing me with a lot of colleagues and

friends who stood by my side when I needed them most. The completion of this

dissertation would not have been possible without the help and support of many people

both in Gainesville and back home in Oman. First I would like to express my gratitude to

my advisor Dr. Dean W. Gabriel for his support, guidance and patience with me during

my time in his lab. I would also like to thank him for providing me financial support

towards the end my PhD. I would like to thank Dr. Jeff Jones for his support,

encouragement, guidance, friendship and listening to me when I was feeling down and

ready to give up. I would also like to thank my other committee members Dr. G. Moore

and Dr. R. Lee for being on my committee, support and correcting this manuscript

I would like to thank Dr. G. Wisler for her support and encouragement. I would

like to give special thanks to Mr. Gary Marlow for his continued help, support, scientific

discussions and just being a good friend that was there for me when I needed him both in

and outside the lab. I want to also thank Dr. Joseph Ready and Dr. Young Duan for their

help, discussions and advice in the lab. I would like to thanks other students in my lab

especially Basma, Adriana and Asha for their help and support. I also thank all former

members of the lab. I thank faculty, staff and students in the PMCB program and the

Department of Plant Pathology who helped and encouraged me in many ways. I would

like to thank everyone in the Department of Plant Industry (DPI) for providing help in









maintenance of citrus plants in the quarantine facility. Special thanks go to my friend

Eduardo Carlos and his family for all their support and encouragement. I also want to

thank my two study partners Mohamed Al-Khairy and Yousef Al-Dligan. I also thank

Mohamed Al-Matar and Fahad Al-Saqqaf.

I would like to recognize two people in the Diwan of Royal Court who have

recently passed away. H.E Said Seif bin Hamed and H.E Mahmood Maki who supported

me coming to the US to get my PhD. Without their support and encouragement I would

have not been able to complete my degree. I would like to thank my advisor and mentor

in Oman, Dr. Ahmed Hamooda, for continuously supporting and encouraging me

throughout my time here. I thank him for being my advocate and believing in me even

when many were ready to give up on me. I cannot say thank you enough to this man who

took me under his wings and guided me through difficult times. I would like to thank

Mr. Yahya Al-Zidjali for his support. Special thanks go to Dr. Yahya Al-Hinai for his

support and friendship. I want to thank Mrs. Nihaya, Dr. Magdy, Dr. Deitz,

Abdulrahman Al-Siyabi, Abduljalil Attiya, Ammer Al-Manthiri and everyone in the

Diwan of Royal Court who helped and supported me.

I thank my parents, Abubaker and Khadija, for unconditional support, love and

encouragement. I also thank my brothers and sisters for their support and encouragement

during my study here. I thank them for making me the person I am. I also like to thank

Yasser Al-Ajmi, Mohamed Al-Balushi, Fida Al-Raissy, Aqeel Abdawani and all my

friends in Oman. I thank and remember my twin brother Abdulrahman who passed away

a few days after our birth. He has always been with me and provided me with strength,









patience and hope. Finally I have to say special thanks to my wife May and son Muadh

for being there for me as they waited patiently for me during my time here.
















TABLE OF CONTENTS

page

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

LIST OF TABLES .............. ......... ... ................. ...................... ..

LIST OF FIGURES ......... ....... .................... .......... ....... ............ xi

A B S T R A C T .............................................. ..........................................x iii

CHAPTER

1 IN TR O D U C T IO N ............................................................. .. ......... ...... .....

C itru s.......... .......................... .................................................. .
Florida Citrus ................................... ................................. ......... 2
C itru s C a n k e r ................... .................................................... ................ .. 3
R resistance to C itrus C anker............................................................... ....................5
C controlling C itrus C anker............................................................................ ....... ...... 5
O objectives ............................................................... ..... ...... ......... 6

2 USE OF TWO DIFFERENT CITRUS HOSTS TO DISTINGUISH ALL FORMS
OF CITRU S CANKER DISEA SE .................................... ............................. ....... 10

Introduction................................... .................................. ........... 10
M material and M methods .................. .................... ............................ .. .............. 11
Strains, Plasm ids and Culture M edia.......................... ......... ...................11
Recombinant DNA Techniques.............................. ........ .......................11
Plant Inoculations ............... .... .... .... .. ..................................... .. 12
In vitro G row th K inetics............................................... ............................. 12
Inplanta Grow th K inetics ........................................................................13
R e su lts .......................... ... .. ...... ................ ................. ................ 13
Pathogenicity Phenotypes of X citri Strains ............................... .................... 13
In v itro G ro w th ............................................................................................... 14
Growth Kinetics in plant .. ............................................................. 14
D isc u ssio n .................................................... ................... ................ 1 5









3 IDENTIFICATION AND CHARACTERIZATION OF HOST RANGE
FACTOR(S) IN CITRUS CANKER STRAINS. ............... .................. ............24

Introdu action ...................................... ................................................. 24
M material and M methods ............................................................... .. ............... 26
Bacterial Strains, Plasmids and Culture Media............................................26
Recombinant DN A Techniques...................................... ......................... 26
V sector Preparation................................................ ............ ..27
Packaging and Transfection ........................................ .......................... 27
Plant Inoculations ......................................... ................... ........ 28
T riparental M atings ......................... .. .................... .. ...... ........... 28
R results ........................... .... ............. ...... ................ ..... ........................... 29
Xanthomonas citri pv citri A 3213 Strain Genomic Library .............................29
Screening of 3213 Library in Xc270 ..........................................................29
D discussion ..................................... .................................. ......... 29

4 SEQUENCE COMPARISON AND CHARACTERIZATION OF FIVE NEW
pthA HOMOLOGS FROM FOUR DIFFERENT Xanthomonas citri STRAINS .....35

Introduction ............ .. ........ ............. ......... ............ .......... 35
M material and M methods ........... .. ........... ..............................................................36
Bacterial Strains, Plasmids and Culture M edia....... .... ................................... 36
Recombinant DN A Techniques...................................... ......................... 37
DNA Library Construction.................... ....... ........................... 37
P lant Inoculations ........................... .................... .. ......... .......... 38
Southern Hybridization Analysis ............................................. ............... 38
Colony Hybridization .............................. .. .. ........ .... .. ................. 38
T riparental M atings ......................... .. .................... .. ...... ........... 39
Sequence A analysis ofpth G enes ........................................ ...... ............... 40
R e su lts .................. ......... .. .. ......................................................................................... 4 1
Southern B lot A nalysis..................... ............................................4 1
Cloning, Characterization and Sequencing ofpthA Homologs from X citri
A *, A ", B and C Strain s ...................... ....... ..... ....... ...... .........................4 1
Inactivation and Complementation of Genes pthB and pthC in X citri pv
aurantifolii ............... ............. .......... .. ............. ......................... 43
None of the pthA Homologs from Group A Strain 3213 Increased the Host
Range of Group A* Strain 270 to Include Grapefruit................................... 44
Sequence Analysis ofpthA Homologs from All Known X citri Groups............44
D isc u ssio n ............... .................................. .................... ................ 4 5

5 SUMMARY AND CONCLUSION .......... ............................................... 60

APPENDIX

A SEQUEN CE OF pthC ................................................................. ............... 63

B SEQUEN CE OF pthAW ........ .............................. ............................ ............... 66









C SEQUENCE OF pthA* .......................... ......................................... 70

D SE Q U EN C E O F p thA *-2 ................................................................ ..................... 74

E ALIGNMENT OF PATHOGENECITY GENES FROM X citri STRAINS ............77

L IST O F R EFER EN CE S ......... ..... .... ........... ................................................87

B IO G R A PH IC A L SK E TCH ..................................................................... ..................95
















LIST OF TABLES


Table p

2-1. Strains and plasm ids used in this study .......................................... ................... 18

2-2. Phenotypic differences among X citri strains................................................. 19

3-1. Strains and plasmids used in this study...................................................... 31

4-1. Strains and plasmids used in this study .......................................... ...............49

4-2. Phenotypic responses of X citri strains in 2 citrus hosts ......................................51

4-3. Amino acid sequence identity between pathogenicity genes from X citri strains...52
















LIST OF FIGURES


Figure p

1-1. W world citrus production. ............................................................ ....................... 7

1-2. Status of citrus canker disease in the state of Florida. ................... ...............8

1-3. Citrus canker sym ptom s in citrus. ........................................................................9

2-1. Inoculation of strains of different citrus canker groups in grapefruit and Key
lim e .......................................................................................... . 2 0

2-2. Inoculation of several different A* strains in Duncan grapefruit.............................21

2-3. Growth of X citri strains in PYGM medium. ................... .................22

2-4. inplanta growth of X. citri strains in Mexican/Key lime and Duncan grapefruit. ..23

3-1. Scheme for cosmid vector preparation and DNA cloning. .....................................32

3-2. DNA fractionation of X citri 3213 genomic DNA ............................................33

3-3. Restriction profiles of random clones from X citi 3213 genomic library ...............34

4-1. Southern Hybridization analysis ofX. citri strains hybridized with the BamHI
internal fragm ent ofp thA ............................................................. .....................53

4-2. Colony Hybridization of E. coli with cloned X0053 A" plasmid DNA fragments
using 32P-labeledp thA .............. .......................... ........................ ............... 54

4-3. Complementation of A strain knockout B21.2 (pthA::Tn5) with pthA homologs
from A strain X 0053 on citrus........................................ ............. ............... 55

4-4. Complementation of A strain knockout B21.2 (pthA::Tn5) withpthA homologs
in citru s ...............................................................................56

4-5. Analysis ofpthA and its three homologs in A* strain Xc270................................57










4-6. Neighbor-joining dengogram of members of avrBs3/pthA genes from different
species and pathovars of Xanthomonas....................... ........ ..... .......... 58

4-7. Sequence alignment of the predicted amino acids encoded in the main variable
portion of the repeat region of all 13 pthA homologs. ..........................................59















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

PHENOTYPIC CHARACTERIZATION AND SEQUENCE ANALYSIS OF pthA
HOMOLOGS FROM FIVE PATHOGENIC VARIANT GROUPS OF Xanthomonas
citri

By

Abdulwahid Al-Saadi

August 2005

Chair: Dean W. Gabriel
Major Department: Plant Molecular and Cellular Biology

Citrus canker is an economically important disease that is caused by five different

groups ofXanthomonas citri strains: three from Asia (A, A* and A") and two from South

America (B and C). In artificial inoculations of grapefruit, only strains of the A and B

groups appear to be virulent; strains of the C and A" group elicit an hypersensitive

response (HR) and the A* strains show various levels of reduced virulence. The tested

A* and A" strains also grew to much lower concentrations in grapefruit compared to the

A strain. The strains from all five citrus canker groups were virulent in Mexican lime,

but the B and C strains elicited a distinctive canker lesion that was almost white in

appearance. Strains from the Asiatic groups grew faster than South American B and C

group strains in artificial media. Mexican lime and grapefruit can be used in artificial

inoculations to readily distinguish all known strains causing citrus canker disease within

10 days without the need for other laboratory tests. Attempts to identify positive host









range determinants from X citri were unsuccessful suggesting the possibility of negative

factors (avirulence) being involved in determining host range ofX. citri.

Every X citri strain carries multiple DNA fragments that hybridize with pthA, a

member of the avrBs3/pthA gene family from X citri group A that is required for

pathogenicity and growth of X. citri in citrus. Three newpthA homologs were cloned and

sequenced from canker groups A" (pthA W) and A* (pthA andpthA *-2), and compared

with pthA, pthB and pthC. Homologs pthA, pthB, pthC pthAWand pthA* all have 17.5,

nearly identical, direct tandem repeats of 34 amino acids and all complemented a

pthA::Tn5 knockout mutation in an X citri group A strain B21.2. Although grapefruit is

a differential host and groups A* and A" are avirulent in grapefruit, none of the pthA

homologs appeared responsible for the avirulence phenotype by cross complementation

tests. Furthermore, none of the fourpthA homologs from the wide host range group A

strain 3213, includingpthA, conferred an increase in host range of group A* or A" strains

to include grapefruit. pthA*-2 carried only 15.5 repeats and did not confer either

pathogenicity or avirulence to B21.2 in any citrus species tested. Phylogenetic studies

separate pthA homologues into two groups, Asiatic and South American groups.

Analysis of the predicted amino acid sequences of all sequenced pthA homologs from X

citri indicated that a specific set of amino acid residues in two variable regions of the 17th

direct tandem repeat may be required for pathogenicity in citrus.














CHAPTER 1
INTRODUCTION

Citrus

Citrus is one of the major fruit crops in the world. It is thought to have originated

in Southeast Asia and India. Citrus was introduced into the new world in the 16th century

by Spanish and Portuguese explorers (Allen, 2000). World production of citrus is

estimated to be about one hundred million metric tons (FAOSTAT data, 2004,

http://apps.fao.org ). World citrus production has seen a substantial increase over the last

40 years (Figure 1-1). Major citrus producing countries include the United States, Brazil,

China, Argentina, Spain and Mexico (Figure 1-1b). Currently, citrus produced in North

and South America account for the majority of citrus production worldwide. Although

citrus is mainly grown for the fresh fruit market, large citrus-based juice industries have

developed in many countries such as Brazil and the United States. Generally citrus is

grown between 400 North and 400 South latitudes where minimum temperatures stay

above 200 240 F (Timmer and Duncan, 1999).

Citrus is a perennial evergreen with an expected economical production expectancy

of about 50 years (Timmer and Duncan, 1999). Originally citrus was grown on its own

root system, but now most citrus production plants are grafted onto various rootstocks.

Rootstocks are selected for their inherent characteristics that affect production, cold

hardiness, salinity tolerance, disease resistance and most importantly compatibility with

scion tissue.









The majority of citrus varieties grown for commercial purposes are in the genus

Citrus including grapefruit (Citrus paradisi Macfad), sweet orange (C. sinensis (L.)

Osbeck), tangerine/mandarin (C. reticulata Blanco), lemon (C. limon Burm), lime (C.

aurantifolia (Christm.) Swingle), pummelo (C. grandis Osbeck) and citron (C. medical

L.). Other citrus relatives that are not in this genus are kumquats (Fortunella spp.) and

trifoliate orange (Poncirus trifoliata). The latter is used only as a rootstock.

Florida Citrus

Total citrus production in the U.S. in 2004 is estimated at 16.2 million tons with an

estimated value of $ 2.4 billion (USDA, 2004). States that produce citrus include Florida,

California, Texas, Arizona, Alabama, Mississippi and Louisiana. Florida produces about

80% of the U.S. citrus, of which 20% 25% is sold for fresh fruit consumption. In 2004,

Florida produced 242 million boxes of oranges and 40.9 million boxes of grapefruit

(USDA, 2004). Citrus is an important economic crop for the state of Florida, as the

worth of the commercial citrus industry in Florida is estimated to be more than $8.5

billion.

Diseases play a critical role in limiting citrus production as citrus is mainly grown

in the same tropical and subtropical areas that also favor the growth of microorganisms.

This provides great challenges to citrus growers since they must balance cost of

controlling diseases against lower projected profit margins. An example of a citrus

disease that is a serious problem for citrus growers is citrus canker disease. Citrus canker

has destroyed many citrus growing areas around the world. Florida authorities are

putting major resources towards completely eradicating this disease. It is important to

gain a better understanding of this disease because of the quarantine of citrus canker as a









pest. Citrus canker it is still spreading, despite $50 million spent between 1996 and 1999

in eradication efforts (Schubert et al., 2001).

Citrus Canker

Citrus canker is one of the major disease problems facing citrus producers in

Florida and many areas of the world (Danos et al., 1981; Elgoorani, 1989; Gottwald et al.,

2001). Citrus canker, also known as bacterial canker, has destroyed large areas of citrus

production (Fegan et al., 2004; Schubert et al., 2001). The pathogen is thought to have

originated in Southeast Asia, from where it has spread to other citrus producing areas.

Asiatic citrus canker was introduced into the United States for the first time in 1912 from

infected nursery material. It took approximately 20 years to eliminate this outbreak of

citrus canker (Loucks and Florida. Division of Plant Industry., 1934). In 1986, citrus

canker reappeared for the second time in both residential and commercial areas around

Tampa, Florida. As a result of this outbreak a new citrus canker eradication program was

initiated (Brown, 2001). Eight years later, Florida declared it had eradicated citrus canker

at a cost of $27 million (Agrios, 1997). Citrus canker reappeared for the third time in

Florida in 1995 in Dade County, and has resulted in the destruction of more than four

million trees in both residential and commercial areas (FDACS data, 2005). Figure 1-2

shows the status of citrus canker disease in the state of Florida in 2004. Currently, citrus

canker has been detected in 20 Florida counties. The total area under quarantine is

estimated at 1,397.82 sq. miles (FDACS data, 2005, www.doacs.state.fl.us). Strong

regulatory and quarantine measures were implemented in the latest effort to eradicate the

disease. Healthy citrus trees anywhere within a radius of 1900 ft from infected trees are

deemed exposed and are destroyed (Gottwald et al., 2002).









Citrus canker is caused by several pathogenic variants of Xanthomonas citri. In

general, five different groups of pathogenic variants are recognized: A, B, C, A* and A"

(Gabriel et al., 1989; Stall et al., 1982; Vemiere et al., 1998). In the literature two other

groups of citrus canker are described: "D" and "E" strains. Although there is a single

extant "D" strain that was reported in Mexico, it is thought that the fungal pathogen

Alternaria limicola was responsible for that disease outbreak (Schubert et al., 2001). The

"E" strain group was found in grapefruit only in nurseries in Florida and was described as

a new "form" of citrus canker. However, strains in this group do not cause hyperplasia

and do not infect fruit or mature citrus in groves. The disease is now recognized as

distinct from citrus canker and was named citrus bacterial leaf spot caused by

Xanthomonas axonopodis pv. citrumelo .

Citrus canker symptoms appear after the pathogen enters the leaves through the

stomata or wounds and multiplies in the intercellular spaces of the spongy mesophyll

(Gottwald et al., 1988; 1989; Graham et al., 1992; Pruvost et al., 2002). The initial

symptom is the formation of water-soaked tissue followed by growth of yellow halos on

the infection margins. As the disease progresses, erumpent necrotic lesions are formed

on leaves, stems and fruits (Figure 1-3). At advanced disease stages, plants defoliate and

fruit can drop prematurely. At the microscopic level, infected cells divide (hyperplasia)

and enlarge (hypertrophy); and the pustules rupture the surface of the leaf tissue and

release bacteria that become a source of inoculum for further infections (Swarup et al.,

1991).

The citrus canker bacterium is transmitted by wind-blown rain, although

machinery, animals and humans can also transmit it (Bock et al., 2005; Danos et al.,









1984). An important factor that contributed to the spread of citrus canker in this last

infection in Florida was the Asian citrus leaf miner Phyllocnistis citrella (Cook, 1988).

The leaf miner is probably not a vector for canker, but instead it provides wounds that

allow entry of bacteria into citrus leaves (Belasque et al., 2005). Although citrus canker

does not cause systemic damage, it results in reduced marketability of citrus fruit

especially those produced for the fresh market.

Resistance to Citrus Canker

Citrus genotypes show differences in susceptibility to this disease. Grapefruit,

sweet orange and Mexican lime are highly susceptible. Sour orange, lemon and tangelo

are moderately susceptible, whereas mandarin, citron and kumquat are less susceptible

(Schubert et al., 2001). It is not clear if resistance in citrus is a result of active defense

responses or if it is due to physical characteristics of different citrus genotypes, e.g.

number of stomata or thickness of the leaf tissue that may influence the number of

bacterial particles entering citrus leaves (Goto, 1969; McLean and Lee, 1922).

Controlling Citrus Canker

The most effective control of citrus canker is application of strict regulatory and

quarantine measures that will protect against the introduction of new infections (Graham

et al., 2004). Most citrus producing areas put many resources into monitoring and

regulating citrus canker. That is because it is so difficult to eliminate the bacteria once it

has become established. Once the disease is established in an area, eradication of both

infected and exposed trees and burning plant material are used to help eliminate and

prevent spread of disease. Multiple applications of copper based compounds were found

to help control the disease to some extent (Hwang, 1949). In some cases pruning infected

branches is used to control and eliminate the source of infection. Since citrus canker









spreads by wind driven rain, wind brakes were found to be useful in controlling this

disease (Gottwald and Timmer, 1995).

Objectives

The aim of this study was to identify host range determinants of canker causing

strains. I was interested in identifying genes that are necessary for increasing the host

range of canker causing strains ofX. citri. These new strains that are limited in host

range were used to screen for genes involved in host range determination. Further

understanding of how host range is determined may provide important tools in

developing control measures. The specific objectives of this work include the following:



Objective 1. Characterizing canker causing Xanthomonas citri A* and A" group strains.

Objective 2. Attempting to identify and characterize positive host range factors) in

canker causing Xanthomonas citri.

Objective 3. Isolating pathogenicity gene (pthA) homologs from A* and A" groups and

characterize their role in host range determination.

















120




10'1




8''


0

S 61-




41'




21'





1961 1965 1970 1975 1980 1985 1990 1995 2000
year








1% 14'oL Brazil
1% U USA
1%- China
1% Mexico
2%- Spain
2% 1 India
0 Iran
2% HINigeria
Italy
Egypt
Argentina
2% 13% Turkey
2% Pakistan
3 South Africa
3 l Japan
3% Greece
4 Morocco
4% 12% Thailand
4% others
6% 6%



B





Figure 1-1. World citrus production. A. world citrus production between 1961 2000

expressed in metric tons. B. Percent production by countries (FAOSTAT data,

2004, http://apps.fao.org).












POUK COUNTY
FBrst detected: May 2005
COaranine area: none
Grrve Itree deCsanyh:TBD
Dooryard vrs destroyed: none


HARDEE COUNTY ,
Firit leected:Mae 2005 a
Quarantine ama: none '
Grove iqel ded~:yefTBD :
Dooryard trees destirye: none

HILLSEHOOUI I COUNTY
First detected: December 201
Quarantine areas none
Grove lreesd dlMrnyl 1 ,2U566
DOearav res ideoBd: 3,fi37 3 ',3

ANATEE COUNTY /
FIrst detecteMa 1997
Quarantine areas: one
Grov iwees destroyed:106.582
Domyard trees destroyed: 6379

SARASOTA COUNTY
First detecred Ocltobe 2002
Quarantine area 4 sq, mi.
GonWe tree destroyed; none
Do
D SOTO COUNTY --
Firt detected; October 2001
Quoarantie a :rea D Sot (26-5 sQ-ile, /
Ven s (19.75 sq. mles)
Grove trees destroyed431,44 /
D -tryrd r" sdstroyed* 1.4A5,

CHARLOTTE COUwrT ---
First detected October M04
Oiearantine wh : Farab ee Grade (11.5 so. mi.: /
Hurrnti Sre (q.25 i4Qiilei
Grve trees destroyed: 14454
DEoyard treesdestroved: t. /b

LEE COUNTY -
Firsu tler. Atr,IMc i'W 12
Qluarantine rea; CpeCp r 6.l (35 sqmi,.t
Plne llard (1.5sq. iles~
Groae trees destroyed:6 t62 /
Doosyardtrees- ivei-erl. 5,0 5

COWEA COUNTY -
First detectedtJune 1998
Quarantinei wrea:4$q. miles
Gove trees destroyed: 299949
loorvard trees destroyed: 309


,/ ,/ I,
I/HEMIM COUNTY
/ t Fest detected: February 1999
ii Quarantiise areas Seaws (2 sQmil; \
I W: e rt llli rGr (;. q qi h ii *
I CIollle(4sq.miles)
Grove treesdestroyedt&85,346
SDooreadtreesdestnryetp964

OI NECHOKE COUNTY
First tf Octt obM 2002
I QLurairwieareaincne
SGrove trees destroyed: none
I Dooryard trees destroyed:

*HIGHLANDS COUNTY
First detected; May2O?2
Quarmli ne are& HNaranja (18 sq. milesi,
Venus 119.75 smniles)
Grove trees destroyed: 71 3,
Doryard trees deirpwd: none


ORANGE COUNTY
First detete d:July 2002
Quaramine areas: SW Orlando
14 sq.nrrns
Groe trees destroyed: 9232
Doorwyard trees destroyed: 35,563

OSCEOLACOIUNTY
First detected: Nowemner 2004
Quarairtin area: none
Gi6,e trees distroed: 0
Dooryard trees desoyed: 1,611

MIEVAlta)COUNTY
First detected:January 202. Feb 200
Qtarmwaiie aree-r nfn
S GMroe trees t.dstr d: tn
I Dooryard trees destroyed: 1,0l

INDIAN MtIEH COUNTY
First detected: Deember 204
l QuaramIinre ar+w: Indrip Road
I 120I q. nile
SGrowe trees destroyed: 2,146
I Dooryard trees destroyed:936

I 'STUCIECDUMTV
First detected: Decemerw2004
SQuBapari e areas: ldria Road
12os sq. mites),
A Rllapattah,.' I .,q 1iIes
SGrve trees destroyed:13.,625
Dooryaird tre estroyed:5,665
I I
MARTIN OUcITYr
First detected: September 001l
Quaratine areas: none
Grove tree destroyed: 101,000
I Dooryard rees destroyed: none

i, IIAMIDADE RIOWAlvID
PALM BEACH COUNTIES
First detected:October 1995
I Qutirts reapp1, 1149 V q milw
I Darydl trees desiryed:669,425
SGrove trees destroyed: 724651

'MOnRODECOURTY
First detected: Big Pine Key-June 20iS
Marathon ey- Jauary2004
Quararire area: KI(t (119 sqrni.
DBoryard trees deslroyed:515
Growe trees destroyed: none


Figure 1-2. Status of citrus canker disease in the state of Florida. (FDACS data, 2005,
www.doacs.state.fl.us)






















Figure 1-3. Citrus canker symptoms in citrus. Citrus canker symptoms on leaves, fruit
and stem. At advanced disease stages, plants defoliate and fruit can drop
prematurely.


6MMOMM














CHAPTER 2.
USE OF TWO DIFFERENT CITRUS HOSTS TO DISTINGUISH ALL FORMS OF
CITRUS CANKER DISEASE

Introduction

Citrus canker disease is caused by several different pathogenic variants of

Xanthomonas citri (ex Hasse) (Brunings and Gabriel, 2003; Gabriel et al., 1989).

Although the taxonomy of these strains is controversial (Gabriel et al., 1989; Vauterin et

al., 1995), five groups of pathogenic variants are recognized, based primarily on field

symptoms: A, B, C, A* and Aw (Gabriel et al., 1989; Stall et al., 1982; Sun et al., 2004;

Verniere et al., 1998). The Asiatic (A) group (X. citri pv citri A) is the most severe and

widely spread throughout the world. The B and C groups (X. citri pv aurantifolii B and

C), which are also known as cancrosis B and cancrosis C, respectively, have been found

only in South America. These South American groups are phylogenetically distinct and

grow more slowly on artificial media than strains from all other groups (Brunings and

Gabriel, 2003; Goto, 1969; Stall et al., 1982). The B and C strains also have a reduced

host range compared to the A group. In addition, the C strains elicit an hypersensitive

response (HR) in grapefruit (Citrus paradise) (Stall et al., 1982). Recently two new

variants of the A group of citrus canker strains were identified and designated A* and A"

(Sun et al., 2004; Vemiere et al., 1998). Both new groups are limited in host range to

Key/Mexican lime (C. aurantifolia); the A" strain causes an HR when inoculated in

grapefruit at high concentrations (Sun et al., 2004). The A* and A" strains ofX. citri are

phylogenetically most closely related to the A group (Cubero and Graham, 2002)









Various diagnostic aids have been used to confirm citrus canker disease, including

PCR (Cubero and Graham, 2002; Mavrodieva et al., 2004), antibodies (Alvarez et al.,

1991) and microscopy. These tests can be critical if the disease appears in regions where

it has not previously been seen or has not been recently observed. Indeed, a fungal

disease was misdiagnosed as citrus canker disease in Mexico (Stapleton, 1986; Stapleton

and Garza-lopez, 1988), and a bacterial leaf spot disease was misdiagnosed as citrus

canker in Florida in 1984 (Schubert et al., 1996). Once confirmation of citrus canker

disease has been made, only host range tests can be used to reliably determine the strain

group or pathovar. Historically, these studies relied on sweet orange, mandarin orange,

lemon, lime and grapefruit (Stall and Civerolo, 1991). In this study, we report the use of

Duncan grapefruit and Mexican lime as differential hosts to differentiate strains from all

variant groups of citrus canker disease.

Material and Methods

Strains, Plasmids and Culture Media

Strains of Escherichia coli, Xanthomonas spp. and plasmids used in this study are

listed in Table 2-1 along with their relevant characteristics and source or reference. E.

coli strains were grown in Luria-Broth (LB) medium at 37 C (Sambrook et al. 1989).

Xanthomonas spp. were grown in PYGM (peptone yeast extract-glycerol-MOPS)

medium at 30 C as described by Gabriel et al. (1989). Antibiotics were used at the

following final concentrations ([tg/ml): rifampin (Rif), 75; spectinomycin (Sp), 35.

Recombinant DNA Techniques

Xanthomonas total DNA was prepared as described by Gabriel and De Feyter

(1992) and also using Amersham Biosciences DNA Isolation Kit as described by the









manufacturer. Plasmids were isolated by alkaline lysis from E. coli (Sambrook et al.,

1989) and Xanthomonas (Defeyter and Gabriel, 1991). QIAGEN's QIAprep and plasmid

midi kits were also used to isolate plasmid DNA from E. coli and Xanthomonas as

described by the manufacturer. Southern hybridizations were performed using nylon

membranes as described (Lazo and Gabriel, 1987).

Plant Inoculations

Duncan grapefruit and Mexican lime plants were grown and maintained under

natural light in the quarantine greenhouse facility at the Division of Plant Industry,

Florida Department of Agriculture, in Gainesville. Temperatures in this greenhouse

ranged from 250 C to 350 C, with 50 % to 100 % relative humidity. All inoculations were

carried out in this facility.

Liquid cultures of the tested strains were grown in PYGM medium at 300 C for

approximately 24 hr. Cultures were centrifuged @ 1000g for 3 min and cells resuspended

in equal volumes of sterile tap water (saturated with CaCO3) and infiltrated into the

abaxial surface of young, freshly flushed partially expanded citrus leaves at two

concentrations (105 cfu/ml for "low" and 108 cfu/ml for "high" levels) using the blunt end

of tuberculin syringe as described (Gabriel et al., 1989). Observations were taken 5-10

days after inoculation.

In vitro Growth Kinetics

Liquid cultures ofX. citri 3213, B69, Xc270 and X0053 were prepared in PYGM

medium and grown at 370 C overnight with slow shaking. The following day, 100 ml of

fresh PYGM was inoculated with 50 [tl of the starter culture. Optical density (OD600)

readings were taken at 0, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38 hr.

This experiment was repeated three times.









In plant Growth Kinetics

For bacterial cell counts, whole leaves were infiltrated as described. For each

strain, three leaves of each host were infiltrated. Bacterial cell counts from the inoculated

leaves were taken at days 0, 1, 6, 10 and 14. A number 7 cork borer (about 1 cm2) was

used to cut one leaf disk from each inoculated leaf per time point. Each treatment, a leaf

disk from each of the three inoculated leaves was placed in a mortar and pestle and

macerated together (in 1 ml sterile tap water saturated with CaCO3). Once homogeneity

was obtained, ten-fold serial dilution were made ranging from 10-1 to 10-9. Ten

microliters droplets of each dilution were spread on PYGM plates without antibiotics and

allowed to grow for 48 hr at 28 C. Colonies were counted from the most readily scored

dilution, and the number of cfu per cm2 of leaf tissue was calculated. The experiments

were repeated three times. Populations were expressed as log cfu/cm2 of leaf tissue.

Results

Pathogenicity Phenotypes of X citri Strains

In addition to the previously described A (3213), B (B69) and C (Xc340) strains,

newly described A* (Xc205, Xc270, Xc280, Xc290, Xc322 and Xc406) and A" (X0053)

strains (Sun et al., 2004; Verniere et al., 1998) were inoculated on citrus. All strains

tested caused hyperplastic lesions in Mexican lime that developed 5- 9 days after

inoculation (Figure 2-1). In Duncan grapefruit (Citrus paradisi) differential reactions

were observed that distinguished each group. Strains from group A elicited typical

canker symptoms in grapefruit within 6 days, but B strains elicited a whitish canker

phenotype within 10 days. C and A" strains elicited a hypersensitive response (HR) in

grapefruit. The A* strains, which were originally isolated from Southwest Asia, also

gave distinct phenotypes in Duncan grapefruit (Figure 2-2). Strains Xc205 and Xc322









did not elicit canker symptoms at low concentration (104 cfu/ml). Strain Xc406 elicited a

weak canker at low concentration, but when inoculated at high concentration, elicited

typical canker lesions. Strains Xc270, Xc280 and Xc290 did not elicit canker in Duncan

grapefruit at either low or high inoculum concentrations. A* strains could be subdivided

into three groups; A*-I, A*-2 and A*-3 (Figure 2-2). Table 2-2 summarizes symptoms

of different X citri groups on grapefruit and lime.

In vitro Growth

In vitro growth of X citri strains in liquid medium was measured by optical density

(OD600) changes recorded over time (Figure 2-3). Strains 3213 (A), X0053 (A") and

Xc270 (A*) were very similar in their growth in PYGM medium. B69 (B) strain was

significantly slower in its growth compared to A, AW and A* strains. On agar plates, the

South American B and C strains grew similarly on a variety of media, and always slower

than the A, A" and A* strains.

Growth Kinetics in plant

Growth kinetics of X. citri strains 3213 (A), 270 (A*) and X0053 (A") were studied

in Duncan grapefruit and Mexican lime leaves. In Mexican lime, growth of all three

strains was similar (Figure 2-4). However the growth kinetics of these strains was

different in Duncan grapefruit (Figure 2-4). Growth of the A" strain X0053 was reduced

by one order of magnitude as compared to A strain 3213. This reduction in growth was

noticeable 6 days post-inoculation and continued through day 14 when the comparison

ended. Growth of A* strain 270 was reduced by at least two orders of magnitude after 6

days post-inoculation as compared to strain 3213. Strain 270 did not continue to increase

after 6 days growth inplanta.









Discussion

Inoculation of different X citri strains on just two citrus host species allowed the

differentiation of all known X citri groups based on their symptoms. All tested strains of

X citri caused canker in Key/Mexican lime. Lime is a very susceptible host compared to

other citrus species or types. The B and C strains elicited a characteristic whitish canker

in key/Mexican lime that is readily distinguished from canker symptoms caused by other

strains. This is probably due to the fact that both strains are phylogeneticlly very similar

and likely share common pathogenicity factors. Indeed, the pathogenicity elicitorspthB

and pthC from the B and C strains were found to be closely related to each other (98%

similarity) at the amino acid level and different from pthA homologues from A, A* and

A" strains. The latter were closely related to each other (Chapter 4).

Strains from different groups ofX. citri exhibited quite different phenotypic

responses when inoculated in Duncan grapefruit leaves. Except for the A and B groups,

which elicited typical canker symptoms, strains from all other groups appeared much less

virulent. X0053 from the A" group elicited necrotic symptoms in grapefruit that took 5

to 10 days to appear. Unlike the Aw strain, the C group strain C340 elicited a relatively

fast HR reaction in grapefruit that took only 3 days to appear. In both cases, the necrosis

and HR, potential avr gene function is indicated. The A* group showed the most within-

group variation among strains in grapefruit; all A* strains were characterized by reduced

virulence as compared to A and B strains and lacked any evidence of eliciting necrosis or

an HR. Strains Xc205 and Xc322 were only capable of causing canker in Duncan

grapefruit when inoculated at high concentrations (108-109 cfu/ml). At (lower)

concentrations that more closely resemble field conditions, no canker symptoms were

observed with these strains. Strain Xc406 elicited a very weak canker phenotype when









inoculated at low concentration, but elicited normal canker symptoms when artificially

inoculated at high concentrations. Strains Xc270, Xc280 and Xc290 were not able to

elicit canker in grapefruit leaves at either concentration. All tested A* strains were

unable to cause canker at low inoculum concentrations or an HR at high concentrations.

Inplanta growth kinetics of strains representing the fast growing A, A* and A"

groups showed interesting differences (Figure 2-4). All three strains appeared to grow

similarly to each other in Mexican/Key lime. Asiatic strain 3213 inoculated in Duncan

grapefruit grew to levels similar to those seen in lime. A" strain X0053, which elicits

necrotic symptoms in grapefruit, grew to a final level that was only one log lower than

strain 3213. A* strain Xc270, which does not cause canker or HR in grapefruit, was

unable to grow well in grapefruit, increasing only 2 logs after inoculation and grew to a

final level that was more than two logs lower compared to strain 3213.

It is possible that A* strains carry an avr gene that specifically triggers grapefruit

defenses, but without an HR. An HR is not always observed with gene-for-gene

resistance (Bendahmane et al., 1999; Goulden and Baulcombe, 1993; Jurkowski et al.,

2004; Lehnackers and Knogge, 1990; Ori et al., 1997; Schiffer et al., 1997; Yu et al.,

2000). Indeed, a Xanthomonas avr gene that elicits host defense without an HR was

recently reported (Castaneda, 2005). An alternative explanation is that this group is

missing a factor or perhaps factors that are specifically required for growth in grapefruit,

such as the extracellular polysaccharides (EPS) and lipopolysaccharides (LPS) that are

needed by X axonopodis pv. citrumelo for virulence on citrus (Kingsley et al., 1993).

Only further experimental testing can distinguish between these explanations.









Although five different citrus host species have traditionally been used to

distinguish pathovars of X citri, all known groups can be readily distinguished by

inoculation of only two host differentials, lime and grapefruit. Even if positive control

cultures are not available, if low inoculations are used, then: 1) only the A strains elicit

green cankers in both lime and grapefruit; 2) only the B strains elicit whitish cankers in

lime and grapefruit; 3) only the C stains elicit whitish cankers in lime and an HR in

grapefruit; 4) only the A* strains elicit green canker in lime and at best very weak

cankers in grapefruit, and 5) only the A" strains elicit green cankers in lime and an HR in

grapefruit.












Table 2-1. Bacterial strains and plasmids used in this study
Strain or plasmid Relevant Characteristics Reference or source
Escherichia coli
DH5a F-, endAl, hsdR17(rkmk-), Gibco-BRL
supE44, thi-1, recAl


Xanthomonas citri
3213
3213Sp

B21.2


B69
B69Sp

C340
Xc205
Xc205Rif

Xc270
Xc270Rif

Xc280
Xc290
Xc322
Xc406
X0053
X0053Rif


Group A, wild type
Spontaneous Spr derivative of
3213, Spr
pthA::Tn5-gusA, marker
exchanged mutant of 3213 Sp,
SprKnr
Group B, wild type
Spontaneous Spr derivative of
B69, Spr
Group C, wild type
Group A*, wild type
Spontaneous Rif derivative of
Xc205, Rif
Group A*, wild type
Spontaneous Rif derivative of
Xc270, Rif
Group A*, wild type
Group A*, wild type
Group A*, wild type
Group A*, wild type
Group A", wild type
Spontaneous Rif derivative of
X0053, Rif


Gabriel et al. 1989
Gabriel et al. 1989

Swamp et al. 1991


Stall et al. 1982
El Yacoubi, 2005

Stall et al. 1982
Verniere et al. 1989
This study

Verniere et al. 1989
This study

Verniere et al. 1989
Verniere et al. 1989
Verniere et al. 1989
Verniere et al. 1989
Sun et al. 2004
This study









Table 2-2. Phenotypic differences among X citri strains.
Mexican Lime Grapefruit
Low High Low High
Strain (104-10O) cfu/ml (10-109) cfu/ml (104-10) cfu/ml (10-109) cfu/ml
A Ca C C C
B Wt Cb Wt C Wt C Wt C
C C C HRc HR
A* -1 C C 0O C
A* -2 C C WCe C
A* -3 C C 0 0
A" C C 0 HR
a=canker, b= white canker, c=Hypersensitive response, d=no canker and e=weak canker




































Figure 2-1. Inoculation of strains of different citrus canker groups in grapefruit and key
lime. A and B strains are able to cause canker in both hosts. A* and A"
strains can only cause canker in Key lime. Note the HR in grapefruit caused
by A"














205


270

280


290


406



322



290



280


270


205


322


406


406


205



270



280



290



322


290


280


406


270


205


10o-109 cfu/ml


Figure 2-2. Inoculation of several different A* strains in Duncan grapefruit. High (left)
and low (right) concentrations of bacteria were used for inoculation.


104 -105 cfu/ml








































0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Time (hr)

Figure 2-3. Growth of X citri strains in PYGM medium. A strain 3213, A* strain Xc270, A" strain X0053 and B strain B69 were
used in this comparison.














10

-9 -3213-KL


8t- X0053-KL .-,,, -O--






















/---------------------
A
6
























0 -----------------------------------------
4 ,




2 -

1 X

A
0 6 10 14
Days after inoculation




9


















grapefruit. A strain 3213, A strain Xc270 and A strain X0053 were used.
5-- ------ -7)7-- ----------------
6--.pfff

4




2

1-

B
0 6 10 14
Days post inoculatic



Figure 2-4. inplanta growth of X citri strains in A) Mexican/Key lime and B) Duncan
grapefruit. A strain 3213, A* strain Xc270 and Aw strain X0053 were used.














CHAPTER 3
IDENTIFICATION AND CHARACTERIZATION OF HOST RANGE FACTORS) IN
CITRUS CANKER STRAINS.

Introduction

Both positive and negative genetic factors have been found to affect the host range

of phytopathogenic bacteria. For example, a given Rhizobium species can only nodulate

a restricted number of hosts, and this specificity is determined by specific signal

molecules that are exchanged between the bacteria and host plants (Fisher and Long,

1993; Kondorosi et al., 1991). Some of the host specific nodulation genes needed to

actively condition the host range include NodD, NodZ, NodW, NolA and NodC (Kamst

et al., 1997). Some negatively acting factors have also been found in some rhizobia and

these have avirulence (avr) function in some hosts. For example, nodFE of R.

leguminosarum by trifolii, which is virulent in white and red clover, condition avirulence

in pea (Djordjevic et al., 1987), and nodQ and nodH were found to confer avirulence to

R. 1. bv. trifolii and R. 1. bv. viceae in their respective hosts, white clover and common

vetch (Debelle et al., 1988; Faucher et al., 1989).

Agrobacterium tumefaciens and A. rhizogenes generally have a wide host range

that includes most dicotyledonous plants. Host range in A. tumefaciens is thought to be

generally determined by positive factors; however some negative factors of host range

determination have also been found (Keen, 1990). For example, certain virulence (vir)

genes on the Ti plasmid condition host range. Progressive deletions of the 3' end of virE

were found to progressively reduce the number of plant species on which crown galls are









formed. virG from supervirulent A. tumefaciens strain extends the host range of certain

Agrobacterium strains (Chen et al., 1991; Hood et al., 1986). On the other hand virA and

virC appear to act as negative regulators of host range in grapevine. virA is involved in

detection of host specific phenolics compounds. virC was shown to act as an avr gene to

trigger host defenses in incompatible interactions and thus limit the number of plants A.

tumefaciens can infect (Yanofsky et al., 1985; Yanofsky and Nester, 1986).

Strains of the genus Xanthomonas are always found associated with plants.

Different xanthomonads attack a very wide range of plant species. However, individual

species show limited host range (Gabriel, 1999b). Members of this genus are divided

into species and pathovars, based on phylogeny, host range and disease symptom

variation. The molecular basis of host range determination at the pathovar level is not

well understood. Azad and Kado (1984) showed that elimination of the HR in tobacco to

Erwinia rubrifaciens did not increase the host range of this pathogen to include tobacco.

Similarly, Swarup et al (1992) showed that elimination of the nonhost HR did not extend

the host range of X. citri. Factors that positively enhance the host range of Xanthomonas

include the extracellular polysaccharide (EPS) and lipopolysaccharide (LPS); the opsX

locus is involved in the biosynthesis of EPS and LPS, and is also needed by X

axonopodis pv. citrumelo for virulence in citrus (Kingsley et al., 1993).

Other positive factors that could influence the host range of pathogens are

suppressors of host defenses (Ponciano et al., 2003). For example, HopPtoD2, from

Pseudomonas syringae, was found to suppress programmed cell death in plants resulting

in infection (Bretz et al., 2003; Espinosa et al., 2003; Hauck et al., 2003). Similarly,

Abramovitch et al. (2003) found that the P. syringae effector, AvrPtoB, induced plant









disease susceptibility by preventing a programmed cell death response from occurring in

tobacco plants. These results suggest that bacterial host range is determined by positive

and negative acting factors.

This chapter describes attempts to identify host range determinants of X citri. A

virulence enhancement approach (Swarup et al., 1991) was used in an attempt to identify

positive factors) required to increase host range of a narrow host range, A* strain Xc270,

to include grapefruit.

Material and Methods

Bacterial Strains, Plasmids and Culture Media

Strains of Escherichia coli, Xanthomonas spp. and plasmids used in this study are

listed in Table 3-1 along with their relevant characteristics, source and/or reference. E.

coli strains were grown in Luria-Broth (LB) medium at 37 C (Sambrook et al., 1989).

Xanthomonas spp. were grown in PYGM (peptone yeast extract-glycerol-MOPS)

medium at 30 C as described (Gabriel et al. 1989). Antibiotics were used at the

following final concentrations ([tg/ml): rifampin (Rif), 75; spectinomycin (Sp), 35;

ampicillin (Ap), 100; gentamycin (Gm), 5.

Recombinant DNA Techniques

Xanthomonas total DNA was prepared as described by Gabriel and De Feyter

(1992). Plasmids were isolated by alkaline lysis from E. coli (Sambrook et al. 1989) and

Xanthomonas (De Feyter and Gabriel 1991). Restriction enzyme digestion was

performed as recommended by the manufacturers. Southern hybridization was

performed by using nylon membranes as previously described (Lazo et al., 1987).









Vector Preparation

To identify genes involved in determining the host range of X citri pv citri,

genomic DNA from the wide host range X citri pv citri A strain 3213 was partially

digested with MboI and size fractioned on a sucrose gradient. Cosmid vector pUFR43

was used to make a DNA library of 3213 DNA fragments. This cosmid vector (Defeyter

et al., 1990) was split into two pools and cut with either EcoRI or Sal restriction enzyme

to produce the two arms and then treated with shrimp alkaline phosphatase. To create

common cloning ends the arms were then cut with BamHI and used for ligations to the 20

- 25kb 3213 DNA fraction (Figure 3-1 and 3-2).

Packaging and Transfection

The recombinant DNA was packaged using stratagene packaging mix

(Gigapack III Gold Packaging Extract), and introduced into E. coli strain DH5 *(mcr)

via transfection as described by the manufacturer protocol. Positive white plaques were

then picked and placed onto LB plates containing the antibiotic Kanamycin (20 [tg/[tl).

Using a 48 pin replicating fork, these colonies were transferred into 96 well microtiter

plates containing liquid LB (with 14% glycerol) and stored at -800 C. At the same time a

replicate of each plate was made and maintained by replicating each plate once every

month. DNA from eighteen randomly selected library clones was extracted and digested

with BamHI and electrophoresed on agarose gels in order to estimate insert size and

evaluate the quality of the library. The total number of cosmid clones required to cover

the entire 3213 genome (N) was determined using the following formula (Clarke and

Carbon, 1976):











N In(1- 0.99)
insert size
STotal genome size)j
Plant Inoculations

Duncan grapefruit and Mexican lime plants were grown and maintained under

natural light in the quarantine greenhouse facility at the Division of Plant Industry,

Florida Department of Agriculture, Gainesville, Fl. Temperatures in this greenhouse

ranged from 25C to 350 C with 50% to 100% relative humidity. All inoculations were

carried out in this facility.

Liquid cultures of all tested strains were grown in PYGM medium at 300 C for

approximately 24 hr. Cultures were centrifuged and resuspended in equal volumes of

sterile tap water (saturated with CaCO3) and pressure infiltrated at appropriate

concentrations (105 for low and 108 cfu/ml for high) into the abaxial surface of citrus leaf

using the blunt end of tuberculin syringes. Observations were taken 5-10 days after

inoculation. For screening of large numbers of clones, colonies were streaked onto

PYGM agar plates incubated at 300 C for approximately 24 hr, resuspended in sterile tap

water (saturated with CaCO3) and pressure infiltrated into citrus as described.

Triparental Matings

To transfer the 3213 library to the limited host range Xc270 (A*) strain, triparental

matings were performed as described by Defeyter et al (1990). Strain pRK2073 was used

as a helper strain. The recipient was concentrated 50 100 fold. Transconjugants were

screened on PYGM plates containing Rif 75[tl g/ml and Gm 3[tl g/ml at 280 C and 2-3

days later colonies were transferred onto new selection plates.









Results

Xanthomonas citri pv citri A 3213 Strain Genomic Library

A genomic library of X citri pv citri A, strain 3213, was made and 18 randomly

picked clones were evaluated for insert size and pattern (Figure 3-3). All 18 clones gave

different restriction patterns indicating random insertions in the vector. The average size

of the inserts was 39 kb. Based on the Clark and Carbon formula (Clarke and Carbon,

1976), 610 clones were required to cover the whole X citri 3213 genome with 99%

probability. Seven hundred and fifty clones were maintained in E. coli strain DH5ca and

stored in 15% glycerol at -800 C.

Screening of 3213 Library in Xc270

Five hundred and fifty clones were transferred from the 3213 library into X citri

pv. citri A*-3 strain Xc270 by triparental mating and transconjugants were individually

screened for symptoms in Duncan grapefruit. No clones were identified that consistently

increased the pathogenicity of Xc270.

Discussion

Attempts to identify positive host range determinants from X citri were

unsuccessful when a library from the wide host range group A strain 3213 was moved

into the narrow host range group A* strain Xc270. Initially six clones (pAW377,

pAW378, pAW380, pAW400, pAW413 and pAW419) seemed to elicit canker-like

symptoms in Duncan grapefruit, but when those clones were re-conjugated into Xc270,

the initial results were not confirmed. The inplanta growth of Xc270 described in

chapter 2 showed that the Xc270 grew poorly in Duncan grapefruit, suggesting that the

only clones that would complement Xc270 and cause canker in grapefruit would be those

that would increase growth. It is likely that inplanta growth requires multiple effectors,






30


and that no individual cosmid would carry enough factors to reveal a strong difference.

Another possibility is that Xc270 may carry avr genes that function in grapefruit and

prevent Xc270 from growing. Avirulence is usually epistatic over virulence and

therefore a screen for positive factors would fail if this were the case. Perhaps a better

approach would be to construct a library of Xc270 DNA and screen in 3213 in order to

identify any avirulence gene function.









Table 3-1. Strains and plasmids used in this study
Strain or plasmid Relevant Characteristics
Escherichia coli
DH5a F-, endAl, hsdR17(rk-mk-), st


Xanthomonas
citri
3213
3213Sp
B21.2

Xc270
Xc270Rif


Plasmid
pRK2013

pRK2073

pURF043

pAW377

pAW378

pAW380

pAW400

pAW413

pAW419


recA1


Reference or source


pE44, thi-1,


Group A, wild type
Spontaneous Spr derivative 3213, Spr
pthA::Tn5-gusA, marker exchanged
mutant of 3213 Sp, SprKnr
Group A*, wild type
Spontaneous Rif derivative of Xc270,
Rif


ColEl, Kmr,Tra helper plasmid

pRK2013 derivative,npt::Tn7,
KmsSpr,Tra+, helper plasmid
IncW, Mob+, lacZa+, Gmr, Nmr, cos,
shuttle vector
Fragment from X citri 3213 library
cloned in pUFR43
15 kb fragment from X citri 3213 library
cloned in pUFR43
Fragment from X citri 3213 library
cloned in pUFR43
Fragment from X citri 3213 library
cloned in pUFR43
24 kb fragment from X citri 3213 library
cloned in pUFR43
Fragment from X citri 3213 library
cloned in pUFR43


Gibco-BRL


Gabriel et al. 1989
Gabriel et al. 1989
Swamp et al. 1991

Verniere et al. 1989
This study


Figurski and Helinski,
1979
Leong et al. 1982

De Feyter and Gabriel,
1991
This study

This study

This study

This study

This study

This study














E=EcoRI
B=BamHI
S=SalI

Sail


S Eos B S



SPhosphotase


S E BS
1 1 V...


E B S

iI,
11 1


E coRI


I '


[P
E BS


BamHI
S E B B S



+ inserts
S E B M MbolBS


I Ligase


ar


Heads



Transfect E. coli
Transfect E. coil


Figure 3-1. Scheme for cosmid vector preparation and DNA cloning.


cos


rI


E



hosphotase

E
I


l















-23kb


23kb V---"'- R`" "
9.4kb
6.5kb

4.3kb












Figure 3-2. DNA fractionation. A). Partial digestion of X. citri 3213 genomic DNA (0.7%
agarose gel). B) Size fractionation of Mbol partial digest of 3213 DNA (0.7%
agarose gel).











1A 1 9 3 4A A 7 R Q 1i 11 19 13 14 15 16 17 1R 1A


-23 kb




-9.4 kb

-6.5 kb


-4.3 kb




-2.3 kb
-2.0 kb


Figure 3-3. Restriction profiles of random clones from X citi 3213 genomic library. DNA
was digested with EcoRI. As a merker, X DNA digested with HindIII (M).














CHAPTER 4.
SEQUENCE COMPARISON AND CHARACTERIZATION OF FIVE NEW pthA
HOMOLOGS FROM FOUR DIFFERENT Xanthomonas citri STRAINS.

Introduction

All strains of Xanthomonas citri cause hyperplastic pustules in citrus that are

dignostic of citrus canker disease (Gabriel, 2001). The Asiatic (A) group (X citri pv citri

A) has the widest host range and is widespread throughout the world. The B and C

groups (X citri pv aurantifolii B and C) have only been found in South America and have

a reduced host range compared to the A groups (Stall and Seymour, 1983). New groups

of X citri pv citri (Aw from Florida and A* from Southwest Asia) were more recently

identified that are primarily restricted in host range to Mexican lime (Citrus aurantifolia)

(Stall et al., 1982b; Sun et al., 2004). Grapefruit (C. paradisi) serves as a differential host

that is resistant to the A*, A" and C strains; the A" and C strains elicit a strong

hypersensitive response (HR) in grapefruit, while some A* strains show reduced growth

in grapefruit (Chapter 2). The molecular basis for avirulence of the A*, A" and C strains

in grapefruit and the wide host range of the A strains is unknown.

Pathogenicity gene pthA encodes the primary causal effector of the citrus canker

disease phenotype (Duan et al., 1999; Swarup et al., 1991; Swamp et al., 1992). All

strains of X citri tested carry pthA homologs (Cubero and Graham, 2002; Mavrodieva et

al., 2004). pthA is capable of conferring ability to cause canker-like symptoms to strains

that cannot otherwise cause canker, such as X campestris pv citrumelo (Swarup et al.,

1991) or even E. coli carrying a functional hrp system (Kanamori and Tsuyumu, 1998).









When pthA is transiently expressed in citrus using either Agrobacterium tumefaciens or

particle bombardment, small canker-like lesions are elicited (Duan et al., 1999).

pthA is the first member of the avrBs3/pthA gene family demonstrated to function

for pathogenicity. The vast majority of cloned or described Xanthomonas avirulence

genes belong to this family; many have demonstrated pathogenicity functions (Leach and

White, 1996). Members of this gene family show very high levels of homology at the

DNA sequence level (De Feyter et al., 1993; Hopkins et al., 1992; Leach and White,

1996; Yang et al., 2000). All members encode more than 11 nearly perfect, 34 amino

acid, leucine rich, tandemly arranged, direct repeats. Swapping repeat regions between

members of the gene family results in chimeric genes that confer the pathogenicity and/or

avirulence phenotypes expected from the source genes (Herbers et al., 1992; Yang et al.,

1994). Although pthA can confer avirulence to other xanthamonads (Swarup et al.,

1992), nopthA homolog from X citri is known to function for avirulence in citrus.

Conversely, although the pthA homolog aplI from X citri pv citri group A has been

suggested as a suppressor of the tobacco defense response (Ponciano et al., 2003), no pthA

homolog from X citri is known to suppress citrus host defenses.

The purpose of this study was to clone, isolate, sequence and characterize pthA

homologs that function to determine pathogenicity from all known X citri groups. A

secondary purpose was to determine if any of these pthA homologs also determined

avirulence in grapefruit or could increase the pathogenicity of an A* strain in grapefruit.

Material and Methods

Bacterial Strains, Plasmids and Culture Media

Strains of Escherichia coli, Xanthomonas spp. and plasmids used in this study are

listed in Table 4-1 along with their relevant characteristics and source or reference. E.









coli strains were grown in Luria-Broth (LB) medium at 37 C (Sambrook et al., 1989).

Xanthomonas spp. were grown in PYGM (peptone yeast extract-glycerol-MOPS)

medium at 30 C as described (Gabriel et al. 1989). Antibiotics were used at the

following final concentrations (tlg/ml): rifampin (Rif), 75; spectinomycin (Sp), 35;

chloramphenicol (Cm), 35; ampicillin (Ap), 100; gentamycin (Gm), 5; kanamycin (Kn),

25.

Recombinant DNA Techniques

Xanthomonas total DNA was prepared as described (Gabriel and De Feyter, 1992).

Plasmids were isolated by alkaline lysis from E. coli (Sambrook et al., 1989) and

Xanthomonas (De Feyter and Gabriel, 1991). Southern hybridization was performed by

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

DNA Library Construction

Genomic DNA from the wide host range Xanthomonas citri pv citri group A strain

3213 was partially digested with MboI and size fractioned on a sucrose gradient. The

cosmid vector pUFR43 was used to make a DNA library of 3213 DNA fragments. This

cosmid vector was split into two pools and cut with either EcoRI or Sal restriction

enzyme to produce the two arms and treated with shrimp alkaline phosphatase. To create

common cloning ends, the arms were then cut with BamHI and used for ligations to the

20 25kb 3213 DNA fraction. Recombinant DNA was packaged using Stratagene *

packaging mix (Gigapack III Gold Packaging Extract), and introduced into E. coli

strain DH5 via transfection as described by the manufacturer protocol. Positive white

plaques were then picked and placed onto LB plates containing the antibiotic Kn (20

[tg/[tl). Colonies were transferred into 96 well micro titer plates containing liquid LB









(with 14% glycerol) and stored at -800 C. DNA from eighteen randomly selected library

clones was extracted, digested with BamHI and run on agarose gels in order to estimate

insert size and evaluate the quality of the library.

Plant Inoculations

Duncan grapefruit and Mexican lime plants were grown, maintained and inoculated

under natural light in the quarantine greenhouse facility at the Division of Plant Industry,

Florida Department of Agriculture in Gainesville, Fl. Temperatures in this greenhouse

ranged from 25C to 350 C with 50% to 100% relative humidity.

Liquid cultures of tested Xanthomonas strains were grown in PYGM at 30 oC for

approximately 24 hr. Cultures were centrifuged @ 1000g for 3 min at room temperature,

and resuspended in equal volumes of sterile tap water (saturated with CaCO3) and

pressure infiltrated at appropriate concentrations (105 for low and 108 cfu/ml for high)

into the abaxial citrus leaf surface using the blunt end of a tuberculin syringe.

Observations were taken 5- 10 days after inoculation.

Southern Hybridization Analysis

Genomic DNA from all canker causing strains were isolated as described, digested

with either EcoRI or BamHI restriction enzyme and the digested DNA was analysed by

electrophoresis on 0.6% agarose gels. DNA was then transferred onto GeneScreen Plus

(DuPont, Wilmington, Delaware) nylon membranes as described by the manufacturer.

Membranes were hybridized with a 32P-labeled BamHI internal fragment ofpthA.

Colony Hybridization

Plasmid DNA from Aw strain X0053 was digested with EcoRI andKpnI and

ligated into shuttle vector pUFR047. Recombinant DNA was transformed into DH5

competent cells, and transformed clones were selected on Apl00 and X-Gal/IPTG in LB









agar. White colonies were transferred onto a registry plate and pZit45 was included at

specific positions as a control. Plasmid DNA from A* strain Xc270 was digested with

EcoRI and HindlII and ligated into shuttle vector pUFR71. Recombinant DNA was

transformed into DH5ca and selected on Cm35 LB and X-Gal/IPTG plates. White

colonies were transferred from registry plates onto Colony/PlaqueScreenTM hybridization

transfer nylon membranes and placed colony side up on plain LB plates and incubated for

2- 4 hr at 37 C. DNA was fixed on membranes as described by the manufacturer and

hybridized with a 32P-labeled BamHI fragment ofpthA. Group B strain B69 plasmid

DNA was digested with EcoRI, and a 23 kb and a 4.3 kb fragment that hybridized with

pthA were cloned in pUFR53 resulting in pQY93.3 and pQY22.1, respectively. A 14kb

HindIII fragment within the pQY93.3 EcoRI fragment was subcloned in pUFR53,

resulting in pQY96. Group C strain C340 plasmid DNA was digested with Sal, and a 20

kb and a 6 kb fragment that hybridized withpthA were cloned into pUFR53, resulting in

pQYC2.1 and pQYC 1.1, respectively. The 6 kb insert from pQYC1.1 fragment was

cloned into the high copy vector pUC 19 resulting in pQY103.5.

Triparental Matings

Clones that hybridized topthA were conjugated into Xanthomonas strain B21.2

(pthA::Tn5) by triparental mating as described by Defeyter et al (De Feyter et al., 1990).

Strain pRK2073 was used as a helper strain. The recipient strain was concentrated 50 -

100 fold for higher conjugation rate. 10 [tl of each recipient, donor and helper were

mixed together on PYGM plate and allowed to grow for 6 hr to overnight at 280 C.

Transconjugants were screened on PYGM plates containing Sp 35 [tl g/ml and Gm 5[tl

g/ml at 280 C. Two to three days later colonies were transferred onto new selection









plates. Successful transconjugants were infiltrated into Duncan grapefruit and

Mexican/Key lime. Southern blot analysis was used to further analyze clones.

Marker Integration Mutagenesis

The mutants BIM2 (pthB::pUFR004) of B69Sp and CIM1 (pthC::pUFR004) of C340

were created by the integration of pYY40.10 (2.0 kb internal Stul-HincII fragment of

pthA in pUFR004), and Cm resistant colonies were selected.

Sequence Analysis ofpth Genes

pthA homologs from A*, A", B and C strains were sequenced using primers based

on the sequence ofpthA (Swamp et al., 1992) and designed to cover the entire gene.

Seven primers were used for sequencing reactions; DG8: gaggtggtcgttggtcaacgc, DG35:

agttatctcgccctgatc, DP35: caggtcactgaagctgcccgc, DP36: gcgggcagcttcagtgacctg, DP37:

ccgaaggttcgttcgaca, DP38:ctgtcgaacgaaccttcg, DP45: gcatggcgcaatgcactgac, and YP03:

tagctccatcaaccatgc. Sequencing was done at the UF ICBR DNA Sequencing Core,

Gainesville, FL. When necessary, fragments were cloned into high copy vectors such as

pUC 119 or pUC19 to obtain larger amounts of DNA.

Sequence information of these genes was used to construct the full DNA sequence

using Vector NTi software (Invitrogen, Carlsbad, California). Nucleotide and predicted

amino acid sequence alignments were carried out with the program CLUSTAL W.

Percent amino acid identity was calculated using the needle program in EMBOSS

package which uses the Needleman-Wunsch algorithm to do global alignment of

sequences. The DNA sequences ofpthA1, pthA2, pthA3 andpthA4 (da Silva et al., 2002)

were taken from GenBank Accessions # NC 003921, NC 003921, NC 003922 and

NC_003922, respectively. The DNA sequences of apll,apl2 and apl3 (Kanamori and









Tsuyumu, 1998) were taken from GenBank Accessions # AB021363, AB021364 and

AB021365, respectively. Dendograms showing phylogenetic relationships of these genes

were generated with TREECON (version 1.3b) (Van de Peer and De Wachter, 1994)

using neighbor-joining algorithm with Poisson correction. RSc1815, an avrBs3/pthA

gene from Ralstonia solanacearum was used as an outgroup for phylogenetic tree

construction. The percentage of trees from 100 bootstrap resamples supporting the

topology is indicated when the percentage is above 70.

Results

Southern Blot Analysis

Southern blot analyses revealed that all tested X citri strains have at least two

BamHI DNA fragments that strongly hybridized to an internal BamHI fragment from

pthA (Figure 4-la; some data not shown). With the exception of group A strains, which

had four BamHI fragments that hybridized withpthA, all other strains from all other

groups, including the A*, A", B and C groups, had only two such BamH1 fragments. All

strains tested appeared to share a 3.4 kb BamHI hybridizing fragment of a size similar or

identical to that ofpthA. Otherwise, each of the different phenotypic groups exhibited

distinct and characteristic banding patterns. Based on the hybridization intensity of both

BamHI and EcoRI digested DNA fragments and other results (not shown), the C and A"

strains appeared to carry their two hybridizing DNA fragments on a single plasmid

(Figure 4-1).

Cloning, Characterization and Sequencing ofpthA Homologs from X. citri A*, AW, B
and C Strains

Using an internal fragment ofpthA as a probe, colony hybridization of E. coli

carrying cloned group A" strain X0053 plasmid DNA revealed eight colonies with









hybridizing inserts (Figure 4-2). The plasmids from these colonies were designated as

pAW5.1 5.8, and all carried hybridizing inserts of identical size (Table 4-1). Four of

these inserts were separately introduced into strain B21.2 (pthA::Tn5) and screened for

pathogenicity. All four clones complemented the knockout phenotype of B21.2 and

restored ability to cause canker in both Duncan grapefruit and Mexican lime (Figure 4-3;

Table 4-2). The pthA homolog encoded on pAW5.2 was sequenced and designated

pthAW.

Similarly, colony hybridization of cloned group A* strain Xc270 plasmid DNA

revealed three hybridizing clones, designated as pAW12.1 12.3. The inserts carried on

pAW12.1 and 12.2 were identical in size; pAW12.3 was smaller. When transferred to

B21.2, pAW12.1 complemented B21.2 and resulted in canker symptoms in both

grapefruit and lime (Figure 4-4; Table 4-2). ThepthA homolog encoded on pAW12.1

was sequenced and designatedpthA *. pAW12.3 did not complement B21.2 in either host

(Table 4-2). The pthA homolog from pAW12.3 was sequenced and designated pthA*-2.

pthA *-2 carried only 15.5 internal repeats. To verify that the lack of evident activity of

pthA *-2 was not due to a cloning artifact, the promoter region and Shine-Dalgarno (SD)

sequence were verified to be present on pAW12.3. In addition, no premature stop codons

or frame shifts were found inpthA *-2.

Colony hybridization of cloned group B strain B69 plasmid DNA revealed several

hybridizing clones of two different sizes. Representative clones of both sizes were

selected for complementation tests. pQY93.3 (23 kb insert) and pQY22.1 (4.3 kb insert)

were mobilized by conjugation into B21.2; only pQY93.3 was found to complement

B21.2, resulting in canker symptoms in both grapefruit and lime (Table 4-2). The pthA









homolog was subcloned from pQY93.3 on pQY96, verified as functional in B21.2

designated as pthB and sequenced.

Finally, colony hybridization of cloned group C strain C340 plasmid DNA revealed

several hybridizing clones of two different sizes, and again representative clones of both

sizes were selected for complementation tests. pQYC2.1 (20 kb insert) and pQYC 1.1 (6

kb) were mobilized by conjugation into B21.2; only pQYC1.1 was found to complement

B21.2, resulting in canker symptoms in both grapefruit and lime (Table 4-2). The pthA

homolog encoded on pQYC 1.1 was designated as pthC and sequenced.

Even when inoculated at high concentrations, none of the pthA homologs (pthA W,

pthA *, pthA *-2, pthB orpthC) in B21.2 elicited an HR in grapefruit.

Inactivation and Complementation of Genes pthB and pthC in X. citri pv aurantifolii

In order to determine the role ofpthB in the pathogenicity of X citri pv aurantifolii

group B strain B69Sp in citrus, marker integration mutagenesis was carried out.

Southern blot analysis showed thatpthB had been interrupted in BIM2 (pthB::pUFR004).

BIM2 was unable to cause canker (data not shown). BIM2 was fully complemented by

pAB2.1, pZit45, pAB18.1 (all carryingpthA), pQY96 (carryingpthB) and pQYC1.1

(carrying pthC) to elicit wild type response in grapefruit and lime (data not shown). In

order to determine the role of gene pthC in the pathogenicity of group C strain C340 in

citrus, marker integration mutagenesis was carried out. Southern blot analysis showed

thatpthC had been interrupted in CIM1 (pthC::pUFR004). CIM1 was unable to cause

typical canker symptom in lime, but elicited an HR in grapefruit that was as strong as the

HR elicited by the wild type strain C340. CIM1 was fully complemented by pZit45

(pthA), pQY96 (pthB) and pQYC1.1 (pthC) to elicit a wild type response in lime (data

not shown).









None of the pthA Homologs from Group A Strain 3213 Increased the Host Range of
Group A* Strain 270 to Include Grapefruit

All fourpthA homologs from group A strain 3213 were isolated and cloned from

the 3213 library by colony hybridization with an internal fragment ofpthA: pAW20.2,

pAW20.4, pAW20.7 and pAW20.11 carry pthA, pthAl, pthA2 and pthA3, respectively

(Figure 4-5). None of these clones complemented B21.2. When these clones were

conjugated into the A* strain Xc270, none extended the host range of the strain to include

Duncan grapefruit. As with Xc270, all three transconjugants elicited cankers in Mexican

lime. When pZit45, which carriespthA from 3213 and complements B21.2 (Swarup et

al., 1992), and pAW20.2 were introduced into Xc270, they similarly did not extend the

host range of Xc270 to include grapefruit (Figure 4-5).

Sequence Analysis ofpthA Homologs from All Known X. citri Groups

The DNA sequences of all 13 available pthA homologs were analyzed and the

predicted amino acid sequences were found to be >75% identical (Table 4-3). With the

notable exception of Apl3, all seven other PthA homologs within X citri pv citri group A

(PthA, PthAl, PthA2, PthA3, PthA4, Apll and Apl2) were more closely related to each

other (> 92% identical), than the active PthA homologs from all X citri groups (PthA,

PthB, PthC, PthAW and PthA*) which were >97% identical (Figure 4-6). Comparative

analysis of the 34 aa direct repeat regions of all thirteen genes revealed three primary

regions of variation within each repeat, at positions 3 and 4 (region 1), positions 11-13

(region 2) and positions 30-32 (region 3) (Figure 4-7). In region 1, no particular set of

amino acids was universally conserved among any of the repeats of activepthA

homologs. However, in regions 2 and 3, and only in repeat number 17 in each gene,









N(12)G(13) in region 2 and Q(31)A(32) in region 3 were correlated with active

pathogenicity gene function.

Discussion

Southern hybridization analyses of a limited number ofX. citri strains revealed a

common 3.4 kb BamHI band shared by all strains examined in all five described groups

of strains; all group A strains tested carried four hybridizing fragments, while all other

strains examined carried only two. Among the 13 sequenced and functionally tested pthA

homologs, including three tested by others [Apl1, Apl2 and Apl3; (Kanamori and

Tsuyumu, 1998)] and the ten tested in this study, only the 3.4 kb fragment appeared to

encode the active pathogenicity gene that is required for elicitation of citrus canker. This

includes genes pthA *, pthA W, pthB and pthC from the A*, A", B and C strains,

respectively, as well aspthA. All five of these genes were found to be fully isofunctional,

and capable of eliciting the typical canker phenotype in grapefruit in B21.2, even though

the source A*, A" and C strains were unable to elicit the canker phenotype in grapefruit.

Furthermore, pthA *, pthA W and pthC did not elicit an avirulence phenotype of any type

in B21.2, despite being members of an avr gene family, and despite the avirulence of the

respective source strains in grapefruit. Indeed, thepthC knockout mutation in CIM1

eliminated pathogenicity in lime, but did not affect the HR in grapefruit, which remained

as strong as that elicited by the wild type. The HR elicited by the wild type C group

strain C340 is therefore independent ofpthC. These results suggest that the A*, A" and

C strains likely carry yet to be identified avr genes that prevent compatible phenotypes

from developing in grapefruit.

The C strain C340 and A* strain Xc270 fragments that hybridized with pthA did

not complement B21.2 to pathogenicity in lime or grapefruit. The sequenced Xc270









homolog that failed to complement, pthA *-2, carried 15.5 repeats and appeared to have

intact promoter, a SD region and an open reading frame. This gene was 97% identical to

PthA2 and Apl2 (Table 4-3) and carried the same number of repeats. All three of these

genes appear intact and yet also appear non-functional in terms of pathogenicity or

avirulence. Although the C340 homolog (on pQYC2.1) that did not complement B21.2

was not sequenced, restriction enzyme analysis (not shown) of the 20 kb insert indicated

that the promoter region was intact, making this homolog unlikely to be responsible for

avirulence in grapefruit.

The other three group A 3213 pthA homologs did not complement B21.2 and also

appeared to be non-functional, confirming and extending the work of Kanamori and

Tsuyumu (Kanamori and Tsuyumu, 1998) on group A strain L-9. However, the fact that

all wide host range group A strains examined carry two additional pthA homologs that are

not present in the more narrow host range B, C, A* and A" strains suggests a potential

role in determining host range. Indeed, Ponciano et al (2003) reported that apll, apthA

homolog that is functionally equivalent topthA but found in a different group A strain,

suppressed tobacco defense response and HR. However, whenpthA or any of its 3213

homologs (pthA1, pthA2 orpthA3) were transferred into Xc270, no increase in host range

of A* strain Xc270 to include grapefruit was observed (Figure 4-5). Although, additional

pthA homologs in a given X citri strain may contribute marginally to pathogenicity

(Kanamori and Tsuyumu, 1998), the primary value of multiple copies of the gene family

in a given strain may be to facilitate recombination and the potential for rapid adaptation

to new hosts (Gabriel, 1999a; Yang and Gabriel, 1995).









All pthA homologs that are required for citrus canker disease from all five known

X citri groups carried exactly 17.5 repeats. All other homologs, even those nearly

identical topthA (e.g., from X citri pv citri group A) were not required for canker and

had a different number of repeats. Interestingly, deletion mutants of various repeats and

numbers of repeats in pthA can result in a gene that confers a weak canker phenotype in

citrus to B21.2 (Yang and Gabriel, 1995). In that study, however, repeat numbers 1-5

and 16,17 were not affected in deletion derivatives capable of conferring canker. This

indicates that while the total number of repeats may be important, the number of repeats

may be less important than the relative location of the specific repeats within the gene.

Surprisingly, sequence variation among these activepthA genes (PthA, PthAW, PthA*,

PthB and PthC) was greater than variation among the pthA homologs within the A group.

Even the nonfunctional homologs were closer to the active genes within the A group than

to active homologs from B and C groups.

The relatively high level of variation within the active homologs from different

phylogenetic groups allowed the possibility of identifying amino acids within the 34 aa

direct repeat that might be critical for pathogenic specificity in citrus. Three somewhat

variable regions were found in each of the repeats, at amino acid positions 3 and 4, 11-13,

30-32. The aligned repeat regions of all active genes revealed that only amino acids

N(12)G(13) in the second and Q(31)A(32) in the third variable regions of the 17th repeat

were conserved. No such conservation of identical amino acids was found in any other

repeat (Figure 4-6). Interestingly, only the 17th repeat of the South American group B

and C strains show a sequence identity to Asiatic strains in the third variable region.

Q(31),A(32) is not seen in any other PthB repeat and in only two other PthC repeats,






48


which favor E(31)Q(32) at that position. In addition, the deletion mutants evaluated by

Yang and Gabriel (Yang and Gabriel, 1995) never affected the 17th repeat. These results

suggest that the 17th repeat may be critical for pathogenicity of X citri.









Table 4-1. Strains and plasmids used in this study


Plasmids
pRK2013

pRK2073

pUC119

pUFR004
pUFR043

pUFR047


Relevant Characteristics


F-, endAl, hsdR17(rk-mk-), supE44, thi-1,
recAl


Strain or
plasmid
Escherichia coli
DH5a


Xanthomonas
citri
3213
3213Sp
B21.2

B69
B69Sp
C340
Xc205
Xc205

Xc270
Xc270Rif

Xc280
Xc290
Xc322
Xc406
X0053
X0053Rif

BIM2

CIM1


Reference or source


Gibco-BRL


Gabriel et al. 1989
Gabriel et al. 1989
Swamp et al. 1991

Stall et al. 1982

Stall et al. 1982
Verniere et al. 1989
This study

Verniere et al. 1989
This study

Verniere et al. 1989
Verniere et al. 1989
Verniere et al. 1989
Verniere et al. 1989
Sun et al. 2004
This study

El-Yacoobi, 2005

This study


Group A, wild type
Spontaneous Spr derivative 3213, Spr
pthA::Tn5-gusA, marker exchanged
mutant of 3213 Sp, SprKnr
Group B, wild type
Spontaneous Spr derivative of B69, Spr
Group C, wild type
Group A*, wild type
Spontaneous Rif derivative of Xc205,
Rif
Group A*, wild type
Spontaneous Rif derivative of Xc270,
Rif
Group A*, wild type
Group A*, wild type
Group A*, wild type
Group A*, wild type
Group A", wild type
Spontaneous Rif derivative of X0053,
Rif
pthB::pUFR004, marker integrated mutant
ofB69Sp
pthC::pUFR004, marker integrated mutant
of C340


ColEl, Kmr,Tra helper plasmid

pRK2013 derivative, npt::Tn7, KmsSpr,
Tra+, helper plasmid
ColE1, M13 Ig, Apr, lacZo

ColE1, Mob+, Cmr, lacZ+
IncW, Mob+, lacZa+, Gmr, Nmr, cos,
shuttle vector
IncW, Mob+, lacZa+, Par+, GmrApr


Figurski and Helinski
1979
Leong et al. 1982

Vieira and Messing,
1987
De Feyter et al. 1990
De Feyter and Gabriel,
1991
De Feyter et al. 1993









Table 4-1. Continued.


Table 4-1. Continued.


Relevant Characteristics


Strain or
plasmid
pUFR053

pUFR071
pYD9.3
pZit45

pAB2.1

pAB18.1

pQY93.3

pQY22.1

pQY99.3

pQY96

pQY103.5

pQYC 1.1

pQYC2.1

pAW5.1- 5.8

pAW12.1-12.2


pAW12.3


pAW20.2

pAW20.4

pAW20.7

pAW20.11


Reference or source


IncW, Gmr,Cmr, Mob+, mob(P), lacZo+,
Par+
IncW, Mob+, Cmr, Gmr, lacZ Par+
pthA in pUC118, Apr
4.5Kb fragment containing pthA from
3213 cloned in pUFR47, Apr
EcoRI/HindIII fragment of pZit45,
containing pthA, in pLAFR3
EcoRI/HindIIl fragment of pYD9.3,
containing pthA, in pUFR47
23 kb EcoRI fragment containing pthB in
pUFR53
4.3 kb EcoRI fragment containing pthBo
(non-functional) in pUFR53
8.8 Kb Sal fragment containing pthB
from B69 was cloned in pUC 119
14 kb HindIII fragment containing pthB
cloned in pUFR53
5 Kb Sal fragment containing pthC from
C340 cloned in pUC119
6 kb Sal fragment containing pthC cloned
in pUFR47
20 kb Sal fragment containing pthCo
(non-functional) cloned in pUFR47
5Kb EcoRI-KpnI fragment containing
pthA Wfrom X0053 cloned in pUFR47
22 kb EcoRI/HindIII fragment containing
pthA from A* group strain Xc270 cloned
in pUFR71
6 kb EcoRI/HindIII fragment containing
pthA *-2 from A* group strain Xc270
cloned in pUFR71
36 kb MboI fragment containing pthA
from 3213 cloned in pUFR43
17 kb MboI fragment containing pthA 1
homolog from 3213 cloned in pUFR43
32 kb MboI fragment containingpthA2
homolog from 3213 cloned in pUFR43
40 kb MboI fragment containingpthA3
homolog from 3213 cloned in pUFR43


El-Yacoobi, 2005

Castaneda, 2005
Duan et al. 1999
Swamp et al. 1992

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study

This study


This study


This study

This study

This study

This study










Table 4-2. Phenotypic responses of X citri strains in 2 citrus hosts
Mexican Lime Grapefruit
Lowa Highb Low High
Strains/Plasmid
3213 +C + + +

B21.2 Od 0 0 0

B21.2/pZit45(pthA) + + + +

B21.2/pQY96(pthB) + + + +

B21.2/pQYC1.1(pthC) + + + +

B21.2/pAW5.2(pthA W) + + + +

B21.2/pAW12.1(pthA*) + + + +

B21.2/pAW12.3(pthA *2) 0 0 0 0
a= 104-10cfu/ml, b=108-109cfu/ml, c= canker, d= no canker,













Table 4-3. Amino acid sequence identity between pathogenicity genes from X citri strains

PthA PthA4 Apll PthAW PthA* PthA*-2 PthAl PthA2 PthA3 Apl2 Apl3 PthB PthC
100 100 100 99 98 92 95 93 92 94 84 87 87
PthA
100 100 99 98 92 95 93 92 94 84 87 87
PthA4
100 99 98 92 95 93 92 94 84 87 87
Apll
100 97 92 95 93 92 93 84 87 87
PthAW
100 92 95 93 92 93 84 87 87
PthA*
100 95 97 97 97 79 82 83
PthA*-2
100 95 95 95 81 84 85
PthAl
100 98 99 79 82 82
PthA2
100 98 79 82 82
PthA3
100 80 82 82
Apl2
100 75 75
Apl3
100 98
PthB
100
PthC







































internal fragment of pthA. A). BamHI restriction digested genomic DNA
from ciri strains. B). EcoRI restriction digested genomic DNA.
-- urn-y








Figure 4-1. Southern Hybridization analysis of X citri strains hybridized with the BamHJ
internal fragment of pthA. A). BamHI restriction digested genomic DNA
from X citri strains. B). EcoRI restriction digested genomic DNA.






54




+ control














Figure 4-2. Colony Hybridization of E. coli with cloned X0053 A" plasmid DNA
fragments using 32P-labeledpthA. pZit45 (pthA) was used as a positive
control.



















2 3213




B21.2/ B21.2/
pAW5.5 pAW5.2


B21.2/
pAW5.2


B21.2/
pAW5.4


B21.2




B21.2/
pAW5.5




p11.2/
p \V% 5.8


B21.2/
pAW5.4


Figure 4-3. Complementation of A strain knockout B21.2 (pthA::Tn5) withpthA
homologs from AW strain X0053 in citrus. pAW5.2, pAW5.4, pAW5.5 and
pAW5.8 carry fragments that hybridized withpthA in grapefruit (left) and
Key lime (right).


































Figure 4-4. Complementation of A strain knockout B21.2 (pthA::Tn5) withpthA
homologs in citrus. pthA W (pAW5.2), pthA (pAW12.1) and pthA *-2
(pAW12.3) in B21.2 andpthA (3213) in grapefruit (left) and Key lime (right).



































Figure 4-5. Analysis ofpthA and its three homologs in A* strain Xc270. Key lime (left)
and grapefruit (right)
















0.1 sutbtitutionsite


apit

170 pMr.4


pIkAW
-prhA W M





MV ITpl2 M7



Mrw8 (XD6861)}
rf L vr(d l627 I Q1)
1a-vrfslr 4x1697E1



.pthN AF2i 6.11
--- rp A 2.4r

aX-- a7 Af26I933B





avzij&13 (A-rl571 I)
pIkB-Xcm (An 23125)


ptIkB-KA
-- c II IqNCOpL 2S5)

Figure 4-6. Neighbor-joining dengogram depicting phylogenetic relationship based on pairwise comparison of neucleotide sequences
of members of avrBs3/pthA genes from different species and pathovars ofXanthomonas. Numbers at the nodes represent
bootstrap values (based on 100 replicates). GenBank Accesions numbers are presented to the right of the gene for genes
not mentioned in material and methods.





























Repeat 1 2 3 4
Gene
PthA PE PE PE PE
PthA4 PE PE PE PE
Apl1 PE PE PE PE
PthA* PE PQ PE PE
PthAW PE PE PE LD
Verable region I PthB PD PA PD PA
PthC PD PD PD PD
PthA* -2 PE PG PE PA
PthA1 PE PD PA PA
PthA2 PE PE PE PE
PthA3 PE PE PE PE
Apl2 PE PE PE PE
Apl3 PE PE PE PE




PthA SNI SNG SNI SNI
PthA4 SNI SNG SNI SNI
Apl1 SNI SNG SNI SNI
PthA* SNI SNG SNI SHD
PthAW SNI SNG SNG SNG
PthB SHD SNG SHD SNG
Verable region II PthC SHD SNG SHD SHD
PthA*-2 SNI SNI SN- SNI
PthA1 SNI SNG SNI SN
PthA2 SNI SHD SNI SHD
PthA3 SNI SHD SNI SHD
Apl2 SNI SNG SNI SHD
Apl3 SNI SHD SNI SHD


PthA COA CQA CQA CQA
PthA4 CQA CQA CQA CQA
Apl1 COA CQA CQA CQA
PthA* COA CQA CQA CQA
PthAW CQA CQA CQA CQA
PthB CEO CEO CEO CEO
Verable region III PthC CEO CEO CEO CEO
PthA* -2 CQA CQA CQA CQA
PthA1 CQA COA COA CQA
PthA2 CQA COA COA CQA
PthA3 COA CQA CQA CQA
Apl2 CQA CQA CQA CQA
Apl3 COA CQA CQA CQA


Figure 4-7. Sequence alignment of the predicted amino acids encoded in the main variable portion of the repeat region of all 13 pthA


homologs. Boxed areas indicate the regions in the 17th repeat that are conserved among all pthA homologs with


experimental evidence of active pathogenic function.


6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23














CHAPTER 5
SUMMARY AND CONCLUSION

The main objective of this dissertation was to study host range determination

factors among all described Xanthomonas citri groups that are known world-wide. Five

variant groups ofX. citri have been described in the literature, and all are known from

field observations to differ in host range and/or pathogenicity. In this study, all known

groups were studied together in lime, grapefruit and sweet orange. All groups were

readily distinguished by inoculation of only two host differentials, lime and grapefruit.

The in plant growth of strains from two different groups that did not elicit an obvious

defense response in grapefruit was found to be poor. This indicated that either these

strains carry negative acting (avirulence) factors that limited growth in grapefruit, or that

they are missing positive acting pathogenicityy) factors that are present in strains from

groups that can attack grapefruit. The lack of a grapefruit defense response that is typical

of bacterial infections limited by avirulence factors led to an attempt to identify positive

pathogenicity factors.

A DNA library of an X citri strain able to attack grapefruit was moved into one of

the strains unable to attack grapefruit in an attempt to identify one or more positive acting

host range factors. Despite using a DNA library that theoretically covered the wide host

range X citri genome with 99% probability, no pathogenicity factors were found, despite

multiple screens of all library clones. It is possible that inplanta growth requires

multiple effectors, and that no individual cosmid would carry enough factors to reveal a

strong difference. Another possibility is that Xc270 may carry avr genes that function in









grapefruit and prevent Xc270 from growing. Avirulence is usually epistatic over

virulence and therefore a screen for positive factors would fail if this were the case.

In addition to the DNA library screen, particular attention was paid to the pthA

homologs from all five X citri strain groups, since pthA is known to be required by at

least three strain groups for citrus canker disease. In this study, pthA was demonstrated

to be required by the remaining strain groups. The fact that all wide host range group A

strains examined carried two additional pthA homologs that were not present in the

narrow host range B, C, A* and A" strain groups suggested a potential role for these

additional homologs in determining host range. However, when pthA or any of its group

A homologs (pthA1, pthA2 orpthA3) were transferred into a narrow host range group

(A*) strain, no increase in host range to include grapefruit was observed.

Three newpthA homologs were cloned, isolated and sequenced from strains of

group A* (pthA and pthA *-2) and A" (pthA W) and functionally compared with pthA

homologs previously isolated from strains of the three remaining groups: A (pthA), B

(pthB) and C (pthC). pthA *, pthA W, pthB and pthC were found to be fully isofunctional

with pthA, and capable of eliciting the typical canker phenotype in grapefruit in

complementation tests using an X citri group ApthA- mutant strain (B21.2), even though

the source A*, A" and C strains were unable to elicit the canker phenotype in grapefruit.

Furthermore, pthA *, pthA W, pthA and pthC did not elicit an avirulence phenotype of

any type in B21.2, despite the fact thatpthA homologs are all members of an avirulence

gene family, and despite the avirulence of the respective source strains in grapefruit.

DNA sequence comparisons of the three newpthA homologs cloned, sequenced

and characterized in this study with ten previously sequenced pthA homologs revealed









that all functional pthA homologs (i.e., those that are required for citrus canker disease in

their respective strains) from all five known X citri groups carried exactly 17.5, 102bp

direct tandem repeats. All other homologs that are not functional for citrus canker

pathogenicity carried a different number of repeats.

Phylogenetic comparisons of the DNA and predicted protein sequences of the

thirteen available pthA homologs revealed the same phylogenetic distinctions that are

found by more general phylogenetic studies. In addition, comparisons of the five

functional pthA homologs from each group against those that were nonfunctional

revealed that amino acids N(12)G(13) in the second and Q(31)A(32) in the third variable

regions of the 17th direct tandem repeat were only conserved in functional genes. These

results suggest that the 17th repeat plays a critical role in citrus canker pathogenicity and

may help explain the origination of new citrus canker strains.















APPENDIX A
SEQUENCE OF pthC

DNA sequence ofpthC:

atggatcccattcgtccgcgcacgtcaagtcctgcccacgaacttttggccggaccccagccggatagggttcagccg

cagccgactgcagatcgtgggggggctccgcctgctggcagccccctgggctgatggcttgccgctcgacggacgatgtcccgaa

cccgtctcccgtctccccctgcccccttgcctgcgttctcagcgggcagtttcagcgatctgctctgtcagttcgatccgttgcttctt

gacacattgctttttgattcgatgtctgccttcggcgctcctcatacagaggctgccccaggagaggcggatgaagtgcaatcgg

gtctgcgtgcagtcgatgacccgcaccccaccgtgcacgtcgctgtgacggccgcgcgaccgccgcgcgccaagccggcgc

cgcgacggcgtgctgcgcacacctctgacgcttcgccggccgggcaggttgatctatgcacgctcggctacagccagcagca

gcaagacgagatcaaaccgaaggcgcgtgcgacagtggcgcagcaccaccaggcactgatgggccatgggtttacacgtgc

gcacatcgttgcgctcagccaacacccggcagccttggggaccgtcgctgtcaagtaccaggccatgatgcgcgttgccgg

aggcgacacacgaagacatcgttggcgtcggcaaacagtggtccggcgcacgcgccctggaagcattgctcacggtgtcgg

gagagttgagaggtccaccgttacagttggacacaggtcaacttctcaagattgcaaaacgtggcggcgtgaccgcggtggag

gcagtgcatgcatggcgcaatgcactgacgggcgctcccctgaacctgaccccggaccaggtggtggccatcgccagccac

gatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccag

gtggtggccatcgccagcaatggcggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaa

catggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggct

gttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggc

gctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccag

caatattggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggac

caggtggtggccatcgccagcaatggcggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgag

caacatggcctgaccccggaccaggtggtggccatcgccagcaatattggcggcaagcaggcgctggagacggtgcagcgg









ctgttgccggtgctgtgcgagcaacatggcctgaccccggcgcaggtggtggccatcgccagcaatggcggcggcaagcag

gcgctggaaacggtgcagcagctgttgccggtgctgtgcgagcaacatggcctgaccccggaccggggggtggccatcgcca

gcaatattggcggcaagcaggcgctggagacggtgcagcggctgttgccggttgcccaggcacatggcctgaccccgg

accaggtggtggccatcgccagcaatattggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcga

gcaacatggcctgaccccggaccagggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagc

ggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggttgccatcgccagcaatggcggcggcaagca

ggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgc

cagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgacccc

ggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgc

gccaggcacatggcctgaccccggcgcaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtg

cagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggccatcgccagcaatggcggcggc

aagcaggcgctggagacggtgcagcggctgttgccggtgctgtgcgagcaacatggcctgaccccggaccaggtggtggcc

atcgccagcaatggcggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgcgccaggcacatggcctg

accccggcgcaggtggtggccatcgccagcaatggcggcggcaggccggcactggagagcatttttgcccagttatctcgcc

ctgatcaggcgttggccgcgttgaccaacgaccacctcgtcgccttggcctgcctcggcgggcgtcctgcgctggaggcagtg

aaaaagggattgccgcacgcgccgaccttgatcaaaagaaccaatcgccgtcttcccgaacgcacgtcccatcgcgttgccga

ccacgcgcaagtggctcgcgtgctgggttttttccagtgccactcccacccagcgcaagcatttgatgaagccatgacgcagttc

gggatgagcaggcacgggttgttacagctatttcgcagagtgggcgtcaccgaactcgaggcccgcggtggaacgctccccc

cagccccgcagcgttggcaccgtatcctccaggcatcagggatgaaaagggccgaaccgtccggtgcttcggctcaaacgcc

ggaccaggcgtctttgcatgcattcgccgatgcgctggagcgtgagctggatgcgcccagcccaatagaccaagcaggccag

gcgctggcaagcagcagccgtaaacggtcccgatcggagagttttcaccggctccttcgcacagcaagctgtcgaggtgc

gcgttcccgaacagcgcgatgcgctgcatttaccccccctcagctggggtgtaaaacgcccgcgtaccaggatcgggggcgg










cctcccggatcctggtacccccatggacgccgacctggcagcgtccagcaccgtgatgtgggaacaagatgcggaccccttc

gcaggggcagcggatgatttcccggcattcaacgaagaggagatggcatggttgatggagctatttcctcagtga

Predicted amino acid sequence ofpthC:

MDPIRPRTSSPAHELLAGPQPDRVQPQPTADRGGAPPAGSPLDGLPARRTMSRTRLPSPPAP

LPAFSAGSFSDLLCQFDPLLLDTLLFDSMSAFGAPHTEAAPGEADEVQSGLRAVDDPHPTVHVAVT

AARPPRAKPAPRRRAAHTSDASPAGQVDLCTLGYSQQQQDEIKPKARATVAQHHQALMGHGFTR

AHIVALSQHPAALGTVAVKYQAMIAALPEATHEDIVGVGKQWSGARALEALLTVSGELRGPPLQL

DTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCE

QHGLTPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLP

VLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNIGGKQALETVQR

LLPVLCEQHGLTPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNIGGKQALET

VQRLLPVLCEQHGLTPAQVVAIASNGGGKQALETVQQLLPVLCEQHGLTPDQVVAIASNIGGKQA

LETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGG

KQALETVQRLLPVLCEQHGLTPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQVVAIASH

DGGKQALETVQRLLPVLCEQHGLTPDQVVAIASHDGGKQALETVQRLLPVLRQAHGLTPAQVVA

IASHDGGKQALETVQRLLPVLCEQHGLTPDQVVAIASNGGGKQALETVQRLLPVLCEQHGLTPDQ

VVAIASNGGGKQALETVQRLLPVLRQAHGLTPAQVVAIASNGGGRPALESIFAQLSRPDQALAAL

TNDHLVALACLGGRPALEAVKKGLPHAPTLIKRTNRRLPERTSHRVADHAQVARVLGFFQCHSHP

AQAFDEAMTQFGMSRHGLLQLFRRVGVTELEARGGTLPPAPQRWHRILQASGMKRAEPSGASAQ

TPDQASLHAFADALERELDAPSPIDQAGQALASSSRKRSRSESSVTGSFAQQAVEVRVPEQRDALH

LPPLSWGVKRPRTRIGGGLPDPGTPMDADLAASSTVMWEQDADPFAGAADDFPAFNEEEMAWL

MELFPQ















APPENDIX B
SEQUENCE OF pthA W

DNA sequence ofpthA W:

atggatcccattcgttcgcgcacaccaagtcctgcccgcgagcttctgcccggcccccaaccggatagggttcagccg

actgcagatcgtggggtgtctccgcctgccggcggccccctggatggcttgcccgctcggcggacgatgtcccggacccggc

tgccatctccccctgccccctcacctgcgttctcggcgggcagcttcagtgacctgttacgtcagttcgatccgtcactttttaatac

atcgctttttgattcattgcctcccttcggcgctcaccatacagaggctgccacaggcgagtgggatgaggtgcaatcgggtctgc

gggcagccgacgcccccccacccaccatgcgcgtggctgtcactgccgcgcggccgccgcgcgccaagccggcgccgcg

acgacgtgctgcgcaaccctccgacgcttcgccggccgcgcaggtggatctacgcacgctcggctacagccagcagcaaca

ggagaagatcaaaccgaaggttcgttcgacagtggcgcagcaccacgaggcactggtcggccatgggtttacacacgcgcac

atcgttgcgctcagccaacacccggcagcgttagggaccgtcgctgtcaagtatcaggacatgatcgcagcgttgccagaggc

gacacacgaagcgatcgttggcgtcggcaaacagtggtccggcgcacgcgctctggaggccttgctcacggtggcgggaga

gttgagaggtccaccgttacagttggacacaggccaacttctcaagattgcaaaacgtggcggcgtgaccgcagtggaggcag

tgcatgcatggcgcaatgcactgacgggtgcccccctgaacctgaccccggagcaggtggtggccatcgccagcaatattggt

ggcaagcaggcgctggagacggtgcaggcgctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtg

gccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggc

ctgaccccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgcc

ggtgctgtgccaggcccatggcctgaccctggaccaggtcgtggccatcgccagcaatggcggtggcaagcaggcgctgga

gacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatagc

ggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtc

gtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccat

ggcctgaccccggagcaggtggtggccatgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgtt









gccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatagcggtggcaagcaggcgct

ggagacggtgcagcgg tgccggttgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagcca

cgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagca

ggtcgtggccatcgccagcaatgcgggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggc

ccatggcctgaccccggagcaggtggtggccatcgccagcaattgcggtggcaagcaggcgctggagacggtgcagcggct

gttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagcaatggcggtggcaagcaggc

gctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccctggaccaggtggtggccatgccagc

aatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggag

caggtggtggccatcgccagcaatagcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccag

gcccatggcctgaccccggaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcg

gctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagccacgatggcggcaagca

ggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcagggtgggccatcgc

ctgcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgacccc

ggagcaggtggtggccatcgccagcaatggcggcggcaggccggcgctggagagcattgttgcccagttatctcgccctgat

ccggcgttggccgcgttgaccaacgaccacctcgtcgccttggcctgcctcggcggacgtcctgcgctggatgcagtgaaaaa

gggattgccgcacgcgccggccttgatcaaaagaaccaatcgccgtattcccgaacgcacatcccatcgcgttgccgaccacg

cgcaagtggttcgcgtgctgggtttttccagtgccactcccacccagcgcaagcatttgatgacgccatgacgcagttcgggat

gagcaggcacgggttgttacagctctttcgcagagtgggcgtcaccgaactcgaagcccgcagtggaacgctccccccagcct

cgcagcgttgggaccgtatcctccaggcatcagggatgaaaagggccaaaccgtcccctacttcaactcaaacgccggacca

ggcgtctttgcatgcattcgccgattcgctggagcgtgaccttgatgcgcccagcccaacgcacgagggagatcagaggcggg

caagcagccgtaaacggtcccgatcggatcgtgctgtcaccggtccctccgcacagcaatcgttcgaggtgcgcgttcccgaa

cagcgcgatgcgctgcatttgcccctcagttggagggtaaaacgcccgcgtaccagtatcgggggcggcctcccggatcctgg









tacgcccacggctgccgacctggcagcgtccagcaccgtgatgcgggaacaagatgaggaccccttcgcaggggcagcgg

atgatttcccggcattcaacgaagaggagctcgcatggttgatggagctattgcctcagtga

Predicted amino acid sequence of PthAW:

MDPIRSRTPSPARELLPGPQPDRVQPTADRGVSPPAGGPLDGLPARRTMSRT

RLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQ

SGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQ

QQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIA

ALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGV

TAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL

TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALET

VQRLLPVLCQAHGLTLDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQV

VAIASNSGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLL

PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN

SGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA

HGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNCGGKQA

LETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTLD

QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQR

LLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIA

SHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIACNGGGKQALETVQRLLPVLC

QAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGR

PALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQA

FDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRA

KPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGP






69


SAQQSFEVRVPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVM

REQDEDPFAGAADDFPAFNEEELAWLMELLPQ















APPENDIX C
SEQUENCE OF pthA*

DNA sequence ofpthA *

atgcggcctcggaagctatgtaggaaccacagaccgctagtctggaggcgaccatgtaaagaggtatgcctgatggat

cccattcgttcgcgcacaccaagtcctgcccgcgagcttctgcccggaccccaacccgatggggttcagccgactgcagatcgt

ggggtgtctccgcctgccggcggccccctggatcttgcccgctcggcggacgatgtcccggacccggctgccatctcccc

ctgccccctcacctgcgttctcggcgggcagcttcagtgacctgttacgtcagttcgatccgtcactttttaatacatcgctttttgatt

cattgcctcccttcggcgctcaccatacagaggctgccacaggcgagtgggatgaggtgcaatcgggtctgcgggcagccga

cgcccccccacccaccatgcgcgtggctgtcactgccgcgcggccgccgcgcgccaagccggcgccgcgacgacgtgctg

cgcaaccctccgacgcttcgccggccgcgcaggtggatctacgcacgctcggctacagccagcagcaacaggagaagatca

aaccgaaggttcgttcgacagtggcgcagcaccacgaggcactggtcggccatgggtttacacacgcgcacatcgttgcgctc

agccaacacccggcagcgttagggaccgtcgctgtcaagtatcaggacatgatcgcagcgttgccagaggcgacacacgaag

cgatcgttggcgtcggcaaacagtggtccggcgcacgcgccctggaggccttgctcacggtggcgggagagttgagaggtcc

accgttacagttggacacaggccaacttctcaagattgcaaaacgtggcggcgtgaccgcagtggaggcagtgcatgcatggc

gcaatgcactgacgggtgcccccctgaacctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggc

gctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccgcagcaggtggtggccatgccag

caatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccgga

gcaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccag

gcccatggcctgaccccggagcaggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcg

gctgttgccggtgctgtgccaggcccatggcctgaccccggcacaggtggtggccatcgccagcaatattggcggcaagcag

gcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgcc

agccacgatggcggcaagcaggcgctggagacggtggcggctgttgccggtgctgtgccaggcccatggcctgaccccg









gaccaggtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgc

caggcccatggcctgaccccgcagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgca

gcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagccacgatggcggcaa

gcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccat

cgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgac

cccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgct

gtgccaggcccatggcctgaccctggaccaggtcgtggccatcgccagcaatggcggtggcaagcaggcgctggagacggt

gcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatagcggtgg

caagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggaccaggtggtggc

catcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcct

gaccccggagcaggtggtggccatcgccagcaatagcggtggcaagcaggcgctggagacggtgcagcggctgttgccgg

tgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgcctgcaatggcggtggcaagcaggcgctggaga

cggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcg

gtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtgg

tggccatcgccagcaatggcggcggcaggccggcgctggagagcattgttgcccagttatctcgccctgatccggcgttggcc

gcgttgaccaacgaccacctcgtcgccttggcctgcctcggcggacgtcctgcgctggatgcagtgaaaaagggattgccgca

cgcgccggccttgatcaaaagaaccaatcgccgtattcccgaacgcacatcccatcgcgttgccgaccacgcgcaagtggttc

gcgtgctgggttttttccagtgccactcccacccagcgcaagcatttgatgacgccatgatgcagttcgggatgagcaggcacg

ggttgttacagctctttcgcagagtgggcgtcaccgaactcgaagcccgcagtggaacgctcccccagcctcgcagcgttgg

gaccgtatcctccaggcatcagggatgaaaagggccaaaccgtcccctacttcaactcaaacgccggaccaggcgtctttgcat

gcattcgccgattcgctggagcgtgaccttgatgcgcccagcccaacgcacgagggagatcagaggcgggcaagcagccgt

aaacggtcccgatcggatcgtgctgtcaccggtccctccgcacagcaatcgttcgaggtgcgcgttcccgaacagcgcgatgc

gctgcatttgcccctcagttggagggtaaaacgcccgcgtaccagtatcgggggcggcctcccggatcctggtacgcccacgg









ctgccgacctggcagcgtccagcaccgtgatgcgggaacaagatgaggaccccttcgcaggggcagcggatgatttcccggc

attcaacgaagaggagctcgcatggttgatggagctattgcctcagtga

Predicted amino acid sequence of PthA*

MDPIRSRTPSPARELLPGPQPDGVQPTADRGVSPPAGGPLDGLPARRTMSR

TRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEV

QSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYS

QQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMI

AALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGG

VTAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH

GLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL

ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQ

VVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRL

LPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIAS

NGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLC

QAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGK

QALETVQRLLPVLCQAHGLTLDQVVAIASNGGGKQALETVQRLLPVLCQAHGLT

PEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETV

QRLLPVLCQAHGLTPEQVVAIASNSGGKQALETVQRLLPVLCQAHGLTPEQVVA

IACNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPV

LCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLG

GRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPA

QAFDDAMMQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMK

RAKPSPTSTQTPDQASLHAFADSLERDLDAPSPTHEGDQRRASSRKRSRSDRAVT






73


GPSAQQSFEVRVPEQRDALHLPLSWRVKRPRTSIGGGLPDPGTPTAADLAAS STV

MREQDEDPFAGAADDFPAFNEEELAWLMELLPQ















APPENDIX D
SEQUENCE OF pthA *-2

DNA sequence of pthA *-2

atgcggcctcggaagctatgtaggaaccacagaccgctagtctggaggcgaccatgtaaagaggtatgcctgatggat

cccattcgttcgcgcacaccaagtcctgcccgcgagcttctgcccggcccccaaccggatagggttcagccgactgcagatcgt

ggggtgtctccgcctgccggcggccccctggatggcttgcccgctcggcggacgatgtcccggacccggctgccatctcccc

ctgcacccttgcctgcgttctcggcgggcagcttcagtgacctgttacgtcagttcgatccgtcactttttaatacatcgctttttgatt

cattgcctcccttcggcgctcaccatacagaggctgccacaggcgagtgggatgaggtgcaatcgggtctgcgggcagccga

cgcccccccacccaccatgcgcgtggctgtcactgccgcgcggccgccgcgcgccaagccggcgccgcgacgacgtgctg

cgcaaccctccgacgcttcgccggccgcgcaggtggatctacgcacgctcggctacagccagcagcaacaggagaagatca

aaccgaaggttcgttcgacagtggcgcagcaccacgaggcactggtcggccatgggtttacacacgcgcacatcgttgcgctc

agccaacacccggcagcgttagggaccgtcgctgtcaagtatcaggacatgatcgcagcgttgccagaggcgacacacgaag

cgatcgttggcgtcggcaaacagtggtccggcgcacgcgccctggaggccttgctcacggtggcgggagagttgagaggtcc

accgttacagttggacacaggccaacttctcaagattgcaaaacgtggcggcgtgaccgcagtggaggcagtgcatgcatggc

gcaatgcactgacgggtgcccccctgaacctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggc

gctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgcacccgggacaggtggtggccatcgccag

caatattggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggag

caggtcgtggccatcgccagcaatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcc

catggcctgaccccggcacaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctgt

tgccggtgctgtgccaggcccatggcctgaccccggcacaggtggtggccatcgccagcaatggcggcaagcaggcgctgg

agacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatat

tggtggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcaggt









cgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggccca

tggcctgaccccggagcaggtggtggccatgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgtt

gccggtgctgtgccaggcccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggtggcaagcaggcgct

ggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggcacaggtggtggccatcgccagcaat

attggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggagcag

gtggtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcc

catggcctgaccccggagcaggtggtggccatcgccagcaatattggtggcaagcaggcgctggagacggtgcagcggctg

ttgccggtgctgtgccaggcccatggcctgaccccggagcaggtcgtggccatcgccagccacgatggcggcaagcaggcg

ctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggctggaccccggagcaggtcgtggccatcgccagcc

acgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccaggcccatggcctgaccccggcac

aggtcgtggccatcgccagccacgatggcggcaagcaggcgctggagacggtgcagcggctgttgccggtgctgtgccagg

cccatggcctgaccccggagcaggtggtggccatcgccagcaatggcggcggcaggccggcgctggagagcattgttgccc

agttatctcgccctgatccggcgttggccgcgttgaccaacgaccacctcgtcgccttggcctgcctcggcggacgtcctgcgct

ggatgcagtgaaaaagggattgccgcacgcgccggccttgatcaaaagaaccaatcgccgtattcccgaacgcacatcccatc

gcgttgccgaccacgcgcaagtggttcgcgtgctgggttttttccagtgccactcccacccagcgcaagcatttgatgacgccat

gacgcagttcgggatgagcaggcacgggttgttacagctctttcgcagagtgggcgtcaccgaactcgaagcccgcagtggaa

cgctccccccagcctcgcagcgttgggaccgtatcctccaggcatcagggatgaagggggccaaacctc tacttcaact

caaacgccggaccaggcgtctttgcatgc attcgccgattcgctggagcgtgacttggcgcccagcccaacgcacgaggg

agatcagaggcgggcaagcagccgtaaacggtcccgatcggatcgtgctgtcaccggtccctccgcacagcaatcgttcgag

gtgcgcgttcccgaacagcgcgatgcgctgcatttgcccctcagttggagggtaaaacgcccgcgtaccagtatcgggggcgg

cctcccggatcctggtacgcccacggctgccgacctggcagcgtccagcaccgtgatgcgggaacaagatgaggaccccttc

gcaggggcagcggatgatttcccggcattcaacgaagaggagctcgcatggttgatggagctattgcctcagtga









Predicted amino acid sequence of PthA*-2

MDPIRSRTPSPARELLPGPQPDRVQPTADRGVSPPAGGPLDGLPARRTMSRT

RLPSPPAPLPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQ

SGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQ

QQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIA

ALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGV

TAVEAVHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGL

HPGQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGKQALETV

QRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVA

IASNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLC

QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK

QALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLT

PAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETV

QRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAI

ASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVL

CQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGG

RPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRT

NRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLF

RRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFAD

SLERDLDAPSPTHEGDQRRASSRKRSRSDRAVTGPSAQQSFEVRVPEQRDALHLP

LSWRVKRPRTSIGGGLPDPGTPTAADLAAS STVMREQDEDPFAGAADDFPAFNE


EELAWLMELLPQ























ALIGNMENT


APPENDIX E
OF PATHOGENECITY GENES FROM X citri STRAINS


L .-.F- LL I' L- 'L L"-
F .-. FELLL-~E:. L 'EL-
I ..F EL '*L E E L"-

I .-.F ELL -' EL' L"E F

EI .F ELLF-' EL ''L L

I .-. FELL-'I E E:'L -
S I .F ELL.-1 I ,i I -
S.-. FELL-L E E'L-
.I .F ELLF-' ''L L"
F.=.F ELL.:.-FF L",-
F H F1. .' '.'F -F 'F-
L -i-LELL' *L 'L* L


F -

' --


S- -



F -
aE-

aE-
aE-




'F F
''F F
''F-
''-


4-I
'F'-,I
'2 -'2 -


T.-.LFI-,
-T.-LF, -.-
-T.-.LFI-'

I T.-.L'I- '

- I .-.L'I- '_,

I T.-.LF-'


- I .-.L'I- '_,
TI .-.LFI-'


T .-.LFI- :


F lE.-.';'.
F F.-.-I


F F.-.I -
F L .-.'1.


F F.-.I -


F F.-.I .

F L .-.'1'

SF ..'
FL '- .-. :


LELL"-.LF.

ELL"-LF.
L LL,, LF
F -- ,I ,- I.F .


ELLII-LE.
FLL"L, L.

LLL"-LF.
LLL"LLF

L LL"-LF,.

LLL"-LF.


MDPIRSRTPSPARELLPGPQPDGVQP TADRGVSPPAGGPLDGLPARRT


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus



PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus



PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Ap13
Apl2
PthB
PthC
Consensus


''LF. E E I


L'LL F' L'FF :

LLL'F E-E
L'LLT -. L'F :



1,L1 F E _- E 1
L'LL F' E L'E :



.L L.F'' F F -

T .i .' F F


LFIiTiLF ,
LFli il LLE '
.LFiT i LF,~

LFliT LFL 1
LFlilT i: LF 1
LFliTii LFi
LEli i LFL',

.LFiT II LF ,
LEIiT'll LF L
LFli'l LF ,I'


LLTLLF[ ,
T.T I 1T T I.Fr r


E-.-..HHTE''
F' .HHTE

FI-.-.HHTE
F,.-.H HTE
EL .-.HHTE
F,-.-.HHTE


F' -.HHTE
F'-.-.HH-iTE

Fl.-.HHTE
F' -.HHTE
.F'-. HTE

F,. HITL
LFI F HT F


MSRTRLPSPPAPSPAFSAGSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTE


101 150


-. Ll I L'L
_-.T Ei i, E
--.T1-Eii[E-

-.T'LUL-Ei
I .T i iLE'
--.T,-Eii E ,
_-.T LUi LL
_-.T,' Ei iLE


S: '- LI--

'1' : '_- LI-
: LI-

'LI' : '_- LI-
**'_.'r'LF-


-- 'Li ILIL ', : '-LI-

_- .T -EiiLE ,l.-LF-

_-. -E-- L '[E '-LF-
-. F L- .- .LF-
_- I L- LIL I-- P_


.L'.-.E L- LT!
. I I.-. I Ti F
L'.-.F F F l IF


.'.-.E I T [ F


L'.-.E I T! [F
I..- F IF FTI IF
.L'.-.LE I T!!
.L,.-.E E 'T! F
. .- F F FTrl IF
.L'.-. F F T !!
LFLFHF'LT H
LLL H HTii


-.F F F.


-.F F F.

-Fb-I-

-F E F.

-F F F.
F .F F F.



-F F F.
-.F E F.


.1 E.-.E F FF.
.1 L- .-. L 1-.



I L- I--F I-
-.1 L-. F F.
.I E..E F F F.
-II IF-I
.1 L -.L F F .

.I E.-.E F F F.


.i F-..F F F F.
.I I.-. FF F.



i 1'_.1 F F F.


-_-.' :L'.-

-_-.' :L'.
_-.'' :L'.
.-... I [
-_-' :L'.-
. I- l -
-_ ''I :L'.-
_-.'' :L'.-


_-.'' :L'.

- HT.[I .
-.HT L':


AATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDAS


I IL'L F
il[ IF i
I ILLE IF
II l. F ;
I ILLE IF I
I ILE IF I
illrIr IF -
I IL[ IF
ill IF i
I ILL- IF :
I ILL- IF :
ill IF I F F
i IL. IF I
IiLL-IF L


F TF LI
FT F T. F
FTFLI
F TF LE
F TF LT. F

FTFLI
F TF T. F

FTF LI
F TF LI
F T F L. F


F TF T. F
F lm 1.1-


(49)
(49)
(49)
(49)
(49)
(49)
(49)
(49)
(49)
(49)
(49)
(51)
(51)
(51)



(99)
(99)
(99)
(99)
(99)
(99)
(99)
(99)
(99)
(99)
(99)
(101)
(101)
(101)


F .-.'_

F .-.,-
F: .-.'_
F: .-.'_
F .f-.-

F : .-.'_
F .f-.-
. :.-.'_,
. .-.'_-
.F .-.,-

E 1.-.*-
.F .-.,


















LLl- 1 L- L 1 I l H
*LELTL.- E'I'II II lL II El

I1.LT F .T '"- ""' i 1I F -I
E'LLF TL'- i' 'Eli iF i

*LIL IL.- I .L I II II -l I
LLF TL'.- i' "-j'"i" I iEl
1-1.F TII i-.1 '" "' F iI 1F i
I_-LF .TL' i EL II iF i


LE, L _- I '" "" i I ElI
LE 1L'- i II El

*L TL -*El 1 l F


* 1I-1- HI
'.*HHL

'.:HHL
-. I l-HHL
'.:HHL
-.'-IH HL

.*:HHL
'.*HHL
-.' l-H L
'.:HHL
-.'I-H l-
.- E. H I
.- E- H
.-- H E H
.- E. H
.-- H E H
.- -, H I


-.H t I IH.
Hi- FT IH
_H' FT IH
Hl- t- I I-.
Hi- FT IH
I H.- t I Ih-
H'-- i- FT H.

'-H'-- FT H.

I.-1l t I H.


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus






PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(149)
(149)
(149)
(149)
(149)
(149)
(149)
(149)
(149)
(149)
(149)
(151)
(151)
(151)






(199)
(199)
(199)
(199)
(199)
(199)
(199)
(199)
(199)
(199)
(199)
(201)
(201)
(201)


--.L,
-.L, -T

-_.L, -T


_-.L,:-T

-.L, -T



_-.L,:-T


_-.L,:-T
-_ .L1-T
i.'i




i.'i


i.'i


/' 'l. II.

' *' ll II


'-L'I II.
'dI ll l
''Ill l

.: .'i II.
'I ill l


'I 'ill

,,-ill
:' L.'i II
'*: J II

l'':-'. 1 i


-.L L-.I-HL-.I
- .LF F-.THF i--
-.LL- IHL- .I
-_.LEE:.THE--.I
-T.LF F-.THF-
-T.F-F THF I-
_-.L L--.IHL-.I
.L EE.TiHEiL-.I
-.LF L- THF --.
.L LE-THL-.I
-T.F-F THF I-
-.F-F THF I

_.LEL IETHEL i
F.FL-.F IHF FI
EL L-IH LLil


'.1''i ~i


, 'aii

'.1'~ai


, 'aii

, 'aii


F- .LEL-.L LT
I I.F- 1...T
F.--.LL-L L I
- I.F L-.L LT
I I.F 1.-.-T'
.F- .LL-.L L


.F-.L L-.L LT I
F I. F 1.- .T
.F-.L--. LLT

F I. F 1.- .T


F I. F 1.- .T
.F -L. LLT
.F--.LL-.LTLT
-.F- -.L L-.LLT


250
.-.--LL
F-..- .
.-.--LL
F-..- .
F-..- .
.-.'-LL
F-..- .
.-.'-LL
F-..- .
F-..- .
.-_LL


.-LL


HPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGEL


F' IF- I- L.'_'LL' ILL. I .-.1I I -
F,- IF I.' .1 1'.I.L1 1 F
F-F FL' LLI'T' LLI I.- -.
F L-F L 'ILLI.'I. 'I ILLI 1.-.I 1-I -11'

F EL'- L'LT LLI I. F'-.'.
F-' F- .L' LL.I LLI .-.I 1- ''
F-- IF I.'' I.'1 -I 1. .-.1 1 '
F'- LE '_' LT'I L l .-.I F'-.-'
F-' F F- .L'LL.I '-' LLI. I 1.-.I 1- ''
F'- LE '_' LT'I L l .-.I F'-.-'
F-' F L' 'I.1 1LLI. .-. I 1- '
F-. L 'F L.' T'_.-'L' .--.1 I -''
F-'_l EE' L I' '-'ELLI I F.- -'_


FL-- H.i iFI IFi
FL-- H.i iFI IFi
L- H.-i i F l i
E I- .i li.

EL-. H.-i iF
E- H.-.ii F i
E-. H-.iiFi.
EL-- -H.-i iF


-. I.-.i F i.
EL-. H.-i iF

E- H.-.ii F i
EL--. -H.-iiF
L--. H..i iF .
L I- i F l i


.LT' -. LIILTE L
.LT' -. IILTEiL'
.LT I.-. LliLT- L' I-


.LT I.-. LliLT- L' I-
.LT-. ELIILTELI-

.LT. -.E LIILT I-
.LTI-.-.F LliLTF1 L'I

F.LT' -. LliLT F LI-
.LT' FLiiLT '-I '

.LTI-.-.- LliLTIF- L"1


RGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVAIA


.i HH 1.:. HIH IET F HI
H H -T. I ,--H FTH F HT


PAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQ


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(249)
(249)
(249)
(249)
(249)
(249)
(249)
(249)
(249)
(249)
(249)
(251)
(251)
(251)























. .-I : [ I I i .-. LI F L






.I.-. Il l.-. L 'F L L


P 1'' H-..-1' T F i E L''





P------------- i '
-------------
-------------
-------------
P 1 -- .-... _-1 T E C F


-------- -..LLI
-------- .LET'
------- -.LLET
-------- ..LET
.I.-. [I-T ''.-.LET'
--------- -.LETlI
-------- -..LET
--------- .L-ET


.I.-. : [i -.L L 'I


l- LLL-
VF LL _F
IF LLLE
'.-.LLE
F'' LL,
'F LL.
F'' LL,
'.-.L L
'.-.LLF
'F LL
-I1.1.1
yFI~ EE


LL z .-11J, L L- L ..
L '.-H,- L- F .-


L,' '.-H,-L L .-.

L'_ :'.- LT L-E .-.
LD_ z-H, L, I:
L .-_ H-- !.LT E I -- .

L, -H1 -L : L ,, -


..LLI I- LL E L' L iL LI .
-.LET F LLE L El H,-LTE1.-1
-..LETl F LLF L lEi-,HLT F[L -
QALETVQRLLPVLCQAHGLTPEQVVA


401






i -. I I '


S.-. H ii .1


I.-. Hi.
i -. -


I '- .-.L. T
! II.-.LET'
1 '.-.LET'
I I.-.LET'
1 .-. L -E T
! *L'.-.LET'
! ''.-.LET'
I .LET
! ''.-.LET'
I ,.-.LET
! '.-. LET
! -''.-.LET'
I ,.-.LET


F1. T. F
Il- LLI-
F1. T. F
'F LLE
-F LLI:
'F LL. F


yF LLE
IF- LL F
F .LL. F
''F LL
'.F LL.
'F LLE-

.F LL. F
i EEL:


E
E

E


E
' I
*L .



'L E'


--H LT L''r
--H' LTF F'
I-ILT El L
-H' LT EI'
-_ I-L T E .-.' '
--H. LTE E
-H' LT .- .'
--H'.LT EL '


H-'LT E L'
--H, LT El '
H- LT I ''

i1 -i'ELT L L'


450
-.. I iI y- -- ELI.-i '-'F
.-.I.-..[ii _,I 1 .-.iLET- I F

-. -. i ----.-I .LET' -F
*.-.i.-. H. *i I .-.LET F
.-. I.-.: [ i1 I -_1 .-.1 I '' F
-. I.-..r i- ,I_-I L.-.LET 'I' F
.-. .-..[i ~II .-.LET I '~' F
- I- il iI --I ,.-iI-.L T ,-,F
.-.i 1.-.: [1/ ,I 1 .LET ''F
-.I.-. .[i I,-- ,- .-.iLET F
i ily ELLI 'I-
-. 1.-.. [i 1 .-.LET F F
- I 1- T, -I -1.-iIT ,-F
I.-..-. HL- l L 1 .-.LETI 'F
'-i H.' L,,I; i 'J L L I' '' F


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus






PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(299)
(299)
(299)
(299)
(299)
(299)
(299)
(299)
(299)
(299)
(299)
(301)
(301)
(301)


301

Ir l- -


1.r L -


1 I IG -T
I ll- -
i ii I -


i ii I -






H[i -1 1



SNIGGK


(305)
(305)
(305)
(305)
(349)
(305)
(305)
(305)
(305)
(349)
(305)
(307)
(307)
(351)






(331)
(331)
(331)
(331)
(399)
(331)
(331)
(331)
(331)
(399)
(331)
(333)
(333)
(401)


IASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQR


.L1 T I!- LLI F L H I.-.I- L T Fi

.LL LL- I- LI -. HI L I- I- -
- - -- - I I- -

















LLL L' l .-I-'-*-LI L- L'
L. T.F l- I -IH LTF F-


LL I F .-- i' T F FL '
LL- L' 'lH- LTF [ L'

LL..F I.' I'.-- ILT F F.-.
LLI L' '1-H- LT .-1 '

LL..F I .-- i LT F L'F-
LLF L, L F L''
L.L.F I l ..-- H-I.T F F---
LLI L, H- LT .-1'F

TI.TI.F I. F I.T r ..-


.-.I.-.. i iI[ I r'I ll 1.-.L IT
.-.I.-..[i iI,-,7li ,.-.LLT
S-.I -. l-.I -- .LET


.I.-. i I L _I ,.-.LET

-..-... i-l I'-l- I.-.LET


_.-I.-.. H I .-.LL ETI
.-.i .-.. H ii ll'l< i I .Ly ETI

.-.I.-. [L I ,,-.LET I
.I... I l-l l LT
.-.I -.: ii L lll 1..L IET
.-.I.-.. il--ll i :.LET


'1- LLL-
F LL F
F LL F
lF- LL-
F LL F
I'F LL -
F LLF
F LLF
lF- LLI
'F LLF
F1- LL
i. .LL F
ll LL l
lI ELL
" i .l m l
Fi .1. 1
IF 1.1.


500
LI l.--l-i'; L I L.--
L '_ --H LT -.-- "
L '--H LT .-'

L' l L--H -LT L-
bL L.-_H': bT E- L'
L, L I L
I. .H I-' LTi-i.-.'


L',l -- L LT L-

L .-'i LT I LE'I '
L' l. L L L- TF '
TL I-I'- T F Il."'
E' *i-F l El ~ r ill "


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(380)
(380)
(380)
(381)
(449)
(380)
(380)
(381)
(381)
(449)
(380)
(383)
(383)
(451)


501
.-.I.-..i -- .-. LET
i. 11 TI I I I .I

".-.I. i.. l i II l-.LET

..I... [7 l I'' 1..LET
.-.I.-..[i I i ll 'l l '.-.LET

.I. li 1 L.e'T

.-.I.-..iI ill I l ..LET

..I... [1i T I'I l .L T

.-.I.-..[i illl .L. 'ET''


yLLL
"IF LLF
'F LLE
-IF LL.F
F LLEF
'F .LL.
'F .LLF
"IF LLF
'F LLEF
"I F LLF
' bF LL
IF LLE


-.i [ill- l 'I F- LLL


L, l.l-H LTF E lI
L'_'.- lLT E''
L, z-.H1 LTE I L

L ll:l.--lH l T F' 1 'l
L' '.-'LTEIL ''
L' z-1H, LT F E'L
L'_ Il.-_i' LTFL"1
Ll z-.Hl LT F El L


L L .-I' iLTi EI ''
L'I LI LT L L'
LL 1'' -i'-LTE F "1


550
i [ I.. LL
-.i -.. [ U-l-ll--li "lll-l E T' "

-.il H L l I ll.l.LET I
-.I. 1-..i l I'' .L



I H Fll I 1" m
.i1 [ ll l ll "ll. LET'

..I. -..i L I l .LET I
-.I. -..i i -- -I .-.LET



..i -. [ ll .llyl E ll.l.L I '


VAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN GGKQALETV


F E l.1- E .1-- I I y- I
F El. E .1-- Il- I
'- LLL L, z.H-1 L I L L',

S'1- LL. L' .- L*- I. L- ,
'F LL L' '.--H-LT F"l--'
iF1.1. EE E 1-lEIL
' 1.1. I'- L,' .- 11-. LTI- L
'F- LLL:L L IH-LTE' L"
'F! LLI- L'I .-I -I-.LT I- L'



* F1.1.1 I': 1- .1T F iii
S1.1.F EL' 1-- .LI L L,
'F 'LLE L,': 'lHi -LTIE_ L"


.-.I .-..H FL i ----------------------
.-.I .-..H L i ----------------------
I--.:H --7-i --


I -..-..HL --* --

.-.I.-.. Hi ,* I -
I 7 *- -1-i i
IL i-iL --
I H .-..i 1 ---I -
I-. i H L"II I I-.-.L E L'' .-. F
.-.I.-.. H F i -------------------- -
-.. 1 -.. ---------------------


(551) QRLLPVLCQAHGLTPEQVVAIASHDGGK


I. I''- -- .LET" --F LL F


LLPVLCQAHGLTPEQVVAIASN GGKQALETVQRLLPVLCQAHGLTPDQV


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(430)
(430)
(430)
(431)
(499)
(430)
(429)
(431)
(431)
(499)
(430)
(433)
(433)
(501)


(480)
(480)
(480)
(481)
(549)
(479)
(479)
(481)
(481)
(549)
(480)
(483)
(483)
















(508) -
(508) -
(508) -
(509) -
(577) -
(507) -
(507) -
(509) -
(509) -
(599) Q
(508) -
(511)
(511)
(601)


650
---- .-.LET
---- -LE T
---- E '?F

---- -.LET





[ i,-, i '': L ET
- --- -.LET
---- -..LET
---- -..LET

QALET


651 700
'F LL L''.- LT .-.I.-. -- '.-.L T F LL L''.- LT b
F LL L .H LTE E .I-. i -''- .LET' 'F LL L''- -I' H.LT
'FLL L''.-. LT '' .-.I.-. -- '.-.L T 'FLLE L''.-. LT b
,_:F LL .-.H.LTE E I .I-. i .- LETi F LL L I-. 'LT
S'FL LL '_''.-.H. LTF E'-' T-I I -- LET' 'F LLF- I ,-.H. LT
'FLLE L' 1.-'LTIE'' -.I.-.H '.-.LET 'FLLE L' ''.-. LT
''F LL L'' .-.H' .LT' FE' "-.- -. I i- .: '.LET LkL *_.-.H' H LT
'FLL L'''.- LT "' .-.I.-. '.-.L T ''FLLb L''.-i LT b
F LL L' ..H.LT [' -I i r-' .LET' IF LL L'' -H. LT
"IF LL- .H LT E' I i I ''-LET F LL L .-.H. i- LT
'FLL L'' L.- LT .-.I.-. -'- '.-.LET FLLE L'' H.-.'LT
F LLE- L,' L',H.LT .-. H- i -l'' .Li"T" LL -L', E'H.LTF
,F LL, 'L E H'-LT .-.'' .-.I.-. ,H -,-_,j -.LETL '" LL, 'L E H'-LT
VQRLLPVLCQAHGLTPEQVAIASNGGGKQALETVQRLLPVLCQAHGLTP




701 750
E' -. I-. HL- I .LET' F LL L ..H.:LTF E .I i-:- l'' I.
' I-.1. H L -I .LET' F LL L ..H.:LTE E .I-. I i -l'' .L
'' -.i -. L".' .LET 'FLL L' 'L'.-.-LT -.I.-. H '..L
' I-.1. HL -I'' LET' F LLF L ..H.:LTE E .I-.' i.:-.l''
'' .-. -. [ '.-.LET FLL L' '.-. LL"T L -.I.-. '.-.L
E' -.T- [i._: -l' F.LET F* LL, Lk ..H.:LTE.,: -' .I.-.}[iI: -,',.
.- -. -. [iI- '.LET" FLL L ..H.:LT E: I -. .-.HL -.L
'' -. -. L"-'I '.LET 'FLL L'' H.- LT E E -. -. 1' '..L
[,,, T1 [i,--,-, :-- .L ET, F L L, L ,-.H LT, F,,-, -.-...}[i.- :-l,_ T.-.T
E''' -. i -. H L" '- '- ..L '' L L'k .-.'-' H LT E '' -.i .-.: HI L"-'-I''..L
E'' -.1I-. H L--I F:..LET :F LL' L .-.H.: LT E': -. i.'- i-:- i .:
[', -I .-. HL-,-I'' :..LET :F L''k -Lk E','H.:LTF'E .-.I.-. i :- '' .:
L"' I -. 1 -.: [i 'l.- .LETI ''FLL L' ''.-.H'.LI L "'' -.i.-. 1 i l-1' '.' -.L
EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus





PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(513)
(513)
(513)
(514)
(582)
(512)
(512)
(514)
(514)
(649)
(513)
(516)
(516)
(651)





(563)
(563)
(563)
(564)
(632)
(562)
(561)
(564)
(564)
(699)
(563)
(566)
(566)
(701)


1- L"--'- --1 -.-.L l 1- L .-.H 1 11-1- '-


















LI I-.H.iL t I- L'
L: : .-.H .:LT E E'i '
I.' ''.-.H :LTF H'i'k
L, I-IH, LI E -I'
L. ,.-.Hi l.LT F E':

L, l :.-.H, LIE L -:
L: ,:'.-.H .:LT 1F ''
L' '-.-.H. :LT F E''
LI I.-.H L I Lli:l
L. .-.H. : LT F ''

L,: I,-H._L I L-I ''
I.- HI-L'T F Fl"'


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus






PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


[i I I I- I I I= I

[ I -- I I -[I .
[i I I I I I I I I II

lii
ii I 'I I I

i i LII'I I'I'II
SI-I- I


[ i' I I III] I 'I


ii I I-I I III
[ i I I I[I I


(613)
(613)
(613)
(614)
(682)
(612)
(611)
(614)
(614)
(749)
(613)
(616)
(616)
(751)






(663)
(663)
(663)
(664)
(732)
(662)
(661)
(664)
(664)
(799)
(663)
(666)
(666)
(801)


ETVQRLLPVLCQAHGLTPEQVVAIASN GGKQALETVQRLLPVLCQAHGL


HI"'II_ I'I'II

[ill'.
H I"'-I I I'III
[ill i Ii



HD

I11 I

HI
SI I_ iI
I-I I i I i i i i
[ i I I I I I


''LLE
F'- LL
:FLLE
'*:F LLF
F LL. F
F LL. F
-:F LLE
F LLL F
'*:F LLF
-F LL F

-F LL. F
:-F LLE
'I-I l k II

''FLEEIiiI
'II I lkIiiI

'II I lkIiiI

''FI iLEE
'II I ilk


L, H-.H- LL I

LI_ I-.IH--.LTLLIF"'

TL i _-. Hi- iT FI -I


I. HI.- LII LF
I H'12 F' F'-
MLI IIII.-.i I_ "'LILLii
I_ ':'.-.H : LT 1 E':'
*L'.-':'.-.H.l: L'T F F-":'
MLII=.-.Il LI LL"'
I* I-':'H' 2T F- I"I'


.L ''e LLE L'- I' I- L L' L II-


TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASN GGKQ


L-' .-.H -LT E El
LI:I:I.-.H._LTF E L:
L,_ ,,.-.Hi--.LT E':'
*LC: :-.H.?LTE E :

L,_ ,,.-.H.:LT F E':'
L' ''.-.H.' LTF.- :

L,_ ,,.-.H.:LT F E'_'

L,_ ,,.-.H.:LT F E''

*L. ELIHILTF [li*lI


.LLI '_ELLE L' ILM'.H ILl L,'


. [i I :- l:-LE IT

. I : Il- I '.--.L ET
. [i ;-:- l .LET
' [I I- I L
. H --I' LE
. [iI_ ,: l : ..L E'T
. [il '-', .-.LET

. [iI_ ,: l : ..L E'T
. :[ '-'_' I .-.L E T

. [iII -'' ET
. H --1-''.LET
H I' FLT


I_'+ LLb


*ilF LL
- .-F LLF
:lF LLE
i--F LLF

i-lF I.F
I*IF LLF
i-lF I.F
:IF LLE
F.iF l.F
"-F LL. F
'I-ilk.


i.'
i.'

i.'

i.'
i.'
- .



i .

iT.*' F
L_ i i
* l_,i_ i i
*LI- i i
* l_,i_ i i
*LI- i i
* l_,i_ i i
I_,I- IlI


.- LLE L.I-.-.H


(851) ALETVQRLLPVLCQAHGLTPEQVVAIASN GGKQALETVQRLLPVLCQAH


ELLE
IF LLb
*F LLEE

IFLLF
'IIF LLF1
'l'FLLE
II[IF LLF
'I'F LL,
'l'FLLE
''F LLE

':'F LLF
II:'F LLF


I' LLb
-F 1.i. F
-F 1.i. F
:FLLE
-F 1.i. F
'FLLE
'F 1. F
-F 1.i. F
'FLLE
F 1.i. F
':FLLE
''F LL.F
-F .i F


800
L '.-.HI-H L
Ii -.H' iL
Ii-i-.H i-L
L' '-.H- L
* i -.H L
L' '-.H' L







IT.- F -H --L.
L' '.L-.H' L

IiI- i-.H iL

L'_ L'.-.i' -*L
i': I-' 'Hi L


F'T'l --IF IT.I. i- 'T. F. H I'T .T F r-'


i-irI --.. T.F T'I ITI T.II F il T. I' F .H -T.


801







IEIt.
'TIF F I-II



I L F L"'
'T F F,-:-

ITIF FI-I


T'r FI-I

'TIF FI-I-


I P LI,


850






iI 'II I'

i"--' i,

HI. L"' ''1
Ii -,-




HL-* :


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(713)
(713)
(713)
(714)
(782)
(712)
(710)
(714)
(714)
(849)
(713)
(716)
(716)


F'i LLb
F LL. F
'*:F LLF
-F LL F
'*:F LLF
-F LL F
-F LL F
'*:F LLF
-F LL F
'*:F LLF
lF LL F


l--.F LLF
:l lk_
l'i ilk'
'I-ilk.
''FLEE.


















I I


I-I I .
HI-I I-
H -I I-


'I- I -
L' I I
'F' LLE
'' F I
'I I I


901







-I I- F




'LTEE

,iTI-II 'I
I I-


i-I' I -I I

i.H -L L- L'F'
.H'-L T L EL''
.H'_LT L-L'T '
.H'_-LT E L''


I.'.. M-I'


.ii' ,.


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(763)
(763)
(763)
(764)
(832)
(762)
(760)
(764)
(764)
(899)
(763)
(766)
(766)
(901)


1000


KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQ


1050
I. L 'T I- L I_ I_' I -. _'I./'L I'F- TI- ,--
I- 1 -- I '.-.LL 'F LLE I' I-.H 'LTE 'F .I.-
- -.L - LL- - -.H'LTE .I.-. ,-i.
ML"_-"1_-! .-.LLT '"ELLLE I _' I.-. I_-/'LT/E '' TI-.- [i'-

- - - - - - -. E: .I.-. [i.
-- LT E iE i- !i,

- - - - - - -LT E : -.I.-. H
_ __-- - - - L T EL I -.I.- i
L ET__ T 1__-_-_ LT E --1 .T HFI

-- F 1l --- -- LT E l -T.I.-. HP
-[il l -ll' l- I LL ETI l I LL I : L I H ,l L lE:l -.I.-. ii
l l 'I LLETI IE LLl i l L H' LTEl -I .. I i
GGKQALETVQRLLPVLC HGLTPEQWVVAIASNG


I _'-I.-.iH I I- LF


I' I-F 'H -I I I I, '


H.-.I: HL"- '- I.-.LL/ 'F1- LLI-


I.-. HL-l ''l- ..LLET ,F LLE

I_.-.;HL- 'I.-.LL/T ..E LLE
T I : -..-.- I -LE T -I F I I-
- - --- --*- l 1-- -
I H I I- I F I I I-
I i I I I F I I- I-


GLTPDQVVAIASHDGGKQALETVQRLLPVLC HGLTPEQVVAIAS


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(809)
(809)
(809)
(810)
(833)
(763)
(795)
(765)
(765)
(949)
(764)
(812)
(812)
(951)


1001


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(809)
(809)
(809)
(810)
(833)
(763)
(795)
(765)
(765)
(999)
(764)
(812)
(812)
(1001)


A H' L- I I'

















1051


.LLI
.LET"
I .FTI
I .FTI
.LET
.LETL
.LET"
.LTL
.LET"
.LET"
-.LET
.LET"
-.LET
.LET"
T .F'T
I.FTI



LLI

I.FTI


,, LLb
*F LLE
-- I- 1. F
OF 11. F
'*'F LLF

O F 1.. F
''FLLE


iF LL. F

'-:'F LLF

iF LL. F
FLL F
'--F LL F
':'FLLE
''F LLF
'-F T.1.


.H.FLT' El
.H.,LT E
.H'.LT EE -
.iHK-'LT F E-_
.H.FLT' El
.H--.LT EE



.H-'1LT F E'
.HILT E E'
.H'-LTEEI


H.'1.1 F E1
H.H'-T F .'


[il ,-- --F



iI-,_-, F



[i, ,-- --F
liJl--,-F
[il-, F
ii, '_' F

[i,--- F
[ii '-F
[I'-'-'*-F
[ii' '-F
[I'-'.-' F


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


1101
L'.-. L--.LITIL-'HL L-.L .' F '- F F E.-.LL-. I LEH-.E


_-.LTI -iL -H L
--.LTI I-IHL
_-.L'I il I-H
_ -. I 1il-l H -I
-.LT I I-IHL
- .LT il'I-iLH
--.LTI -i'HL
-.LT I il'H -iL

- Eli H T-
--.LTI -'IHL
- .LT, i H -iL
--.LTI IL-HL
:-.LTI il-1-iL


-' -F
L'-,_-'-





L_-,i-F
L'_- F



L.:-.:-F
L'_-,i- F




L'_l'_ F
L:--F
L':_,':_ F


I '-L. H -.I

S'1. F i--.F
-IE --_.E


'-LEN-.E
S'1.F H_-.F
i' l-I H -.

S'1.F i--.F

i' -IF H-.
'-LE-.F
S'1. F H_-.F
_I l- H_-.
' -LE 1--.


1150
LI I F TIIF F I EF T


.-.Li I
.-.L 1
.-.LI ,.
.-.LI








T 1 1
.LI
Ll.




LII
TLII


FT!iFF
F TI F F



FTIIFF
F TI IF F
FTIIFF
F TIIF- F
F TI IIFF
FTIIFF
F' T'IIF F

FTIlFF


I F F FT
I E I- T
I F- F T
I F- F T


I E F- T
TPEFT
I F F T
I E L- I
I F F T
I F Fl-
L EFT
LEFLI-T


DPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTS


1151


F,,, H
F-,-, H
F'-I- H


F-_-_ H

F,-,I H

F'-I- H




F-,-,,- H


1200
F H. --L.': F F- -.
F H 1.1. : L F F- T
F H -LL.':'LEF F I
F H .LL '.L F- '_F I
F H'-LL'_LEF F ':- I'


F H. 1.L.L .EF F IT
F H.:LL: LEF F F F Y
F H,-1.T I.- .TF F F I
F H' LL.L'LEFF F '
F H,-1.I.-_- E F F 'T
F H,-1.I.-_- E F F 'T
F H' LL.' LEF F F T
FM-EiL 'L FLl- '-i I


HRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVT


(845)
(845)
(845)
(846)
(846)
(776)
(808)
(778)
(778)
(1049)
(777)
(848)
(848)
(1051)


GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRP


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(895)
(895)
(895)
(896)
(896)
(826)
(858)
(828)
(828)
(1099)
(827)
(898)
(898)
(1101)


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(945)
(945)
(945)
(946)
(946)
(876)
(908)
(878)
(878)
(1149)
(877)
(948)
(948)
(1151)


1100


-I


-i.
-i.


-i.


-i.
-i.


-i.


















F i i' l-I L'.-. il F 1-. F E I

: F i i I ,F I 1.-.. I F -i F E. I

'F iMF IL L'.-.-I -1 F L F I


'F i il-F I -1.. I F1 F E I
'F i iLF I L' I'.-. II F-. F I
,-F i i LF I -L, -. .- 11i F -. F ET
F: i iLF I L ,L .-..,-! I F_-.i E ET
*:'F'i- iL'FI/ L 'l'.-.. '- l I F-_-.i F .- : T
: F i i L,!F I ,2.-..:,-! i 'F_-.J E E T


1250
I'T E [ .-. : LH.-.E.-.L L' L
T :TFL" LH-E-.* L
T T'I F -. I H.-. .-. I
T 'IT F I '-. i H.-. .-.L' L
IT E L":'. LH.-.-. L L
T'I F [.-. I H-. F-. I L

T :TE "'.:-. LH.-._.-.L' L
I ''I F .-. I.H.-. F.-. I' i.
T:TE "'.-. LH-.E-. L L
TI'I F .'-. LH.--.F.-.L' L
T ITL":' L- L. LHE. L
T 'T F [ -. i .- '-.L' :
-'' F L".-. LH.-. F-.E .L
-,-,'T'F -r .l I.H F- -I'- T.


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus






PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(995)
(995)
(995)
(996)
(996)
(926)
(958)
(928)
(928)
(1199)
(927)
(998)
(998)
(1201)






(1045)
(1045)
(1045)
(1046)
(1046)
(976)
(1008)
(978)
(978)
(1249)
(977)
(1048)
(1048)
(1251)


1300
- F i[ FF [I : -. TI- E .- L*. l EE F--.F E'F [ -I .


-F IF F .I DF
-F F F F i'F
F IF .F DFI
-F IF F F
F i'F : F : L'F
F I F F .I LF
-F iF F i -F
F F F .f F

-F F .F .r ,F

F iF F
* F iF :F : 1


'I .-- -

"T.:-
"T. --

' '-I F
T.:- F


T.:-F


T -. F

TI,' !-


I I- I I -

.F I' 'F L -
F I F- F -

F- I' F- L,-

F EF L-
I- I EI I -


F Fl -


'F 'F
EFE'F L.-.


ERDLDAPSPTHEGDQRRASS RKRSRSDRAVTGPSAQQSFEVRVPEQRDA


i F F '-I- F I _
1 'I-'-I-L LE 'T TI.-_


I'- L ELFTE T.- I
i ---L F F -' F I _

I, -L F LEF '--' F T.I

I'-i-L ElE T.--_

I'---LLLL'-IL TIT-
I '-I-I-L F 1,F '- F I._


I .'.'.L F ,'F '- F

I -, L F F ,-' F i LL-
l' ',:L P- L'1', P T !L F.-.


1350
T l IF E'_'L' LI IE.-i.-
TI l F ''L' L I'FE.-.-
IT l IF E'I_'LIL LI 'F I I -.* I

T IF FE'l L',F E-.- -
TI I F E 'ILL L'F I F -,--.-

T -1 IF E L'L'lF E .-

TI l F F L' L IE .- .- .-
T 1 F El L L' L'''- I -.I .
T 1 IF ''L'lE F E .-' -

_T I IF EL''L'L L'L L -,--
T IF F E' F E, IF -I .
T -I F E L ,L L, -
I i riF ', ,- I, I. .
TI 11 iE- .-' L'[: .- .-
T 1 F1i E- [ 1.[ E_


LHLP LSWRVKRPRTSIGGGLPDPGTPTAADLAASSTVMREQDEDPFAGA


1201

ELE -.F -
EL E--.F
F -.F-.F
E L E-. F
ELF -.F
EL EF-. F
LF --.F
L T-.F-F
E L -. F
F T. F-F


FT.LL -F
F I.F ',


' ILE L.-
' L.FF.-
IT.FF- I
'TLE E.-.
ILT.F- I
'ITLE E.
TT.FF.-
'T' FF.-
'TLE E..
I' L P I-.
'Ti FF.-
-.TLE E


ELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSL


ETHI E -I*1F F F- :-.

IT HEI' EI'-'FF_
F T HEI- FF -F F_-.':
E TH EF -L*,F F--.T

F T H E-, F .F F -..:

F Ti-iE H. : F F -.':

E TH -E ,L* ITF --.
F T H E- F. F F -..T
F T H E -.*,F F--.T
F Ti iEH. FFF F '-.':


F i ["T '- .-,L' :
I T I F--- --


1251
ELF L[LL'[.-.
EF F iT. .-.F
ELF L'LL' .-.
EF L. ..-.F
EF IL.L ..-.F
EFL LLL' .-.
EF F .T. -. F
EFL LLL'.-.
EF F .T. -. F
EF T...-. F

EF LE L-.

E-F EL L'..


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(1094)
(1094)
(1094)
(1095)
(1095)
(1025)
(1057)
(1027)
(1027)
(1298)
(1026)
(1098)
(1098)
(1301)


1301
.HL.F- .1 iiF
.H1.F- .1 iiF
LHL -L iiF
.H1.F- .r iiF
LHLF -L iiF
L.H1.F- L.iiF
L.H1.F- L.iiF
LHL -iL iiF
L.H1.F- iiF
LHL -L iiF
L.H1.F- .1iiF
I.HL.F F L. ii,
LHLE F L ii'


I I- L- I- T
IIFF FF-T
SIF F F- T'


IFFFFT!
IF F F' T
iF FFT':
iF F FT'
FFFT!:

iF FFT'

iF FFT'

IF FFT' F
1 I F T F-


T .F F.-. F :Fii- HF I L .-..HI IIIF--. E
_TL' F .-. :F i i F I I L:. I-..- !i F-.E
,_-' F [ -:IE l- H I l 'I -. l -! i F_-
















1351


PthA
PthA4
Apll
PthAW
PthA*
PthA*-2
PthAl
PthA2
PthA3
Apl3
Apl2
PthB
PthC
Consensus


(1143)
(1143)
(1143)
(1144)
(1144)
(1074)
(1106)
(1076)
(1076)
(1347)
(1075)
(1148)
(1148)
(1351)


1371


-.LL[E L ---.FliELLL-.iiLi IELL L
-IF.L I I.FF-.TFIIFFF i L.- iI. I F.
.-.L*L.EF.-.I I -EEL.iL EI LFT.T F
.-. E T..F- IiEEE i-.ii Li i LL 'E,
.-.LLFF L.-.FIIFF F L..i- L F IFLL-. F-
.-. LF- -. iEEIi L.I-.ii L IiLL F -
.-.LL -FF-.-.FlIF FF ..i-i i.IFLL. F-'
I.lFI-.FIIFIF L.i-.iLFIF.LLFk''
.-.,FE---.FliEE Ei L.-.ii Li iELLL,
.-.LL -FF-.-.FlIF FF ..i-i i.IFLL. F-'
.--. ,~-E -..E iEE EL.-.iiL Li iELL ,-
.-.LL L--.-.FlIFF -F .i L IFLL -FF;
-IF.L~FI-F-.FiFFFI -.iiLF IELtFF'
ADDFPAFNEEELAWLMELLPQ