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Genetic and Molecular Analysis of Pathogenicity Genes in Xanthomonas citri Subsp. Citri

Permanent Link: http://ufdc.ufl.edu/UFE0024195/00001

Material Information

Title: Genetic and Molecular Analysis of Pathogenicity Genes in Xanthomonas citri Subsp. Citri
Physical Description: 1 online resource (163 p.)
Language: english
Creator: Figueiredo, Jose
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: citri, expression, hypersensitive, response, secretion, system, three, transient, type, xanthomonas
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, relies on a type III secretion system (T3SS) to successfully develop disease. Various plant pathogenic bacteria carrying a T3SS inject more than 40 different effector proteins (T3-effectors) into the plant cells via this apparatus. In Xanthomonas, the genes for the T3SS are regulated by HrpX, which is an AraC-type transcriptional regulator. The possible roles that nineteen candidate T3-effector genes play in the ability of Xcc to cause citrus canker were investigated through the use of site-directed mutagenesis. The candidate genes were selected on the basis of having promoter features similar to gene regulated by HrpX or by sequence similarity to known T3-effectors in other plant pathogenic bacteria. Inoculation in grapefruit revealed that none of these mutants were visually impaired for disease development. Unlike the hrpW null mutant, deletion in the harpin domain from hrpW resulted in the loss of Xcc pathogenicity symptoms, while not affecting the ability of of the bacteria to multiply in the plant. The mutants were also assessed for the ability to elicit hypersensitive response (HR) in non-host plant tomato. The Xcc hrp- mutants, in contrast to many pathogenic xanthomonads, retained the ability to trigger HR in the nonhost. Using subcloning procedures and homology search, three candidates open reading frames, XAC3857, XAC3858 and XAC3859, were identified in Xcc that might be responsible for this T3SS-independent HR elicitation. Experiments were also performed to further characterize a new avirulence gene, avrGf1, isolated from Xcc strain Aw, which induces HR in grapefruit. The avrGf1 gene was demonstrated to encode a protein that is translocated into plant cells via the T3SS. Additionally, a transient expression on grapefruit leaves was devised using an Agrobacterium-mediated delivery system. Different avrGf1 deletion mutants in the N- and C-terminal coding regions were tested using the system, and the results showed that the first 116 amino acids in the N-terminal and the last 83 amino acids in the C-terminal were crucial for HR elicitation in grapefruit. In summary, this study reported the presence of candidate T3-effector genes that did not affect the disease progress under the experimental conditions and a harpin domain of hrpW gene is essential for symptom development. Additionally, the development of an efficient transient expression in grapefruit leaves revealed specific regions of avrGf1 are required for defense activation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jose Figueiredo.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Graham, James H.
Local: Co-adviser: Jones, Jeffrey B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024195:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024195/00001

Material Information

Title: Genetic and Molecular Analysis of Pathogenicity Genes in Xanthomonas citri Subsp. Citri
Physical Description: 1 online resource (163 p.)
Language: english
Creator: Figueiredo, Jose
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: citri, expression, hypersensitive, response, secretion, system, three, transient, type, xanthomonas
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, relies on a type III secretion system (T3SS) to successfully develop disease. Various plant pathogenic bacteria carrying a T3SS inject more than 40 different effector proteins (T3-effectors) into the plant cells via this apparatus. In Xanthomonas, the genes for the T3SS are regulated by HrpX, which is an AraC-type transcriptional regulator. The possible roles that nineteen candidate T3-effector genes play in the ability of Xcc to cause citrus canker were investigated through the use of site-directed mutagenesis. The candidate genes were selected on the basis of having promoter features similar to gene regulated by HrpX or by sequence similarity to known T3-effectors in other plant pathogenic bacteria. Inoculation in grapefruit revealed that none of these mutants were visually impaired for disease development. Unlike the hrpW null mutant, deletion in the harpin domain from hrpW resulted in the loss of Xcc pathogenicity symptoms, while not affecting the ability of of the bacteria to multiply in the plant. The mutants were also assessed for the ability to elicit hypersensitive response (HR) in non-host plant tomato. The Xcc hrp- mutants, in contrast to many pathogenic xanthomonads, retained the ability to trigger HR in the nonhost. Using subcloning procedures and homology search, three candidates open reading frames, XAC3857, XAC3858 and XAC3859, were identified in Xcc that might be responsible for this T3SS-independent HR elicitation. Experiments were also performed to further characterize a new avirulence gene, avrGf1, isolated from Xcc strain Aw, which induces HR in grapefruit. The avrGf1 gene was demonstrated to encode a protein that is translocated into plant cells via the T3SS. Additionally, a transient expression on grapefruit leaves was devised using an Agrobacterium-mediated delivery system. Different avrGf1 deletion mutants in the N- and C-terminal coding regions were tested using the system, and the results showed that the first 116 amino acids in the N-terminal and the last 83 amino acids in the C-terminal were crucial for HR elicitation in grapefruit. In summary, this study reported the presence of candidate T3-effector genes that did not affect the disease progress under the experimental conditions and a harpin domain of hrpW gene is essential for symptom development. Additionally, the development of an efficient transient expression in grapefruit leaves revealed specific regions of avrGf1 are required for defense activation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jose Figueiredo.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Graham, James H.
Local: Co-adviser: Jones, Jeffrey B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024195:00001


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1 GENETIC AND MOLECULAR ANALYSIS OF PATHOGENICITY GENES IN X anthomonas citri subsp citri By JOS FRANCISCO LISSONI FIGUEIREDO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLM ENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Jos Francisco Lissoni Figueiredo

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3 To my wife Tania and my kids Jo o Pedro and Antonio, for the love and s upport allowing me to accomplish my dreams

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4 ACKNOWLEDGMENTS I would like extend my gratitude to Dr. Jeffrey B. Jones, who served as my co advisor. I am really grateful to him to accept as student in his laboratory. I appreciate all the unconditional sup port and encouragement during my graduate studies. I am also grateful to Dr. James H. Graham, who served as my committee chair and for providing the financial support for all th ese years. I really thank Dr. Frank F. White for invited me to work with him at Kansas State University and to give me the opportunity to start my Ph.D. studies in the U S A. I also appreciat e the valuable help from the members of my committee ; Dr. Robert E. Stall for provide plant material and support with the plant assays, and Dr. James Preston for reviewing the manuscript. I would like to thank Jerry Minsavage for all the time spent to teach me new techniques, discussing all the problems with this study and for his unconditional help and support with my research. Without him this research would not have been completed. I thank the faculty and staff of the Department of Plant Pathology; th ey were very supportive since I started my program here. For all my friends at the plant pathology department s at University of Florida and Kansas State University that helped me with valuable discussions and friendship, I would like to give a hearty "Muito Obrigado" I e special ly thank my wife for her patience and courage in all these years. This dissertation is also dedicated to my kids who were always waiting for me with a smile on their face. I am thank to my precious family. My father, mother, sister, brother, nephews and my loved uncle for all the love along these years E ven as I am so far away they never doubt ed my capacity. Last but not least, I thank all my fellow Brasile i ros who live here in U S A or in Bra zil for the ir sympathy, support and friendship.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 ABSTRACT ........................................................................................................................................ 11 CHAPTER 1 THE WEAPONRY INVOLVED ON PLANT PATHOGEN INTERACTION .................... 13 Introduction ................................................................................................................................. 13 Plant Pathogen Translocon Pathways ................................................................................. 14 Type I secretion system ............................................................................................... 14 Type II secretion system .............................................................................................. 15 Type III secretion system ............................................................................................. 16 Type IV secretion system ............................................................................................ 18 Hypersensitive Response and Pathogenicity ( hrp ) Genes ................................................ 19 Harpins a HR elici tor protein ...................................................................................... 22 T ype Three Secretion System Dependent Effectors ......................................................... 23 Project Goals and Objectives ...................................................................................................... 25 2 MUTATIONAL ANALYSIS OF TYPE III EFFECTORS GENES FROM Xanthomonas citri subsp. citri .................................................................................................... 27 Introduction ................................................................................................................................. 27 Mate rials and Methods ................................................................................................................ 28 Bacterial Strains and Plasmids ............................................................................................ 28 Media and Growth Conditions ............................................................................................ 28 Recombinant DNA Techniques .......................................................................................... 29 DNA Amplification ............................................................................................................. 30 Mutation of Candidate Genes by Homologous Recombination ....................................... 30 Complementation of hrpG and hrpX Deficient Mutants of Xcc Strain 306 .............. 31 Plant Material and Inoculations .......................................................................................... 32 Results .......................................................................................................................................... 33 Analysis of HrpXRegulon Candidate Genes .................................................................... 33 Mutations of the regu latory and structural genes of Xc c 306 hrp pathway cause loss of pathogenicity ................................................................................................................ 33 Phenotypic Analysis of Nineteen Candidate Effector and Hrp -Regulon Associated Genes. ............................................................................................................................... 34 Discussion .................................................................................................................................... 34

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6 3 IDENTIFICATION OF A HRP INDEPENDENT HR ELICI TOR FROM Xanthomonas citri subsp. citri ............................................................................................................................ 50 Introduction ................................................................................................................................. 50 Materials and Methods ................................................................................................................ 52 Bacterial Strains and Plasmids ............................................................................................ 52 Media and Growth Condition s ............................................................................................ 53 Recombinant DNA Techniques .......................................................................................... 53 DNA Amplification ............................................................................................................. 54 Mutagenesis of hr pG and hrpX in X. citri subsp. citri 306 ............................................... 54 Subcloning of HR Elicitor Candidate from Xaa Library .................................................. 55 Plant Material and Plant Inoculations ................................................................................. 57 Measurement of Electrolyte Leakage ................................................................................. 58 Protein Thermal Stability .................................................................................................... 58 Results .......................................................................................................................................... 58 Unusual Incompatible Responses of Mutants 306:: hrpG and 306:: hrpX in Tomato .............................................................................................................................. 58 Subclone of X. f. pv. aurantifolli Confers the Ablilty to Trigger an HR in Tomato ....... 59 Sequencing Anal ysis Revealed Novel Genes Controlling Nonhost HR Elicitor ............ 60 Discussion .................................................................................................................................... 62 4 GENETIC CHARACTERIZA TION OF HRPW OF Xanthomonas citri sub sp. citri ............ 76 Introduction ................................................................................................................................. 76 Material and Methods ................................................................................................................. 78 Bacterial Strains an d Plasmids ............................................................................................ 78 Media and Growth Conditions ............................................................................................ 78 Recombinant DNA Techniques .......................................................................................... 78 DNA Sequence Alignment and Phylogenetic Analysis of hrpW Gene ............................ 79 Generation of hrpW Deletion Mutants ............................................................................... 80 Construction of hrpW Harpin Domain ............................................................................... 81 Complementation Tests ....................................................................................................... 82 Plant Material ....................................................................................................................... 83 Plant Inoculations ................................................................................................................ 83 Bacterial Populations ........................................................................................................... 84 Translocation Activity in 306:: harpin Mutant ................................................................. 84 Construction of hrpW ::avBs2 Fusion ................................................................................. 85 Results .......................................................................................................................................... 85 Xanthomonas citri subsp. citri hrpW Phylogeny ............................................................... 85 Total Deletion of hrpW Gene Is Irrelevant for Xcc Pathogenicity and HR in Non Host Plants ........................................................................................................................ 86 Harpin Domain in hrpW Gene is Required for Pathogenicity .......................................... 87 Complementation of HrpW harpin Mutant In -Cis ........................................................... 88 Translocon Machinery, T3SS, Is Not Affected by harpin Mutant in Xcc ..................... 88 HprW is not Translocated into the Mesophyll of Plant Cell ............................................. 89 Discussion .................................................................................................................................... 89

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7 5 CHARACTERIZATION OF avrGf1 FROM Xanthomonas citri subsp. citri STRAIN AW .............................................................................................................................................. 117 Introduction ............................................................................................................................... 117 Materials and Methods .............................................................................................................. 118 Bacterial Strains and Plasmids .......................................................................................... 118 Media and Growth Conditions .......................................................................................... 118 Recombinant DNA Techniques ........................................................................................ 118 Plant Material and Plant Inoculations ............................................................................... 119 Agrobacterium -mediated expression of avrGf1 ............................................................ 120 Bacterial Expression of avrGf1 ........................................................................................ 122 Results ........................................................................................................................................ 123 Induction of Hypersensitive Response by Transient Expression of avrGf1 Within Ci trus ............................................................................................................................... 123 The N -terminal and C -terminal Are Required for HR Elicitation .................................. 124 X anthomonas citri subsp. citri Harboring avrGf1 Induces a H ypersensitive Reaction in Grapefruit ................................................................................................... 124 AvrGf1 I s Translocated Inside the Host Plant Cell ......................................................... 125 Discussion .................................................................................................................................. 125 6 OVERALL SUMMARY AND DISCUSSION ...................................................................... 135 APPENDIX: SEQUENCE AND ALIGNMENT .......................................................................... 139 LIST OF REFERENCES ................................................................................................................. 142 BIOGRAPHICAL SKETCH ........................................................................................................... 163

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8 LIST OF TABLES Table page 2 1 X anthomonas citri pv. citri genes used in the study. ........................................................... 38 2 2 Bacterial strains and plasmids used in the study. ................................................................. 39 2 3 Oligonucleotides sequence used in this study. ..................................................................... 42 2 4 Candidate PIP box ( -like) and 10 boxlike sequences of the proposed hrpX regulons and putative effector/avirulence genes of Xcc. .................................................................... 44 2 5 X anthomonas citri pv. citri mutants pathogenicity phenotype in grapefruit leaveas and HR elicitation in tomato leaflets. .................................................................................... 45 3 1 Bacterial strains and plasmids used in the study. ................................................................. 64 4 1 Bacterial strains and plasmids used in the study. ................................................................. 93 4 2 Oligonucleotides sequence used in this study. ..................................................................... 95 5 1 Bacterial strains and plasmids used in the study. ............................................................... 128 5 2 Oligonucleotides sequence used in this study. ................................................................... 130

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9 LIST OF FIG URES Figure page 2 1 Genetic organization of the hrp genes of X. citri subsp. citri .............................................. 46 2 2 Proposed model for hrp gene regulation cascade in X. citri subsp. citri ............................ 47 2 3 Comparison of the bacterial pathogenicity and HR elicitor in grapefruit leaves and tomato leaflets, respectively .................................................................................................. 48 2 4 Integration by site -specific recombination using one single target sited ............................ 49 3 1 Phenotype of grapefruit leaves were used to distinguish among the strains ...................... 66 3 2 Phenotype of 306 wild type, 306:: hrpG and 306:: hrpX in leaflet of tomato cv Bonny Best .............................................................................................................................. 67 3 3 Hypersensitive response assay in tomato cv. Bonny Best and pepper cv. ECW 20R ....... 68 3 4 Electrolyte leakage in tomato plant is changed in WT::pLAFR3, compatible interaction, and WT:: 450, incompatible interaction ........................................................... 69 3 5 Subclone, B38, triggers hypersensitive response (HR) independent of T3SS in X. perforans in non -host plants, tomato cv. Bonny Best and pepper cv. ECW20R ............... 70 3 6 Protein alignment of XAC3857 gene and Xanthomonas spp .............................................. 71 3 7 Protein alignment of XAC3858 gene and Xanthomonas spp .............................................. 72 3 8 Protein alignment of XAC3859 gene and Xanthomonas spp .............................................. 73 3 9 Tra nscriptional organization of the ORFs XAC3857 to XAC3859 region in Xanthomonas citri subsp. citri ............................................................................................... 75 4 1 Neighbor joining analyses using full nucleotide sequence of hrpW gene from X. c subsp. citri strain 306 ............................................................................................................. 96 4 2 Sequence alignment of Xcc strains collected world -wide ................................................... 97 4 3 Sequence alignment of Xcc strain 306 wild type (W1); W:: Harpin mutant (W2); and 306:: hrpW HrpW (W3) .............................................................................................. 112 4 4 Phenotype assay in grapefruit leaves .................................................................................. 113 4 5 Populations of Xcc 306 (Xcc306) ....................................................................................... 114 4 6 Canker symptoms on abaxial leaf surface o f grapefruit .................................................... 115

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10 4 7 PCR amplification of genomic DNA of Xcc 306 .............................................................. 116 5 1 Diagram of AvrGf1 and truncated versions used to test regions of the protein for elicitor activity ...................................................................................................................... 131 5 2 Agrobacterium -mediated expression of avrGf1 in grapefruit leaf tissue ......................... 132 5 3 Hypersensitive reaction induced by X. citri subsp. citri 306 expressing avrGf1 ............. 133 5 4 Phenotypes elicited on pepper plants cv. ECW20R 24 h after inoculation ...................... 134 A 1 hrpW nucleotide sequence and deduced amino acid sequence. ........................................ 139 A 2 Nucleotide sequence of AvrGf11106::AvrBs262574 fused protein ..................................... 140 A 3 Nucleotide sequence of HrpW1109::AvrBs262574 fused protein ........................................ 141

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11 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 GENETIC AND MOLECULAR ANALYSIS OF PATHOGENICITY GENES IN X anthomonas citri subsp citri By Jos Francisco Lissoni Figueiredo May 2009 Chair: James H. Grah am Cochair: Jeffrey B. Jones Major: Plant Pathology Xanthomonas citri subsp. citri (Xcc), which causes citrus canker, rel ies on a type III secretion system (T3SS) to successfully develop disease. Various p lant pathogenic bacteria carrying a T3SS inject mo re than 40 different effector proteins (T3 -effectors) into the plant cells via this apparatus. In Xanthomonas t he genes for the T3SS are regulated by HrpX, which is a n AraC -type transcriptional regulator. The possible roles that nineteen candidate T3 effe ctor genes play in the ability of Xcc to cause citrus canker were investigated through the use of site -directed mutagenesis. The candidate genes were selected on the basis of having promoter features similar to gene regulated by HrpX or by sequence similar ity to known T3 -effectors in other plant pathogenic bacteria. Inoculation in grapefruit revealed that none of these mutants were visually impaired for disease development. U nlike the hrpW null mutant, deletion in the harpin domain from hrpW resulted in the loss of Xcc pathogenicity symptoms while not affect ing the ability of of the bacteria to multipl y i n the plant. The mutants were also assessed for the ability to elicit h ypersensitive r esponse (HR) in non-host plant tomato The Xcc hrpmutants in contr ast to many pathogenic xanthomonads, retained the ability to trigger HR in the nonhost Using subcloning procedures and homology search, three candidates open reading frames, XAC3857,

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12 XAC3858 and XAC3859, were identified in Xcc that might be responsible for this T3SS independent HR elicitation E xperiments were also performed to further characterize a new avirulence gene, avrGf1 isolated from Xcc strain Aw, which induces HR in grapefruit The avrGf1 gene was demonstrated to encode a protein that is translo cated into plant cell s via the T3SS. Additionally, a transient expression on grapefruit leaves was devised using an Agrobacterium mediated delivery system. D ifferent avrGf1 deletion mutants i n the N and C terminal coding regions were tested using the syst em, and the results showed that the first 116 amino acids in the N terminal and the last 83 amino acids in the C terminal were crucial for HR elicitation in grapefruit. In summary, this study reported the presence of candidate T3 -effector genes that did not affect the disease progress under the experimental conditions and a harpin domain of hrpW gene is essential for symptom development. Additionally, the development of an efficient transient expression in grapefruit leaves revealed specific regions of avrGf1 are required for defense activation.

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13 CHAPTER 1 T HE WEAPONRY INVOLVED ON PLANT PATHOGEN INTERACTION Introduction Many complex steps are involved i n the interaction of plants and their pathogens Although plants are exposed to attack by many bacteria, d isease development is a relatively rare event. Usually, plant defenses retard or stop bacterial in gress without the development of disease symptoms. The term i nnate immunity, which is observed in both plants and animals, is often used to refer to the defen ses after the physical barrier s are broached by microbe s (Menezes et al ., 2000). Microbe invasion commonly induces a series of biochemical -changes that may lead to resistance to disease development, such as production of the salicylic acid (SA), deposition of lignin and callose in the cell wall, transcription of pathogenesis related (PR) genes, and production of antimicrobial compounds (Loake and Grant, 2007). The plant may also recognize specific pathogen effectors that interact directly or indirectly, wi th resistance (R) proteins, leading to the activation of a rapid defense response and accompanying hypersensitive re action (HR), which is also called effector triggered immunity (ETI) (Jones and Dangl, 2006). Basal plant defenses are also triggered by the recognition of common microbial molecules called pathogen associated molecular patterns (PAMPs). This recognition, which is called PAMP triggered immunity (PTI), involves recognition of PAMPs by pattern recognition receptors (PRRs), which are receptor like kinases (Jones and Dangl, 2006; Block et al ., 2008). The major known elicitors of PTI include flagellin peptide (flg22) (Zipfel et al ., 2004), lipopolysacharides (LPS) derived from pathogen cell walls (Keshavarzi et al ., 2004), peptidoglycans, microbial c ell wall fragments, phospholipids, proteins, double stranded RNA and methylated DNA (Ingle et al ., 2006; Iriti and Faoro, 2007). Activation of basal defense response s by PTI results in rapid increases of the Ca2+ flux, nitric oxide and reactive oxygen spec ies (Nurnberger et al ., 2004).

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14 P hytopathogenic proteo bacteria (Gram -negative bacteria) rely on different weapons to neutralize the host defenses and promote the coloniz ation of the host tissue The weapons include as several protein secretions systems, T 3 effector proteins, toxins, small molecule suppressors, cell wall degradation enzymes, and genes involved in flagellar and biofilm biosynthesis (Shiraishi et al ., 1997; Gross et al., 1999; Alfano et al ., 2002; Wolpert et al ., 2002; Moissiard and Voinnet, 2004; Hommais et al., 2008). Pathogens use this diversity of weapons to undermine core components of plant defenses, such as the HR, cell wall -based defenses, jasmonic acid signaling, systemic acquired and induced resistance and expression of defense gene s (Abromovitch and Martin, 2004). Most phytopahogenic bacteria carry four secretion systems, although new studies have demonstrated the presence of up to six secretion systems (Yahr, 2006). F our of the secretion systems have been well characterized in plan t pathogenic bacteria and are fundamental for the ingress, colonization, suppression of host defenses and disease development. Approximately 20% of all polypeptides synthesized by bacteria are located partially or completely outside of cytoplasm and this arsenal of proteins makes use of these systems for secretion and translocation into the apoplast and into plant cell s (Pugsley, 1993; Salmond and Reeves, 1993). In general p athogenicity in proteobacteria is dependent upon secretion machiner y that mediate s the transport and injection of toxic molecules into target tissues or cells. These secretion systems are classified into six types (I to VI ) and a summary of the four major types are presented here. Plant Pathogen Translocon Pathways Type I secretion syst em The route that each protein will be translocated through bacterial membranes is dependent on the substrate. Translocation of most proteins, in general, is catalyzed by the secretory ( Sec ) apparatus although some exported proteins are translocated indepe ndently of the core Sec -

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15 translocon, named sec -independent (Danese and Silhavy, 1998; Cristobal et al., 1998). This system, which is sec independent and ABC -transporter dependent, is specific for a particular protein or a family of closely related proteins (Higgins, 1995). The proteins are exported by ABC (A TP -b inding c assette) transporters, which are involved in the import or export of a great variety of substrates such as different small peptide signaling molecules, bacteriocins and amino acids, through th e outer membrane (Gray et al ., 1989; Wandersman, 1992; Higgins, 1995; Young and Holland 1999). Basically, the type I translocator is assembled on two dedicated inner membrane proteins, first, the ABC transporter (provide energy and it is the initial chann el across the inner membrane), second, the membrane fusion protein (MFP), which starts in the periplasm and form a channel to the surface with an third protein, TolC, in the outer membrane protein (Holland et al ., 2005). The proteins that are translocated by this system, usually, carry the secretion signal at the C -terminal and is not removed during transport (Holland et al., 2005). The type I translocon system plays an important role in both animal and plant bacteria pathogens. A type I mutant in Actinobac illus pleuropneumoniae which has hemolytic activity, is defective in protein secretion and the strain is non -hemolytic (Kuhnert et al., 2005). The importance of the type I system is demonstrated in plants as well. Rice plants that carry the Xa21 resistanc e gene, which mediates recognition of bacterial strains expressing AvrXa21 activity, are resistant to Xanthomonas oryzae pv oryzae although plants lacking Xa21 are susceptible (Gomez Gomez and Boller, 2002). Mutational analysis of X. oryzae pv oryzae genes involved in type I secretion revealed that the strain is no longer recognized by Xa21 (da Silva et al., 2003). Type II secretion system Of the six secretion systems reported in phytopathogenic bacteria, the most prolific pathway is the type II secretion system from which most proteins are secreted, including elastase, lipase, phospholipases and exotoxins (Filloux et al., 1998; Ball et al ., 2002). For these proteins to

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16 reach their final destination, they may be delivered through two routes that are via sec machinery or Tat export pathways (Douglas et al ., 1987; Voulhoux et al ., 2001). The proteins translocated through the type II -dependent system are first translocated across the cytoplasmic membrane via the sec machinery. The next step in the secretion pro cess involves the mature peri plasmic protein being transported across outer membrane, which requires the function of at least 12 gene products of a specialized apparatus called the secreton (Pugsley, 1993; Sandkvist 2001). Depending on the species, betwee n 12 and 15 genes (named by letters A through O and S) are essential for type II secretion and minor differences in genetic organization of this cluster in closely related species have been observed (Pugsley, 1997; Sandkvist 2001). Studies on secretion genes in Erwinia chrysanthemi and Erwinia carotovora, which cause soft rot diseases on various plants, showed that one of the genes, outN is missing in E. chrysanthemi whereas its presence in E. carotovora is required for functioning of the system ( Lindeber g et al ., 1996). da Silva et al. (2002) describe the presence of two type II secretion systems in Xanthomonas citri subsp citri (Xcc) and Xanthomonas campestris pv campestris, that are involved in the secretion of degradative enzymes and toxins. Although HrpX is known as type III secretion system regulator in Ralstonia solanacearum (Genin et al ., 1992) and Xanthomonas spp (Wengelnik and Bonas, 1996), it also regulates some type II secretory proteins in Xanthomonas oryzae pv oryzae causal agent of bacteri al leaf blight (Furutani et al ., 2004). This broad regulation is also observed in X. citri subsp citri where HrpG, which regulates HrpA and HrpX (Brito et al ., 1999; Wengelnik et al., 1996b), also regulates 11 type II secretion system related proteins ( Yam azaki et al., 2008). Type III secretion system A major breakthrough in the plant pathology field was the elucidation of T3SS, which is used for most Gram negative pathogenic bacteria and, it is conserved among animal and plant

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17 bacteria (Cornelis and Van G ijsegem, 2000). Species in the genera Yersinia Shigella flexneri, Salmonella typhimurium Salmonella typhimurium enteropathogenic E. coli Erwinia amylovora, Pseudomonas syringae Pseudomonas solanacearum and Xanthomonas spp rely on the T3SS for successf ul colonization of their respective hosts (Hueck, 1998; Cornelis and Van Gijsegem, 2000). Phytopathogenic bacteria such as Pseudmonas Rasltonia Xanthomonas Erwinia and Pantoea in vade their hosts by using natural openings, such as stomata, or wounds. When bacteria ingress the host they promote the secretion and translocation a diverse group of bacterial virulence factors through T3SS, refered to here as T3 -effectors (Cornelis and Van Gijsegem, 2000). The first demonstration of the translocation of T3-effector s was observed with Yersinia effector proteins (Rosqvist et al., 1994). Once these T3-effector s are inside the host cell, it is expected to modulate plant physiology providing an environment beneficial to sustain the growth of the pathogen outside the cell and suppress host defenses (Mudgett, 2005). The identification of the P syringae hypersensitive response and pathogenicity ( hrp ) gene cluster suggest that the expression of these genes results in the formation of a T3S S structure or hrp pilus, which creates a bridge between pathogen and host cell for protein delivery (Aizawa et al ., 1998; Kimbrough and Miller, 2000). All proteins secreted and/or translocated from T3S system are delivered from the bacterial cytoplasm to the host cell interior passing between the bacterial and host membranes/cell wall through this channel (Whittam et al., 2004). The remarkable achievement related with T3S system in mammalian and plant bacterial pathogenesis is the finding that a defect in this system often leads to comp lete loss of bacterial pathogenicity ( Lindgren et al., 1986; Zischeck et al ., 1987). Despite the obvious importance of the T3SS, which functions in delivering the type three effectors (T3 -effectors) into plant cells (Galan & Collmer, 1999; Kjemtrup et al ., 2000), not all plant pathogens bacteria rely on this particular system, including

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18 one of major pathogens of citrus trees in Brazil, Xylella fastidiosa and the causal agent of citrus variegated chlorosis (CVC) (Dow and Daniels, 2000). Type IV secretion system Type IV secretion (T4S S ) systems mediate the transport of toxic molecules to host cells and DNA or protein -DNA complexes across the cell envelopes of Gram negative and Gram positive bacteria (Cascales an d Christie, 2003; Grohman et al., 2003). This system is involved in the bacterial conjugation process, where two bacteria exchange genetic material, transmission of DNA among diverse species of bacteria, and also able to deliver DNA to fungal, plant, and m ammalian cells (Bundock et al., 1995; Baron et al ., 2002). Consequently, it plays crucial roles in the spread of antibiotic resistance genes and other fitness traits among different species (Christie et al ., 2005). Studies have demonstrated that T4S Ss are able to deliver not only DNA but also effector proteins into the cytosols of eukaryotic cells, which in turn induce physiological changes to aid in establishment of pathogenic or symbiotic relationships (Fisher et al ., 2002; Ding et al ., 2003). Several phy topathogenic bacteria carry the T4S S system but the significance of this system for the plant pathology is that T4S S in Agrobacterium tumefaciens holds the ability to transfer DNA to different kingdoms. T4S S system, in Gram -negative, is a multisubunit cell-envelope spanning structure composed of a transenvelope channel or pilus or other surface filament that spans both membranes, the periplasm, and the cell wall (Christie, 2004; Lawley et al ., 2003). T4 S S systems have been classified as type IVA, IVB, or ot her (Christie and Vogel, 2000). Basically, the composition of type IVA shows similarity of the archetyp ic A. tumefaciens VirB/D4 system, which is responsible for the synthesis this secretion machinery (Christie, 2004). In addition, a subunit of genes terme d Mpf proteins is required for assembly of the channel or pilus for protein secretion (Haase et al., 1995). A T4 S S system also found in Xcc is involved in

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19 the adhesion of this pathogen to plant cells through the fimbriae, but the fimbrillin genes, which ar e responsible for encoding the proteins that build the fimbriae structure, are different from other Xanthomonas species (Cao et al ., 2001; da Silva et al ., 2002). Hypersensitive Response and Pathogenicity ( hrp ) Genes The T3S S is crucial in the process of colonization and establishment of favorable environment for plant pathogenic bacteria in the host, thus, failure or malfunction in the system severely impairs disease development. The first clue about the existence of h ypersensitive r eaction and p athog enicity ( hrp ) genes was their requirement for elicitation of h ypersensitive r eaction (HR) in nonhost plants and disease stimulation in susceptible plants (Lindgren et al. 1986). The correlation between hrp genes and secretion of bacterial proteins was firs t observed some time later (Collmer et al ., 1993; Rosqvist et al ., 1994, ; Wei and Beer, 1994). The first hrp genes described were in P. s. pv. phaseolicola and have more recently been identified in most gram -negative phytopathogenic bacteria, including P. syringae (Cuppels, 1986; Lindgren, 1997; Huang et al ., 1988), Xanthomonas campestris (Bonas et al ., 1991), X. oryzae pv. oryzae (Kamdar et al., 1993), Ralstonia solanacearum (Boucher et al .,1987), Erwinia amylovara (Barny et al ., 1990), E. chrysanthemi (Ba uer et al., 1994), E. carotovora (Cui et al., 1996) and Pantoea stewartii (Frederick et al ., 1991). Usually the hrp cluster is composed of ~ 20 genes of which nine genes have been shown to share highlevel of homology among species, and as a result those nine genes have been named hrc (for hrp gene conserved ; Bogdanove et al., 1996; Hueck, 1998). Moreover, these genes may be clustered in the chromosome as part of a large pathogenicity island (PAI), which are characterized by having a G + C content different from the rest of the chromosome and /or flanked by remnants of mobile DNA elements (Hacker and Kaper, 2000). The Xcc strain 306 hrp cluster is composed of 26 genes extending from hpa2 to hrpF (da Silva et al., 2002). As a result of structural studies, the structure or T3SS pilus consists

PAGE 20

20 of two outer rings that interact with the outer membrane, two inner rings that interact with the cytoplasmic membrane, and an extracellular needlelike extension that is ~ 8 nm in diameter and 80 nm in length in animal path ogenic bacteria (Aizawa et al ., 1998; Kimbrough and Miller, 2000). In plant pathogens, the T3SS pilus is much longer (several m) but has the same diameter (Roine et al ., 1997). Although this system is conserved among proteobacteri a the genes responsible for the Hrp pilus for T3SS pilin subunits are hypervariable in their sequence, even within pathovars. For example, the major subunit of the T3SS pilus of P s pv tomato is the 11kDa HrpA protein, which shares only 30% identity with the HrpA protein of P. s. pv syringae (Deng et al., 1998). Mutational analyses on hrp genes have demonstrated that disruption in core genes in this system leads to failure to cause disease or multiply in susceptible plants, as well in the ability to trigger HR on nonhost plants (Lindgren et al ., 1986). T3SS gene expression in phytobacteria is induced in the plant apoplast, in close contact with host cells or some formulations of minimal med ia (Schulte and Bonas, 1992; Galan and Collmer, 1999; Alfano et al ., 2000). The T3SS of Xanthomonas spp. is regulated by two major regulators, HrpG and HrpX, which are transcribed from a divergent promoter region (Oku et al., 1995; Wengelnik et al., 1996; Wengelnik and Bonas, 1996). HrpG is a member of the OmpR subclass of twocomponent regul atory systems (Wengelnik et al., 1999). HrpX is another hrp regulatory gene, which belongs to the AraC family of regulatory proteins, and responsible for the transcriptional activation of genes in the HrpA to HrpF operons. The hypothesis behind the hrp sys tem regulation is that HrpG is phosphorylated by sensing an unknown environmental signal, the phosphorylated HrpG regulates expression of the hrpX and hrpA and HrpX subsequently activates transcription of the remaining hrp genes and some genes encoding T3effector proteins (Wengelnik et al., 1996; Wengelnik and Bonas, 1996). For HrpX -dependent activation the

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21 presence of a PIP -box (plant -inducible promoter box, consensus TTCGC N15TTCGC) in the promoter region of each HrpX -regulated gene is required (Fense lau and Bonas, 1995; Wengelnik and Bonas, 1996). Additionally, the HrpX inducibility requires a cis acting element (YANNRT), which is present in the 10 promoter region (Ciesiolka et al ., 1999; Cunnac et al., 2004). The hrp regulatory genes in P. syring ae are dependent of the activator HrpL which is a member of the extra cytoplasmic functions (ECF) family of response regulators (Xiao et al ., 1992; Morett and Segovia, 1993; Xiao et al ., 1994). hrpL expression is controlled by HrpR and HrpS which are rel ated to the NtrC class of response regulators and, synergistically activate expression of hrpL in response to plant signals (Albright et al., 1989; X. Tang et al ., 2006).. The mechanism of regulation of the hrp genes in R solanacearum is similar to Xanthomonas A signal is sensed by an outer membrane receptor PrhA and the signal is transferred by the putative transmembrane protein PrhR and the ECF sigma factor PrhI. Thus, the activated PrhI controls expression of the regulators PrhJ, HrpG and HrpB. HrpB i s 40% identical and 58% similar to HrpX in X. c. pv. vesicatoria and functions as a positive regulatory protein that activates the expression of hrp transcriptional units in R. solanacearum (Genin et al ., 1992; Marenda et al., 1998; Gurlebeck et al ., 2006). HrpA in P. syringae pv. tomato is the major structural component of a pilus -like structure, and consequently, inactivation of hrpA leads to the termination of T3 -effectors flux out of the bacteria, and disease progress (Roine et al ., 1997). In X anthomonas HrpE is the structural component of the Hrp pilus and shows no sequence similarity to other T3SS pilin genes. Models for X. c. pv vesicatoria HrpF protein proposed that the protein produces a pore in the plant cell membrane and facilitates protein tr anslocation into plant cells (Rossier et al., 2000; Buttner et

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22 al ., 2002). However, HrpF plays a role in protein translocation and pathogenicity in vivo, it is dispensable for type III mediated protein secretion in vitro (Buttner et al ., 2002). Harpins a HR elicitor protein The hrp cluster of phytopathogenic bacteria carry genes that encode proteins able to elicit HR and have been named harpins. Harpin or harpin like proteins in general are glycine -rich heat -stable proteins that are secreted through T3 S S system and trigger the elicitation of HR in plant tissues. H owever, harpins do not contain a n amino terminal signal peptide similar to T3 effectors (Wei et al., 1992; Alfano and Collmer, 1997; Zhu et al ., 2000; Zou et al., 2006). In addition, evidence s uggests that HR elicitation by harpins does not always resul t in a visible HR it may trigger o the r plant defense mechanism such as systemic acquired resistance (SAR) or expression of defense related proteins (Peng et al., 2004; Liu et al., 2006; Wang et al ., 2008). The first harpin isolated was HrpN from E. amylovora Ea321 (Wei et al., 1992). Purified HrpN was able to elicit a HR on incompatible tobacco plants and the E. amylovora hrpN mutant was no longer able to induce HR on tobacco plants or cause dis ease on susceptible apple plants (Wei et al., 1992). Based on homology searches, hrpN homologs were found in other Erwinia species including E. chrysanthemi (Bauer et al ., 1995) and E. carotovora showing the same features (Cui et al., 1996). In 1998, a sec ond Harpin protein was isolated from E. amylovora (Kim and Beer, 1998). The novel harpin, named HrpW, shows a C terminal domain that is homologous to fungal pectate lyases (Pels) and an N terminal similar to harpin protein. Contrasting with hrpN h rpW is n ot required for HR and pathogenicity although it still elicits an HR in its purified form and enhances the HR activity of a hrpN mutant (Kim and Beer, 1998). Harpins are not exclusive to Erwinia species. An homologous protein, HrpZ, has been isolated form P. syringae pathovars (Lindgren, 1997; He et al., 1993). Like HrpN, p urified HrpZ induce s a HR in tobacco and tomato plants and hrpZ mutants show slight difference in virulence

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23 when compared with the wild type strain (Preston et al ., 1996). HrpW was al so isolated in P. s pv tomato DC3000. L ike harpins the protein is acidic and glycine rich lacks cysteine and is deficient in aromatic amino acids ( C harkowski et al ., 1998). In agreement with the finding by Kim and Beer (1998) the protein sequence revealed two distinct domains, an N terminal harpin like domain and a C -terminal pectic lyase (Pel) domain similar to Nectria haematococca mating type IV protein ( Fusarium solani f. sp. pisi), bacterial Pels in E. carotovora, E. chrysanthemi and Bacillus subtilis (Liu et al ., 1994; Guo et al., 1995; Shevchik et al., 1997; Charkowski et al., 1998). Additionally, the gene is preceded by a consensus hrp promoter, PIB box, followed by a rho -independent transcription terminator which is characteristic of a monocist ronic operon (Charkowski et al., 1998). More recently, genome sequencing projects have identified hrpW homologs in X. citri subsp citri (strain 306) and X. campestris pv campestris (strain 33913) (da Silva et al., 2002). Type Three Secretion System -Depend ent Effectors The mechanism that controls secretion of the T3 -effectors to plants is still unclear However, the secretion signals are conserved among plant and animal pathogenic bacteria. X c pv. v esicatoria is able to secrete T3SS heterelogous proteins such as PopA from R. solanacearum AvrB from P. syringae and YopE from Yersinia pseudotuberculosis inside of host cells, showing that the secretion signal is conserved among the T3-effectors (Rossier et al., 1999). Studies demonstrated that the secretion signal for T3 -effectors is in the N terminus, e. g. the first 11 17 amino acids of Yersinia outer proteins (Yops) are sufficient to induce its secretion through T3SS (Sory et al.,1995; Lloyd et al ., 2001). T he secretion signal of AvrBs2 protein from X. c pv. vesicatoria is present in the first 58 residues (Mudgett et al ., 2002). In addition, the N terminal sequences of the T3 -effectors show some similarity such as presence of hydrophilic amino acids, absence of acidic residues in the first 12 amino acids r esidues and high content of

PAGE 24

24 serine residues (Guttman et al., 2002; Petnicki Ocwieja et al., 2002; Gurlebeck et al., 2006). The serine content in the first 25 amino acids of X. c pv. vesicatoria T3 S S substrates also present certain level of variation (Gurl ebeck et al ., 2006). Studies have been reported that some animal and plant pathogenic T3 -effectors require specific chaperones for secretion (Bennett and Hughes, 2000; Gaudriault et al., 2002). In X. c pv. vesicatoria the protein HpaB, a so -called hr p as sociated protein, is a T3 S S chaperon that interacts with more than one effector and inhibits the translocation of noneffector ptoteins such as HrpF A hpaB mutant was shown to strongly reduce secretion and translocation of effector proteins and enable th e translocation of HrpF (Buttner et al ., 2004). The T3 -effectors inhibit different defense mechanism s in host cells in various ways. One mechanism is the suppress ion of plant innate immunity by removing or inactivat ing a defense signalling component. A hi gh number of T3-effectors are cysteine proteases. For example, AvrXv4 from X. c pv. vesicatoria and a member of the AvrRxv/YopJ family, is a small ubiquitin -related modifier (SUMO) protease that blocks ubiquitination and subsequent proteolysis (Orth et al ., 2000; Roden et al 2004) AvrPphB from P. syringae is a papain like cysteine protease and cleaves the Arabidopsis protein kinase PBS1, which forms a complex with RPS5 (resistance protein) The role that the protease plays in defense suppre ssion is unkn own in this case. However, cleavage of this complex initiates ETI (Shao et al ., 2003; Ade et al., 2007); AvrRpt2, another cysteine protease from P. syringae cleaves RIN4, an interactor of the matching RPS2 resistance protein and a negative regulator of PTI (Kim et al ., 2005; Kim et al., 2005b); AvrPtoB, in P. syringae is a ubiquitin E3 ligase and participates in the degrad ation of the tomato protein kinase Fen, which promotes the ETI activation by the interaction between Fen and

PAGE 25

25 AvrPtoB AvrPtoB also suppr esses PTI induced by MAPK pathways in Arabidopsis (He et al., 2006; Rosebrock et al ., 2007). Another strategy of phytopathogenic bacteria to overcome host defense mechanisms is to deliver T3 -effectors that induce alterations in host transcription that resu lts in increase in host susceptibility (Block et al ., 2008). Part of that group is AvrBs3 protein family, which in Xcc is represented by the PthA protein that plays a role in pathogenicity by promoting cell hypertrophy, division and, finally, death (Swarup et al ., 1991, Duan et al ., 1999). T hree other AvrBs3 like members from X. o pv. oryzae PthXo1, PthXo6 and PthXo7, that are transcription activator like T3 effectors increased the expression of rice genes Os8N3 OsTFX1 (a bZIP transcription factor) and O sTFIIA1 (a small subunit of the transcription factor IIA), which increase disease susceptibility ( Yang et al, 2006; Sugio et al ., 2007). AvrBs3 was recently shown to bind to upa20 promoter, which encodes a transcription factor that regulates cell size. Th us, induces gene expression and causes hypertrophy of plant mesophyll cells by mimicking eukaryotic transcription factors (Kay et al., 2007). Today more than 40 effectors have been analyzed and characterized although the complete understanding of the compl ex interaction of plant pathogen still has gaps. Project Goals and Objectives The aim of this project was to screen putative Xcc T3 effector genes that may be involved with Xcc pathogenicity and virulence by mutagenesis. Additionally, a transient expressio n assay in citrus leaf tissue was developed to better characterize bacterial proteins recognized by citrus plants and which activate defense mechanism s The objectives were to (I) us e a molecular mutagenesis tool s to screen for candidate HrpXregulon genes ; (II) identify an unknown T3SS independent HR elicitor in Xcc; (III) characteriz e hrpW and its role in pathogenicity ; and (IV)

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26 i dentif y core regions of avrGf1 which is linked with hypersensitive r eaction in grapefruit plants using a transient expressio n procedure

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27 CHAPTER 2 MUTATIONAL ANALYSIS OF TYPE III EFFECTORS GENES FROM X anthomonas citri subsp. citri Introduction Most phytopathogenic bacteria rely on complex gene networks to penetrate the defenses of their host plants (Nurnberger and Lipka, 2005). Xanthomonas citri subsp. citri (Xcc) is the causal agent of the citrus bacterial canker and gains access into the host through wounds or natural openings (Brunings and Gabriel, 2003). Once inside, the bacteri um inject s a collection of proteins, called t ype III effectors (T3 -effectors) into the plant cells. T3 -effectors are delivered by a conserved type III secretion system (T3SS), and are indispensable for disease in most situation and condition the host cell environment in favor of bacterial growth (But tner and Bonas, 2002; Alfano and Collmer, 2004). The T3SS system is encoded by h ypersensitive r eaction and p athogenicity (hrp ) genes (Cornelis and Van Gijsegem, 2000; Alfano and Collmer, 2004; Collmer et al., 1993). Little is understood about the activitie s and targets within the host cells of many T3 -effectors but undobutely more than one T3 effector is expressed by a given pathogenic strain (Cunnac et al., 2004; Schechter et al ., 2004; Abramovitch et al., 2006). On the host side, many plant species have e volved mechanisms to recognize specific T3 -effec t ors, and the genes that control recognition specificity are known as resistance ( R) genes (Flor, 1971). This specific recognition is called effector -triggered immunity (ETI), which generally culminates in the induction of a form of rapid cell death known as the h ypersensitive r eaction or HR (Greenberg and Yao, 2004; Block et al., 2008) According to the genome sequence of Xcc strain 306, the hrp gene cluster is composed of six hrp operons ( hrpA hrpB hrpC h rpD, hrpE and hrpF ), six hpa (hpa2, hpa1, hpaP hpaA hpaB and hpaF ) genes and nine hrp conserved genes hrc (hrcC hrcT hrcN hrcJ hrcU, hrcV hrcQ, hrcR and hrcS ) (Figure 2 1; da Silva et al., 2002). Expression of the hrp genes is up-

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28 regulated duri ng growth in the host. Induction is believed to be initiated upon phosphorylation of the hrpG gene which is promoted by a yet unidentified environmental stimulus. Phosphorylated HrpG, which is a member of the response regulator of two-component regulatory systems, in turn, activates the transcription of hrpX and hrpA (Wengelnik and Bonas, 1996). Consequently, HrpX, which is a member of the AraC family of transcription factors, activates the expression of hrpB to hrpF operons ( Figure 2 2 ). The genes in the hrpB hrpC hrpD hrpE and hrpF operons carry a promoter element known, in xanthomonads as a PIP (plant inducible promoter) -box, which is represented by the consensus sequence TTCGC N15TTCGC and the binding site for the HrpX protein (Wengelnik and Bonas, 1996; Wengelnik et al ., 1996a; Wengelnik et al ., 1996b ). Additionally, another motif known as the 10 box-like motif (YANNNT; Y: C/T; N: A/T/C/G) is localized 30 32 base pair s downstream of the PIP box and has been proposed to be necessary for HrpX -medi ated regulation (Cunnac et al ., 2004). Our laboratory is interested in elucidating the function of proteins secreted through the hrp system in the X. citri supbs. citri citrus interaction. T he genome sequence analysis of Xcc strain 306 revealed greater th an 30 candidate effector genes based on sequence relatedness to known effectors and promoter elements ( Table 2 1 ; da Silva et al., 2002). Therefore, a targeted mutational analysis of the HrpX regulon genes was undertaken Materials and Methods Bacterial St rains and Plasmids The bacterial strains and plasmids used in this study and their sources are described in Table 2 2. Media and Growth Conditions Escherichia coli DH5 and C2110 were cultivated in Luria Bertani broth (LB; 1% bacto tryptone, 0.5% bacto ye ast extract, 1.0% sodium chloride) or on LB agar (2% agar) plates at

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29 37oC with the appropriate antibiotics. For solid media BD Bacto agar was added at 15 g l1. Strains of Xanthomonas were routinely grown on nutrient agar (NA) medium or on tryptone sucrose agar at 28oC (Zhu et al., 2000). All bacterial strains used in this study were stored in 20% glycerol in sterile tap water and maintained at 80oC. Triparental conjugations were performed on NYG agar (Daniels et al ., 1984). Antibiotics were used at the fo llowing concentrations: ampicillin (Amp) 100 g ml1; chloramphenicol (Cm) 100 g ml1; kanamycin (Kn) 50 g ml1; spectinomycin (Sp) 100 g ml1; rifampicin (Rif) g ml1; and tetracycline (Tc) 12.5 g ml1. Recombinant DNA Techniques Genomic and plasmid DNA were collected as described by Sambrook et al (1989). Restriction enzymes and T4 DNA ligase were obtained from, Promega (Madison, WI, USA) and used following the manufacturers recommendations. Chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), and Fisher Scientific (Hampton, NH, USA). Polymerase chain reaction (PCR) was performed with GoTaq Flexi DNA Polymerase (Promega, Madison, WI, US) and the PCR Express Thermal Cycler (ThermoHybaid, Ashford, UK).PCR products were cloned with a TA cloning kit (Invitrogen Corp., Carlsbad, CA, USA). Individual constructs were transfected into E. coli DH5 cells as described by Sambrook et al. (1989). Generation of Xcc mutants was made using single homologous recombination methodology as described Sugi o et al (2005) The selected plasmids were introduced into X c c strain 306 by e lectroporation of Xcc competent cells using a BioRad GenePulser II Electroporator The conditions applied were 200 OHMs (resistance), 25 FD (capacitance) and 2.5 Volts. After t he pulse, cells were diluted by addition of 250 L of NB media. The cells were incubated for 1 h at 28oC with constant shaking and plated on NA containing kanamycin at 50 g ml1. Plates were incubated at 28oC for 3 days.

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30 Generation of Xcc 306 mutants was made using single homologous recombination methodology as described by Sugio et al (2005) Triparental mating, as described by Daniels et al. (1984), was also used as a technique to introduce plasmids into Xcc 306. DNA sequencing on both strands of DNA f ragments was completed at DNA Sequencing Core Laboratory of the Interdisciplinary Center for Biotechnology Research, University of Florida Gainesville, FL (UF ICBR). DNA Amplification Individual sets of oligonucleotide primers were selected from the nucl eotide sequence of each gene of Xcc strain 306. The oligonucleotide primer sequences used in this experiment are shown in Table 2 3 F1 and R1 primers were used to amplify a partial fragment from each gene. A set of upstream primers (Up primers) were appli ed to confirm the insertional mutation in each gene. DNA was amplified in a total volume of 25 L. The reaction mixture contained 5 L of 5x buffer, 0.7 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate (Promega), 2 pmol of each primer, and 0.04 U of T aq polymerase. The amount of template DNA added was 100 ng of purified total bacterial DNA or 25 ng of a plasmid preparation, unless otherwise stated. A total of 30 amplification cycles were performed in an automated thermocycler. Each cycle consisted of 1 min of denaturation at 95oC, 1 min of annealing at XoC ( X = the annealing temperature was used in accord with the primer), and 1 min per kb of extension at 72oC The last extension step was extended to 10 min. Amplified DNAs were detected by electrophore sis in 1.0% agarose gels in TAE buffer. The gel was stained with 0.5 g of ethidium bromide per ml and then photographed over a UV transilluminator. Mutation of Candidate Genes by Homologous Recombination A partial fragment, amplified by PCR and having an average size between 300 900 bp, from Xcc genomic DNA was cloned in pCR2.1 TOPO, containing kanamycin and ampicillin

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31 resistance genes. The plasmids containing the amplified fragment were used for transformation of E. coli DH5 cells. The cells were plated onto LB agar plates containing both kanamycin and ampicillin. The plasmid DNA extracted from DH5 transformants were restricted with EcoR I, under conditions specified by the manufacturer, for insertion confirmation, and M13 forward and M13 reverse primers were used to sequence the inserts in pCR2.1. The resultant plasmids with partial sequence were introduced into Xcc 306 cells by electroportation as described above. The pCR2.1 plasmid cannot replicate in Xcc strain 306, and cells containing the plasmids i ntegrated into the Xcc strain 306 genome putatively by a single homologous recombination event, were selected on NA plates containing kanamycin. After 3 days of culture, a number of the transformed colonies were tested by PCR, using a primer flanking the upstream region of the target gene combined with a pCR2.1 internal primer, to confirm an insertion event ocurred the chromosomal gene of interest. Because of the presence of the linearized plasmid in the gene, the insertional mutation was deemed to cause a disruption of the transcription of inserted gene (Figure 2 3 ). A sequence analysis of the PCR fragment was also performed on each transformant for validation of the insertion (Altschul et al ., 1997). The insertional mutants were then tested for pathogenic ity and induction of the HR on appropriate plant cultivars and species. Complementation of hrpG and hrpX Deficient Mutants of Xcc Strain 306 For the complementation of hrpG and hrpX mutants a pLAFR3 clone pL22, from X. citri pv. citri strain Aw (XccAw) containing hrpG and hrpX was used to complement both mutants (Rybak et al., 2009). pL22 was transferred to the two Xcc mutants, 306:: hrpG and 306:: hrpX by biparental mating (Daniels et al., 1984). The transconjugants were then used for phenotype analys is.

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32 Plant Material and Inoculations Grapefruit cv. Duncan ( Citrus paradisi ) plants were grown from seed in 15 cm plastic pots in Terra -Lite agricultural mix (Scott Sierra Horticultural Products Co. Marysville, OH). The plants were kept in the glasshouse o f the University of Florida in Gainesville, Florida at temperatures ranging from 25 30oC. Tomato plants ( Solanum lycopersicum ) cvs. Bonny Best were planted from seeds in Plugmix (W. R. Grace & Co., Cambridge, MA, USA). After two weeks, the seedlings were t ransferred to Metromix 300 (W. R. Grace & Co) in 10 cm plastics pots. The plants were kept in the glasshouse of the University of Florida in Gainesville, Florida at temperature s ranging from 25 30oC. For preparation of bacterial suspensions, 18 h cultures were harvested from the NA plates and suspended in sterile tap water, and standardized to an optical density at 600 nm (OD600) = 0.3 (5 x 108 CFU/ml) with a Spectronic 20 Genesys spectrophotometer (Spectronic -UNICAM, Rochester, NY, USA). For disease sympto m assays, bacterial suspensions of the strains used in this study were infiltrated at 5 x 108 CFU/ml into abaxial surface of citrus leaves by using hypodermic syringe and needle, and the symptoms were assessed up to 10 days after inoculation. Following inoculation, p lant responses were evaluated after 3 4 days for water -soaking and 6 7 days for pathogenicity. For HR tests, bacterial suspensions adjusted to 5 x 108 CFU/ml in sterile tap water were infiltr ated into leaflets of tomato cv Bonny Best with a hyp odermic syringe and needle. The inoculated plants were incubated in a growth room at 24 28oC and assessed for HR elicitation 24 h after infiltration for HR induction. Disease scores are the means of those for three leaves. All the experiments were repeated at least three times.

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33 Results Analysis of HrpX -Regulon Candidate Genes HrpX controls expression of the operons hrpB to hrpF by binding to the PIP -box of the hrp genes (Wengelnik and Bonas, 1996). In this study, DNA sequence analysis revealed 19 putative H rpX regulon genes in Xcc strain 306 based on the presence of the motif TTCGC N15TTCGC. Six genes were present with a perfect PIP -box sequence (TTCGC N15/16TTCGC). Eleven genes had imperfect PIP box elements (mostly with one base change in the third base TTCG N -N15TTCG N ). Several genes were chosen simply on the basis of homology to other T3 -effectors despite the absence of the PIP box element such as avrPphE2 and xopX (Table 2 4 ). The promoter regions were also analyzed for the presence of the 10 boxlike motif YANNNT (A and T are conserved, Y represents C or T). In total, 11 putative 10 box-like sequence motifs were found among the 19 genes (Table 2 4 ). Mutations of the regulatory and structural genes of Xcc 306 hrp pathway cause loss of pathogenicity Mutations were constructed in hrp mutants, 306:: hrpG 306:: hrpX and 306:: hrpA in order to compare the range of host response from wild type to nonpathognenic as well as to determine the effectiveness of the single recombination mutagenesis strategy in Xcc306. Mutations were generated by single recombination in the regulatory genes hrpG and hrpX and the structural gene hrpA (hrcC ). The virulence of the mutants w as determined by observing the response in inoculated grapefruit leaves at high bacterial concentration (5 x 108 CFU/ml) in comparison to the response of the wild-type strain Xcc 306. All three mutants were nonpathogenic ( Figure 2 3 ). Complementation assays of 306:: hrpG and 306:: hrpX mutant s were performed using pL22 cosmid containing hrpG a nd hrpX genes from XccAw which was introduced, individually, into each mutant by conjugation. Consequently, full recovery of the Hrp

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34 phenotype was observed with pL22 ( Figure 2 3 ). The hrp mutants were also tested for their ability to elicit a nonhost HR on tomato. Mutants 306:: hrpG and 306:: hrpX did not lose the ability to trigger HR in non -host tomato plants at a 5 x 108 CFU/ml inoculum concentration In addition, the HR induction observed for 306:: hrpG and 306:: hrpX mutants was with similar intens ity and speed compared with the HR triggered by the Xcc wild type strain (Figure 2 3 ). One possible explanation for the observed phenotypes was that a T3SS independent protein is recognized by a tomato resistance protein. Phenotypic Analysis of Nineteen C andidate Effector and Hrp -Regulon Associated Genes. To evaluate the role in pathogenicity of nineteen Xcc T3 -effector candidate genes we mutated each gene, individually, using insertional mutagenesis procedure (Figure 2 4 ). The insertion event on each mut ant was analyzed by PCR and the resulting fragment was sequence d to confirm the mutagenic plasmid has inserted in the proper position in the Xcc 306 genome. The virulence assay in grapefruit leaves with all generated Xcc 306 mutants showed that all ninetee n mutants retained the ability to produce disease symptoms. Discussion In an attempt to isolate and characterize new pathogenicity genes from X. citri subsp. citri strain 306, we have analyzed, using mutagenesis tools, 19 putative HrpX regulated and/or ca ndidate effector genes, a hrpA gene that is expressed independent ly of the HrpX regulon, and two hrp regulatory genes hrpX and hrpG as revealed by genomic sequence data (da Silva et al ., 2002). N ot all of the Xcc HrpX -regulon candidate genes carry the co nsensus PIP box motif upstream of the start codon and the associated 10 boxlike sequence (Table 2 4 ). This motif has been proposed to function as another cis element of HrpX regulon, and has been identified in several HrpX dependent expression genes (Noel et al ., 2001; Furutani et al ., 2006, Koebnik et

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35 al ., 2006; Furutani et al ., 2009). Such motifs are found in most of the HrpX regulated genes in Xanthomonas spp. (Wengelnik and Bonas, 1996; Wengelnik et al., 1996a; Wengelnik et al., 1996b ; Cunnac et al ., 2004). Additionally, both motifs have been reported to be required for HrpX -dependent expression and base substitution in either motif can affect the expression of the genes (Tsuge et al ., 2005; Furatani et al ., 2006; Furatani et al ., 2009). And here we identified 11 genes preceded by both perfect/imperfect PIP -box and 10 boxlike motifs in each promoter region, six genes only with perfect/imperfect PIP -box motif and two genes, avrPphE2 and xopX with the absence of both motifs ( Table 2 4 ). T he absence of PIP -box region in avrPphE2 has been reported (da Silva et al ., 2002) and the xopX gene has been identified in X. oryzae pv. oryzae and X. campestris pv. vesicatoria which possess a imperfect PIP box and 10 boxlike region ( TTCTT N15TTCGC N30-CA AAT G ) (Koebnik et al ., 2006; Salzberg et al ., 2008). Recently studies have demonstrated that a perfect/imperfect PIP -box es and 10 like box motifs may be not sufficient for HrpX dependent expression of genes and in xanthomonads, example, xopB and hrpF genes la cking the PIP -box are expressed under HrpX -regulatory protein (Furutani et al., 2004; Furutani et al., 2006). Moreover, base substitution i n the conserved region may reduce the promoter activity but when substitution affect s several bases such as TTCGB N15TTCGB, TRCGBN15TTCGB, TTCTB N15TTCGB, TTCGB N15-VTCGB, TTCGB N15TRCGB, TTCGBN15TTCHB (B: C/T; H: A/C/T; R: A/G; V: A/C/G) it can ha ve a negative effect on gene activities (Furutani et al ., 2006). Pathogenicity experiments to assess the influence of each gene for disease development were carried out. Surprisingly, none of the 19 mutants generated in this study was observed to affect the ability to incite disease symptoms in susceptible grapefruit leaf. Neither mutated homologous genes, which have bee n reported to be required for pathogenicity, such as XAC 0661

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36 (endopolygalacturonase) and XAC2374 (polygalacturonase) that are similar to E. carotovora subsp. carotovora PehA polygalacturonase, a cell wall degrading enzyme and HR elicitor in Arabidopsis (K ariola et al., 2003). However, X. o. pv. oryzae T3 effectors suppress a plant basal defense response elicited by T2 -secreted plant cell -wall -degrading enzymes (Jha et al., 2007). In addition, Yamazaki et al (2008) suggested that Xcc may suppress the polyg alacturonase mediated plant defense by coregulating polygalacturonase isozymes. The pathogenicity assay with 306:: hpa1 and 306:: hpaF mutants did not show any macroscopic change in the symptoms compared with the Xcc wild type strain contrasting with the results reported in X. o. pv. oryzae and X. a pv. glycines where hpa1 and hpaF are required for full virulence in (Zhu et al., 2000; Kim et al., 2003; Cho et al ., 2007). In the class of Xanthomonas outer proteins two mutants were assessed 306:: xopX (c arrying an insertion in xopX gene) and 306:: xopQ (carrying an insertion in xop Q gene) for pathogenicity, neither one demonstrated canker symptoms However, xopQ and xopX ha ve been reported to be highly conserved among Xanthomonas spp. (Roden et al., 2004; Furutani et al ., 2009). In Xcv, XopX contributes to the virulence on host pepper and tomato plants and is T3SS -dependent (Metz et al ., 2005). When the avirulence genes, 306avrXacE1 avrXacE2 avrXacE3 and avBs2 were mutated in Xcc strain they produced t ypical canker symptoms on grapefruit leaves S imilar results were reported previously with avrXacE1 and avrXacE3 homologs in Xcv xopE1 and xopE2, respectively, mutants when tested in susceptible pepper plants ECW (Thieme et al ., 2007). However, avrBs2 phenotype observed here contrast s with the phenotype demonstrated with avrBs2 mutant from X. c. pv. vesicatoria which is required for full virulence (Swords et al.,

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37 1996). In conclusion these avirulence genes do not appear to have a pronounced effect on X cc fitness in disease development in grapefruit. We demonstrated above that a putative pathogenicity Xcc genes tested are not required for disease development in greenhouse conditions. These genes were identified, based o n sequence analysis, by da Silva e t al (2002) and they are considered genes that are regulated by HrpX Our results indicate that the putative hrpX regulon and effector/avirulence genes do not have any direct role in disease development (Table 2 5) However, we can not conclude that they are not important for bacteri al surviv al In xanthomonads hrp activation cascade, the H rpG upregulates expression of hrpA and hrpX genes, and then HrpX activates the transcription the hrpB to hrpF operons (Wengelnik and Bonas, 1996; Wengelnik et al., 1996a; Wengelnik et al., 1996b ). In our experiments, the disruption of either hrpG or hrpX severe ly disrupts the induction of any symptoms in grapefruit leaves but, unexpectedly, both mutants retained the ability to trigger HR in non -host tomato plants. Simila r results were reported by Marutani et al. (2005), where a P. syringae pv. tabaci hrp regulatory hr cC mutant also was able to trigger HR in non -host tomato plants in the hrp independent manner (Chapter 3) Additionally, a hrp X. perforans mutant harbori ng a X. fuscans pv. aurantifolli clone was able to trigger HR in similar manner. Based on these results, we speculate that an unknown T3SS independent gene in Xcc is a HR -elicitor. To deepen our understanding the role of the HrpX -regulon genes for pathogen icity and the model of action of T3SSindependent HR -elicitor proteins, more detailed mutagenesis and protein secretion analysis are required.

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38 Table 2 1. X anthomonas citri pv. citri genes used in the study ORF Number Relevant characteristics Hrp Gene Cluster XAC0416 hpa1 XAC0415 hrpA ( hrcC ) XAC1265 hrpG XAC1266 hrpX Extended Hrp Conserved Regulon XAC0277 Conserved hypothetical protein XAC0661 endopolygalacturonase XAC1706 Alkanal monooxygenase XAC1886 K adipate enol lactone hydrolase XAC2374 Polygalacturonase XAC2534 Conserved hypothetical protein XAC3309 aminopeptidase Additional Hrp Regulon Candidate XAC2370 Endopeptidase XAC2922 hprW XAC3230 Actin ADP ribosylating toxin domain Putative Ef fector/Avirulence XAC0286 avrPphE1 XAC3224 avrPphE2 XACb0011 avrPphE3 XAC0393 hpaF XAC3090 Leucine rich protein XAC0076 avrBs2 Xanthomonas outer protein XAC0543 xopX XAC4330 xopQ All the genes show n in this table were identified by da Silva e t al. (2002)

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39 Table 2 2. Bacterial strains and plasmids used in the study Strain or plasmid Relevant characteristics Source Strains Xanthomonas citri subsp. citri 306 Wild type, Asiatic strain, isolated in Brazil, Rif R DPI a 306:: avrBs2 av rBs2 single recombinant of pCR avrBs2 KnR, AmpR This study 306:: 2277 Conserved hypothetical protein, single recombinant of pCR2277, Kn R Amp R This study 306:: avrPphE1 avrPphE1 single recombinant of pCR avrPphE1, KnR, AmpR This study 306:: hpaF hp aF single recombinant of pCR hpaF Kn R Amp R This study 306:: hrcC hrcC single recombinant of pCR hrcC Kn R Amp R This study 306:: hpa1 hpa1 single recombinant of pCR hpa1 KnR, AmpR This study 306:: xopX xopX single recombinant of pCR xopX Kn R Amp R This study 306:: 0661 Xac0661 single recombinant of pCR0661, Kn R Amp R This study 306:: hrpG hrpG single recombinant of pCR hrpG Kn R Amp R This study 306:: hrpX hrpX single recombinant of pCR hrpX Kn R Amp R This study 306:: 1706 Xac1706 single recombinant of pCR1706, KnR, AmpR This study 306:: 1886 Xac1886 single recombinant of pCR1886, Kn R Amp R This study 306:: 2370 Xac2370 single recombinant of pCR2370, KnR, AmpR This study 306:: 2374 Xac2374 single recombinant of pCR2374, K n R Amp R This study 306:: 2534 Xac2534 single recombinant of pCR2534, Kn R Amp R This study 306:: hrpW hrpW single recombinant of pCR hrpW Kn R Amp R This study 306:: 3090 Xac3090 single recombinant of pCR3090, Kn R Amp R This study 306:: avrPphE2 avrPphE2 single recombinant of pCR avrPphE2, KnR, AmpR This study 306:: 3230 Xac3230 single recombinant of pCR3230, Kn R Amp R This study 306:: 3309 Xac3309 single recombinant of pCR3309, Kn R Amp R This study

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40 Table 2 2 Continued Strain or plasmid Relevant characteristics Source 306:: xopQ xopQ single recombinant of pCR xopQ Kn R Amp R This study 306:: avrPphE3 avrPphE3 single recombinant of pCR avrPphE3 Kn R Amp R This study Escherichia coli DH5 F recA BRL b PIR Host for pOK1; Sp R oriR6K, K2 replicon UB c Plasmids pCR2.1 TOPO Phagemid, Cb R Kn R Invitrogen pLAFR3 Tc R rlx + RK2 replicon pRK2013 Kn R tra + mob + Daniels et al (1984) pOK1 Suicide vector, SacB Huguet et al (1998) pUFR034 Kn R Tn903, IncW, Mob + De Feyter et al (1990) pCR avrBs2 avrBs2 partial fragment in pCR2.1 TOPO This study pCR0277 Conserved hypothetical protein partial fragment in pCR2.1 TOPO This study pCR avrPphE1 avrPphE1 partial fragment in pCR2.1 TOPO This study pCR hpaF hpaF partial fragment i n pCR2.1 TOPO This study pCR hrcC hrcC ( hrpA ) partial fragment in pCR2.1 TOPO This study pCR hpa1 hpa1 partial fragment in pCR2.1 TOPO This study pCR xopX xopX partial fragment in pCR2.1 TOPO This study pCR0661 Endopolygalacturonase partial fragment in pC R2.1 TOPO This study pCR hrpG hrpG partial fragment in pCR2.1 TOPO This study pCR hrpX hrpX partial fragment in pCR2.1 TOPO This study pCR1706 Alkanal monooxygenase partial fragment in pCR2.1 TOPO This study pCR1886 K adipate enol lactone hydrolase pa rtial fragment in pCR2.1 TOPO This study pCR2370 Endopeptidase partial fragment in pCR2.1 TOPO This study pCR2374 Polygalacturonase partial fragment in pCR2.1 TOPO This study pCR2534 Conserved hypothetical protein partial fragment in pCR2.1 TOPO This st udy pCR hrpW hrpW partial fragment in pCR2.1 TOPO This study pCR3090 Leucine rich protein partial fragment in pCR2.1 TOPO This study pCR avrPphE2 avrPphE2 partial fragment in pCR2.1 TOPO This study

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41 Table 2 2. Continu ed Strain or plasmid Relevant charac teristics Source pCR3230 Actin ADP ribosylating toxin domain partial fragment in pCR2.1 TOPO This study pCR3309 Aminopeptidase partial fragment in pCR2.1 TOPO This study pCR4074 Ribonucleotide disphosphate reductase partial fragment in pCR2.1 TOPO Thi s study pCR4090 3 oxoacyl [ACP] reductase partial fragment in pCR2.1 TOPO This study pCR xopQ xopQ partial fragment in pCR2.1 TOPO This study pCRb0011 avrPphE3 partial fragment in pCR2.1 TOPO This study pL22 X cc strain A w pLAFR3 cosmid that contains hrp X hrpG and hsp90 Xo homologues Rybak et al (2009) pO Harpin H rpW Rif R This study pO HrpW Harpin Rif R This study pCR3309 Aminopeptidase partial fragment in pCR2.1 TOPO This study pCR4074 Ribonucleotide disphosphate reductase partial fragment in pC R2.1 TOPO This study a BRL, Bethesda Research Laboratories, Gaithersburg, MD. b UB, U. Bonas, Martin Luther -Universit t, Halle, Germany.

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42 Table 2 3. Oligonucleotide sequences used in this study Gene Primer name Primer sequence length avrBs2 avrBs2F1 5 CGCATCATCTTCAATCTGCAGC 3 22 avrBs2R1 5 CCGTGTAGTACTTGGCATAGAC 3 22 UpavrBs2F1 5 ACCTGGTCTATCTGTCGATC TC 3 22 Xac0277 0277F1 5 CAGAACGCTTTCAGCAACGTC 3 21 0277R1 5 CGCTTTCCTTCAATGACTCC 3 20 Up0277F1 5 GCTCATCGACCTGTTGCTCAAG 3 22 avrPphE1 0286F1 5 GCAGATAACGAACGTCTGTG AC 3 22 0286R1 5 CTTCAGCCGTGTGTGAGAAG T 3 21 Up0286F1 5 GCTTCATCCAGCACGATTTC 3 20 hpaF 0393F1 5 C TGAAGACAGGCCTTCACATC AG 3 23 0393R1 5 GGCCGATGTTCTCAGGTAAC TC 3 22 Up0393F1 5 CATGCTTGCAATGAAGACGG TC 3 22 hrcC 0415F1 5 CCAGGGTATGTGGATACCGT TTC 3 23 0415R1 5 GATGATGGTGGCATCGATCT GCAG 3 24 Up0415F1 5 GACTTCTTCACTTGCCTAGC AG 3 22 Hpa1 0416F1 5 CTTTGAACACACAGCTCGGC 3 20 0416R1 5 CTGCTCGGCATTGTTGCTCT 3 20 Up0416F1 5 TGAATACAGGTCTCCAGGTGAG 3 22 xop X xopXF1 5 GACATCCATACCGACAATGCTG 3 22 xopXR1 5 CAGATGCCGAGAGCGTCATCAC 3 22 UpxopXF1 5 GCAGCGGAAGGAATATTGCTGTAG 3 24 Xac0661 0661F1 5 CACTTGTCAGCCGTCCTTGA AG 3 22 0661R1 5 TGATCAGGTTGACGTCGTAG AG 3 22 Up0661F1 5 GTTGTTGACGGTCGTGACGAG 3 2 1 hrpG 1266F1 5 GATCTTCGATGCCAGCTATG TC 3 22 1266R1 5 ACTTGTAGCCGTGCGAATAG AC 3 22 Up1266F1 5 GATCGAGAGAAGCAGACATGACAC 3 24 hrpX 1265F1 5 CATGAGCGACCATGTGTTCT G 3 21 1265R1 5 CACTTCGTTGATCGACAGAT CC 3 22 Up1265F1 5 GATCCGCTGCATACAATCGT TTG 3 23 Xac1706 1706F1 5 CTTCGCCAACATGCTCGATC TG 3 22 1706R1 5 CTTTGCTGCTGGGTAGTGAA C 3 21 Up1706F1 5 CAGGTCGTACAACTCGAAGAAC 3 22 Xac1886 1886F1 5 CAGCACTACCAGGTCATCGT TC 3 22 1886R1 5 GATCAGCTCGTAATCGCAGC AG 3 22 Up1886F1 5 GAACTTCGATCAGATC CAGGAG 3 22 Xac2370 2370F1 5 CCTAGCCTTCCTAGTGATTA CC 3 22 2370R1 5 CAGACGCTCCTACAGTATTC GAC 3 23 Up2370F1 5 TCCTGCAACTCTTTGGTGTC TG 3 22 Xac2374 2374F1 5 GTGGGATCTTGCCTATCTCA AC 3 22

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43 Table 2 3 Continued Gene Primer name Primer sequence length Xac2374 2374R1 5 GTTCTCGAAGCTGATGTCGT AG 3 22 Up2374F1 5 CACAGGTTCGCTAGCTGTGATAC 3 22 Xac2534 2534F1 5 GTAATCCGCTGTATGCCTAT GG 3 22 2534R1 5 ATAGCTCTGCGCGTTCAGAT AC 3 22 Up2534F1 5 GTCATCTACGACAGTCGCTG 3 20 hrpW 2922F1 5 CGACGGACACAACAATCT CTAC 3 22 2922R1 5 GACAGTGCCTCCACTTGATG 3 20 Up2922F1 5 CACCTAGAACTGCTGACAAA CGAC 3 24 Xac3090 3090F1 5 G ACCAGGCATTCCGTCTTTCT C 3 22 3090R1 5 C AGTCTGGAGAGATTGACGCA AC 3 23 Up3090F1 5 CTGACCAGATATGTGCCTGG TGAC 3 24 avrPphE2 3224F1 5 GTGTCG TAAATGGGCTGTTGAG 3 22 3224R1 5 CTAGGACGATGGTCTTGTGA TG 3 22 Up3224F1 5 GCATTCTGCGCTACAACAAT TC 3 22 Xac3230 3230F1 5 CGGGTTCACCTGAGCATTAT 3 20 3230R1 5 GAGAACTTCCTGCTCATTCG GACG 3 24 Up3230F1 5 GTTGGAACACAAGCACGAAC AC 3 22 Xac3309 3309F1 5 CTTCACTGGGTTTCGACAAG 3 20 3309R1 5 GATCACGTTGTAGGAGGTGG 3 20 Up3309F1 5 CAGTTCCTGATCATCGCCTTC 3 21 avrPphE3 0011F1 5 GTCACTGCAGGAGATCAAGA TG 3' 22 0011R1 5 CGAGTCGAGGACTACAGGTG ATTC 3 24 Up0011F1 5 CACGACGCCCATGCGAAATC GTTG 3 24 xopQ 433 3F1 5 GACGATGTCGTGACCTACAC C 3 21 4333R1 5 GTAGGCAGCTTCCTTGGTCA G 3 21 Up4333F1 5 GCTTCTGAGACAGTCACAAT GAG 3 23

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44 Table 2 4. Candidate PIP box ( like) and 10 box-like sequences of the proposed hrpX regulons and putative effector/avirulence genes of Xcc ORF number (Putative) ORF Product PIP box sequence a Distance (bp) b 10 sequence c XAC0076 avrBs2 TTCGC N 15 TTCGC XAC0277 Conserved hypothetical protein TTCG G N 15 TTCGC 30 TA GCA T XAC0286 avrPphE1 TTCG T N 15 TTCG G 30 CA AAC T XAC0393 hpaF TTCGC N 16 TTCGC 30 TA GCC T XAC0416 hpa1 TTCGC N 15 TTCGC 31 TA CTG T XAC0543 xopX None XAC0661 endopolygalacturonase TTCGC N 15 TTCGC 30 TA GAG T XAC1706 Alkanal monooxygenase TTCG T N 15 TTCG T XAC1886 K adipate enol lactone hydrolase TTCG T N 15 TTCG T XAC23 70 Endopeptidase TTCGC N 15 TTCGC 30 TA TAG T XAC2374 Polygalacturonase TTCG G N 15 TTCG T 32 A A GCT T XAC2534 Conserved hypothetical protein TTCGC N 15 TTCG T 30 TA TGG T XAC2922 hprW TTCGC N 15 TTCG G 31 G A TGA T XAC3090 Leucine rich protein TTC CA N 15 TTCG C XAC3 224 avrPphE2 None XAC3230 Actin ADP ribosylating toxin domain TTCG T N 15 TTCG G 30 CA AAC T XAC3309 aminopeptidase TTCGC N 15 TTCGC XAC4330 xopQ TTCG T N 15 TTC A C 31 TA ACG T XACb0011 avrPphE3 TTCGC N 15 TTCG G a PIP box sequence and the distance in base pairs between the two conserved motif. Nucleotides deviating from the consensus are underline. b Distance in base pairs between the end of PIP box and the 10 promoter motif c 10 promoter motif sequence and 10 conserved base pairs are shown in bold. N ucleotides deviating from the consensus are underline.

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45 Table 2 5. X anthomonas citri pv. citri mutants pathogenicity phenotype in grapefruit leaveas and HR elicitation in tomato leaflets Designation (Putative) ORF Product Grapefruita Tomatob XCC 30 6 Wild type strain + + 306:: avrBs2 avrBs2 + + 306:: 2277 Conserved hypothetical protein + + 306:: avrPphE1 avrPphE + + 306:: hpaF hpaF + + 306:: hrcC hrpA + 306:: hpa1 hpa1 + + 306:: xopX xopX + + 306:: 0661 endopolygalacturonase + + 306:: hr pG hrpG + 306:: hrpX hrpX + 306:: 1706 Alkanal monooxygenase + + 306:: 1886 K adipate enol lactone hydrolase + + 306:: 2370 Endopeptidase + + 306:: 2374 Polygalacturonase + + 306:: 2534 Conserved hypothetical protein + + 306:: hrpW hprW + + 306:: 3090 Leucine rich protein + + 306:: avrPphE2 avrPphE2 + + 306:: 3230 Actin A DP ribosylating toxin domain + + 306:: 3309 aminopeptidase + + 306:: avrPphE3 avrPphE3 + + 306:: xopQ xopQ + + a Bacterial Pathogenicity: (+) positive for symptoms development, ( -) no symptoms. b HR induction: (+) positive for HR, ( ) negative for HR.

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46 Figure 2 1. Genetic organization of the hrp genes of X. citri subsp. citri The arrows indicate the orientation of the six hrp operons, hrpA to hrpF The boxes correspond to open reading frames (ORFs). hrc genes encode proteins conserved among type II I secretion system; hrp and hpa genes encode non -conserved proteins (da Silva et al. 2002; Dunger et al ., 2005).

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47 Figure 2 2. Proposed model for hrp gene regulation cascade in X. citri subsp. citri The model was formulated based on the X. axonopodis pv. citri srain 306 genome sequence (da Silva et al. 2002) and the model presented by B ttner and Bonas 2002 for hrp gene regulation in X. campestris pv. vesicatoria The cascade is initiate d with an uncharacterized signal in the bacterial envelope (indic ate by a question mark) that senses the outside stimuli and activates hrpG Consequently, HrpG activates the expression of hrpX and hrpA and HrpX activates the expression of hrpB -hrpF xop genes, hrpW (which is part of the hrp cluster although is located downstream) and other hrpX regulon genes.

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48 Figure 2 3 Comparison of the bacterial pathogenicity and HR elicitor in grapefruit leaves and tomato leaflets, respectively. The strains were infiltrated into the leaves at concentrations of 5 x 108 CFU/mL. Lab eling: (1) 306:: hrpG comlemented; (2) 306:: hrpG ; (3) Xcc strain 306 wild type; (4) 306:: hrpX ; (5) 306:: hrpX comlemented; (6) 306:: hrpA ; (7) 306:: hrpG The grapefruit leaves were photographed 7 days after infiltration and the tomato leaf (right ) was photographed 24 h after in filtra tion.

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49 Figure 2 4 Integration by site -specific recombination using one single target sited. Integration of the complete linearized plasmid is mediated by single crossing -over event. Amp = Ampicillin resistance cass ette, Km = kanamycin resistance cassette.

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50 CHAPTER 3 IDENTIFICATION OF A HRP INDEPENDENT HR ELICI TOR FROM Xanthomonas c i tri subsp. citri Introduction Plants are frequently under attack by pathogens. P lants are resistant to most pathogens, and t he respo nses to pathogen attack involve different innate immunity responses. Innate immunity responses can be initiated by a local infection where the defense response extends to unaffected parts, a response called induced systemic resistance (ISR) or systemic acq uired resistance (SAR) (Hunt et al ., 1996; Hammerschimidt, 1999). The elicitation of plant innate immunity by bacteria is sometimes divided, conceptually, into two type of elicitation. PAMP triggered immunity (PTI) involves the induction of defense response by a variety of common molecular components of bacteria known collectively as pathogen associate molecular patterns. Effector triggered immunity (ETI) involves the triggering of defense by effector proteins of the type III secretion pathway, which are p resent in many plant and animal pathogenic proteobacteria. Innate immunity involves the activation of mitogenactivated protein kinase (MAPK) cascades and synthesis of hormones such as salicylic acid (SA) or jasmonic acid (JA) (Block et al., 2008). The bac terial pathogens also trigger the production of an oxidative burst, which, ultimately, limits pathogen spread by induction of host cell death (Baker and Orlandi, 1995; Gozzo, 2003; Block et al ., 2008) and leads to changes in the cell wall composition and t he de novo synthesis of antimicrobial compounds such as phytoalexins and pathogenrelated (PR) proteins (Bestwick et al ., 1998; Hammerschimidt, 1999b; Innes, 2001). Other responses include closure of stomata in response to bacterial attack (Melotto et al ., 2006); and the h ypersensitive r eaction (HR) around the initial infection sites isolating the pathogen in dead cells, consequently, preventing the spread throughout the plant (Alfano and Collmer, 2004).

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51 Type III secretion systems (T3SS) function to transl ocate a collection of effectors (T3 effectors) into the plant cells, where the T3 -effectors modulate the cell environment and enhance the conditions for bacteria l multiplication (Cornelis and van Gijsegem, 2000). The genes encoding most of the components f or the T3SS are clustered into the hypersensitive reaction and pathogenicity ( hrp ) gene regions (B ttner and Bonas, 2003). Basically, hrp gene clusters in plant pathogenic bacteria, including Xanthomonas species, are regulated and dependent on the host int eraction. In Xanthomonas species, two key regulatory proteins are HrpG and HrpX (Wengelnik and Bonas, 1996). I n R solanacearum the hrp system is regulated through a HrpG homolog and, downstream, a HrpX homolog originally named HrpB. HrpB and HrpX belong to to the AraC family of transcriptional regulators (Wengelnik and Bonas 1996; Brito et al 1999). P. syringae possesses three regulatory genes hrpR hrpS and hrpL The products of two genes, HprR and HrpS positively regulate the expression of hrpL whi ch encodes an alternative sigma factor responsible for transcriptional activat ion of other hrp genes (Xiao et al. 1994). In addition, homologs of hrpS and hrpL also occur in Erwinia amylovora (Sneath et al 1990; Wei et al. 1992). In 1971, Flor demonstr ated that a component from the bacteria, avirulence ( avr ) gene, when recognized by the host triggered a defense response. Later, studies showed that T3SS was the system used by the bacteria to inject the avr genes into the host cells (T3 -effectors) and the recognition was performed by host resistance protein. A malfunction of the T3SS generally results in the complete loss of disease development and the loss of ETI or Hrp-dependent elicitation of resistance responses due to the inability to transport the av r protein into the host (Jones and Dangl, 2006; Block et al ., 2008). The first avirulence gene, avrA was isolated from P. s. pv. phaseolicola in 1984 (Staskawicz et al ., 1984). More than 40 Avr proteins have since

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52 been identified. The first direct interac tion between an Avr protein and the cognate R protein was demonstrated with the tomato bacterial speck bacterium P. s. pv. tomato harboring the gene avrPto whose product is recognized by the tomato resistance protein Pto (Tang et al., 1996; Mucyn et al., 2006). Additionally, Pto also recognizes another distinct Avr protein from P. s. pv. tomato known as AvrPtoB (Abramovitch et al ., 2003,Wu et al ., 2004). AvrXa27 in X. oryzae pv. oryzae also induces resistance in rice plants carrying the cognate R gene Xa 27 but the resistance is induced only in the presence of the pathogen (Gu et al., 2005). PTI, on the other hand, is the recognition of PAMPs by receptor -like kinases, which are localized in the extracellular matrix (Jones and Dangl, 2006). PTI response is not generally considered to be a race -specific defense. Therefore, ETI requires the T3 -effectors and is essentially T3SS dependent, and PTI is T3SS independent and is generally not accompanied by an HR. In chapter 2 was demonstrated that hrpG and hrpX muta nts of Xanthomonas citri subsp. citri strain 306, the causal agent of citrus canker (Stall and Civerolo, 1991), retained the capability to trigger an HR in the non -host plant tomato, indicating that the HR was T3SS independent. Similarly, Xanthomonas fuscans subsp. aurantifolli (Xfa) strain C, the causal agent of citrus canker in Key lime (Schubert et al ., 2001; Schaad et al ., 2005), also triggered an HR i n tomato in a T3SS -independent manner as (data unpublished). In order to identify the elicitor of HR a cloning strategy using restriction enzymes was initiated to identify the genes controlling the T3SS independent HR response in tomato. Materials and Methods Bacterial Strains and Plasmids Bacterial strains and plasmids used in this study are listed in Ta ble 3 1

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53 Media and Growth Conditions Escherichia coli was grown in Luria Bertani (LB) medium (Sambrook et al., 1989) at 37oC with appropriate antibiotics. For solid media BD Bacto agar was added at 15 g l1. Strains of Xanthomonas were cultured on nutrient agar (NA) medium and on tryptone sucrose agar at 28oC (Zhu et al ., 2000). All bacterial strains used in this study were stored in 20% glycerol in sterile tap water and maintained at 80oC. Triparental matings were performed on nutrient -yeast extract glyc erol (NYG) agar (Daniels et al ., 1984). Antibiotics were used in the following concentrations: ampicillin (Amp) 100 g ml1; kanamycin (Kn) 50 g ml1; rifampicin (Rif) 100 g ml1; and tetracycline (Tc) 12.5 g ml1. Recombinant DNA Techniques Standard DN A manipulations were performed as described by Sambrook et al. (1989). Restriction enzymes, T4 DNA ligase, and GoTaq Flexi DNA Polymerase were used according the recommendations of the manufacturer (Promega, Madison, WI, USA). Chemicals were purchased fr om Sigma -Aldrich (St. Louis, MO, USA), and Fishers Scientific (Hampton, NH, USA). The polymerase chain reaction (PCR) was performed in a PCR Express Thermal Cycler (ThermoHybaid, Ashford, UK). The Topo TA cloning kit was used for cloning of PCR products (Invitrogen, Carlsbad, CA, USA). All the enzymes and kits were used according to specifications of the manufacturers. Constructs were transformed into competent Escherichia coli DH5 cells described by Sambrook et al (1989). Generation of Xcc 306 mutants wa s made using single homologous recombination methodology as described by Sugio et al (2005) The selected plasmids were introduced into X c c 306 thorough e lectroporation. Aliquots of Xcc competent cells were mixed with plasmid DNA and electroporated using a BioRad GenePulser II Electroporator instrument The conditions applied were 200 OHMs (resistance), 25 FD (capacitance) and 2.5 Volts. After the pulse, the cells were immediately diluted by addition of

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54 250 L of NB medium. The cells were incubated for 1 h at 28oC with constant shaking, and plated on NA containing kanamycin at 50 g ml1;, then incubated at 28oC for 2 days. Triparental mating, as described by Daniels et al (1984), was used as a technique to introduce plasmids into Xcc 306. Sequencing was performed at DNA Sequencing Core Laboratory of the Interdisciplinary Center for Biotechnology Research, University of Florida Gainesville, FL (UF ICBR). DNA Amplification For mutagenesis analysis of hprG and hprX gene s in Xcc 306, the custom primer pairs of 1266F1 5 GATCTTCGATGCCAGCTATGTC 3 and 1266R1 5 -ACTTGTAGCCGTGCGAA T AGAC 3' and 1265F1 5 CATGAGCGACCATGTGTTCTG 3 and1265F1 5 CATGAGCGAC CATGTGTTCTG 3 and 1265R1 5 GATCG -AGAGAAGCAGACATGACAC 3 were designed to amplify a partial fragment of hprG and hprX genes, respectively from Xcc 306 genomic DNA. For subsequent validation of gene insertion, the primers Up1266F1 5 GATCG A AGAGAAGCAGACATGACAC 3 Up1265F1 5 GATCCGCTGCATACAATCGT TTG 3 were designed upstream start codon of hrpG and hrpX respectivel y, and were used in combination with the standard flanking vector M13FOR and M13REV primers of the TOPO vector pCR2.1. The parameters used for PCR were as follows: Step 1. 90oC for 5 min; step 2. 95oC for 1 min; step 3. annealing for 1 min (the annealing t emperature was calculated in according to the C/G content of the primer); step 4. 72oC for 1 min per Kb, 30 cycles from step 2 to step 4; step 5. 72oC for 10 min. Mutagenesis of hrpG and hrpX in X. citri subsp. citri 306 A partial fragment of each gene was amplified by PCR using Xcc genomic DNA as template and the appropriate primer pair. The PCR fragments were purified using QIAquick PCR Purification Kit purification system (QIAGEN) and cloned into pCR2.1. The constructs were

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55 transformed into E. coli DH5 and the transformants containing the single homologous recombination were selected by plating onto LB media containing kanamycin 50 g ml1. Transformants were analyzed by restriction enzyme digestion profiles and sequencing. The derivative plasmids with e ither a portion of hrpG or hrpX respectively, were introduced into Xcc 306 by electroporation. Treated strains were spread on NA plates containing Kanamycin 100 g ml1 and incubated at 28oC for 2 3 days. PCR amplification with upstream primers and the st andard flanking vector M13FOR and M13REV primers were used to confirm if colonies contained a mutation in the appropriate gene as a result of a single crossover and insertion of the pCR2.1 vector into the wild type gene. The appropriate wild type gene in p L A FR3, pVX9, was re introduced into the respective mutant (306:: hrpG and 306:: hrpX ) and tested for complementation of the mutant phenotype. Wild type, mutant, and complemented strains were then tes ted for pathogenicity on citrus and their ability to elic it an HR in the non host plants tomato and pepper. Subcloning of HR -Elicitor Candidate from Xaa Library The genomic library used in this study was made using genomic DNA from X a. pv. aurantifolli C strain. The DNA was partially digested with Sau3A and fr agments in the 25 to 30kilobase (Kb) range were isolated from the gel following gel electrophoresis. DNA was ligated with Bam HI -digested pLAFR3, and transformed and maintained in E. coli DH5 (Staskawicz et al ., 1984). The cosmids were introduced into a hrpmutant of X perforans strain 91118 (91:: hrp ) by triparental mating. Mixing 91:: hrp mutant cells as recipient with cosmid as donor and with pRK2073 as the helper plasmid on NYG agar. The ratio of recipient to donor to helper was 2:1:1 (vol/vol/vol) After 24 h of incubation at 28oC, the mating mixtures were resuspended in sterile tap water, and aliquots were spread onto NA plates containing rifamycin

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56 and tetracycline (100 g ml1 and 12.5 g ml1, respectively) for selection of transconjugants. Indi vidual colonies were selected and subsequently inoculated in tomato plants to screen for HR induction after 24 h ( Gerald V. Minsavage personal communication). Sequencing analysis of the pL450 clone, which was identified as HR positive in the tomato scree n, was performed using c uston primers, NP5 (5 CCCTTCACCAAGTTCGACGACA 3) and NP3 (5 GCGGGTGCCGTGCTCGTGTT 3), used to sequence both ends of the 450 clone. By sequencing the ends of the cosmid we generated a restriction map to facilitate subclon ing the HR -elicitor. The subcloning of the 450 clone was performed by restriction digestion of the original 450 clone with Bam HI enzymes and purification of the excised fragments from an agarose gel by using the QIAquick Gel Extraction Kit purification system (QIAGE N). The fragments were ligate d individually into Bam HI digested pUFR034 using T4 DNA ligase according to the manufacturers instructions. Ligation products were transformed into competent cells of E. coli DH5 produced by the calcium chloride procedure as described by Sambrook et al (1989). Triparental matings were performed with Xcv (wild type), 91:: hrp mutant, opportunistic xanthomonads T55 and INA42 as recipients with plasmid as donor and with pRK2073 as the conjugational helper on NYG agar. Individual transconjugant colonies were injected into tomato leaflets to detect subclones carrying the HR -elicitor genes. A 4.5 Kb HR positive subclone, B38, w as digested with Bam HI and Eco RI enzymes, and purification was performed as described above. Two fragments, 3.0 kb and 1.5 kb, were ligated into Bam HI and Eco RI digested phagemid vector pBluescript II/KS (Stratagene, La Jolla, CA) using T4 DNA ligase used according to the manufacturers instructions. Ligation and triparental matings in X. perforans (wild type) and 91:: hrp mutant were performed as described above. Individual transconjugant colonies were injected into tomato leaflets to detect which fragment

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57 was carrying the HR -elicitor gene (s). For sequence analysis, sequencing of the 3.0 kb fragment subclone ( B38.3) was performed using the standard flanking vector R24 and T7 primers. DNA sequencing was conducted at the ICBR sequencing facility (University of Florida, Gainesville, FL) with the Applied Biosystems model 373 system (Foster City, CA). Plant Material and Plant Inoculations Grapefruit cv. Duncan ( Citrus paradisi ) plants were grown from seed in 15 cm plastic pots in Terra -Lite agricultural mix (Scott Sierra Horticultural Products Co. Marysville, OH). The plants were kept in the glasshouse of the Univer sity of Florida in Gainesville, Florida at temperatures ranging from 25 30oC. Tomato plants ( Solanum lycopersicum ) cv Bonny Best and pepper plants ( Capsicum annuum ) cv. ECW and its NIL ECW20R containing the Bs2 resistance gene were planted from seeds in Plugmix (W. R. Grace & Co., Cambridge, MA, USA). After two weeks, the seedlings were transferred to Metromix 300 (W. R. Grace & Co) in 10 cm plastics pots. The plants were kept in the glasshouse of the University of Florida in Gainesville, Florida at temp erature ranging from 25 30oC. For preparation of bacterial suspensions, 18 h cultures were harvested from the NA plates and suspended in sterile tap water, and standardized to an optical density at 600 nm (OD600) = 0.3 (5 x 108 CFU/ml) with a Spectronic 20 Genesys spectrophotometer (Spectronic -UNICAM, Rochester, NY, USA). For disease symptom assays, bacterial suspensions of the strains used in this study were infiltrated at 5 x 108 CFU/ml into abaxial surface of citrus leaves by using hypodermic syringe and needle, and the symptoms were assessed up to 10 days after inoculation. Plant responses were evaluated after 3 4 days for water -soaking and 6 7 days for pathogenicity. For HR tests, bacterial suspensions adjusted to 5 x 108 CFU/ml in sterile tap water wer e infiltrated into leaflets of tomato cvs. Bonny Best and pepper plants cv. ECW and ECW20R with a hypodermic syringe and needle. The inoculated plants were incubated in a growth room at 24 28oC and assessed for

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58 HR elicitation 24 h after infiltration for HR induction. Disease scores are the means of those for three leaves. All the experiments were repeated at least three times. Measurement of Electrolyte Leakage Electrolyte leakage assessments were determined for different strains to evaluate disease progres s and amount of tissue damage after inoculation. Bacterial suspension, adjusted to 3 x 108 CFU/ml in sterile tap water, was injected into tomato leaflets. Inoculated plants were maintained at 25 28oC in the growth room. Following inoculation and 24 and 48 h after inoculation, 3-cm2 of inoculated leaf tissue were transferred to 3 ml of de ionized water in 16 x 100mm test tubes and the initial conductivity reading T he samples were then placed under vacuum for 1 min, shaken for 1 h at 28oC, and then vortexed briefly before a second (final) electrolyte reading was made. Leakage was reported as the difference in the two readings for each sample as micro omhs. The readings were recorded for each sample using a Model 31 conductivity bridge (YSI Instrument Co., In c., Yellow Springs, OH, USA). Protein Thermal Stability To estimate thermal stability of the elicitor activity due to the B38 subclone in 91 118, sterile tap -water bacterial suspensions of 91::B38 and 91:: hrp mutant harboring B38 clone (91 hrp ::B38) at 5 x 108 CFU/ml were boiled in hot water for 10 min. The boiled bacterial suspensions were infiltrated into tomato plants with hypodermic syringe and needle. For control, part of the bacterial suspensions listed above was left untreated and infiltrated into tomato leaves at the same time. The plants were kept in the growth room for 24 to 72 hr at 28oC. Results Unusual Incompatible Response s of Mutants 306:: hrpG and 306:: hrpX in Tomato The wild -type Xcc 306 and the mutants 306:: hrpG and 306:: hrpX were infiltrated at 5 x 108 CFU/ ml in grapefruit leaves. Water soaked lesions were observed in the leaves inoculated

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59 with Xcc 306 wild -type 3 4 days after inoculation (DAI), while after 6 8 days raised lesions typical of a compatible interaction were observ ed (Figure 3 1). In contrast, the infiltrated areas where the 306:: hrpG and 306:: hrpX were injected were without any sign of citrus canker symptoms ( Figure 3 1). Additionally, the ability to incite citrus canker symptoms was complemented by the complementation with the wild type genes. The 306:: hrpG and 306:: hrpX mutants were inoculated at 5 x 108 CFU/ ml into tomato cultivar Bonny Best leaves and pepper cultivars ECW and ECW20R leaves. Neither mutant lost the ability to trigger HR in tomato ( Figure 3 2) 24 h after inoculation. The HR induced by the mutants was compared with the HR generated by the Xcc wild-type and no macroscopic difference was observed (Figure 3 2) Subclone of X. f. pv. aurantifolli Confers the Ablilty to Trigger an HR in Tomato In order t o investigate genes invo lved in non -host defense activation we constructed a genomic library of the wild type X. f. pv. aurantifoli and it was conjugated into Xanthomonas perforans strain 91 118. All the transconjugants were inoculated on tomato to find HR positive clones. Subseq uently, the HR positive clones were then conjugated into X. perforans strain 91 118 hrp (91:: hrp ) mutant to identify genes that were able to trigger HR in the T3SS independent manner. Clone pL450 when expressed in 91:: hrp mutant retained the ability to induce cell death in tomato plants. The 91:: hrp mutant was selected for use in this experiment because the mutant was non -pathogenic on tomato cv. Bonny Best due to core deletions in the hrp cluster. After screen ing for HR positive transconjugants in toma to plants, one clone (designed pL450) was selected and used as a model to isolate the HR -elicitor in Xcc for further experiments. The 91:: hrp mutant harboring pL450 clone (91 hrp ::450) was infiltrated into tomato plants at concentration of 5 x 108 CFU/ml. On Bonny Best plants a typical HR was noted

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60 for the 91 hrp ::450 mutant 24 h after infiltration ; as expected, the 91:: hrp mutant did not show any signs of compatible or incompatible interaction ( Figure 3 3). When pepper plants (cultivars ECW and ECW 20R) were challenged with 91 hrp ::450 mutant at the same bacterial concentration used above, neither, ECW or ECW20R infiltrated leaves developed HR 24 h after inoculation ( Figure 3 3). Electrolyte leakage in tomato plants was performed to measure, quantitativel y, the amount of tissue damage caused by each strain. In the first experiment, we compared the 91 118 wildtype (WT) by itself and carr yi ng pLAFR3 and pL450. The electrolyte leakage patterns for 91::pLAFR3 and the WT were similar but the 91 118 carrying pL 450 showed high electrolyte leakage 48 h after infiltration compared with the controls ( Figure 3 4a ). We analyzed the second group, 91 hrp ; 91 hrp ::pLAFR3; 91 hrp :: 450; 91 hrp ::XV9, hrp mutant complemented with pXV9 clone. The electrolyte leakage patterns of 91 hrp ::XV9, although higher, and 91 hrp ::pL450 were similar and characteristic of an HR, while the pattern for 91 hrp and 91 hrp ::pLAFR3 were similar to a compatible interaction ( Figure 3 4b). Sequencing Analysis Revealed Novel Genes Controlling N onhost HR Elicitor The pL450 clone was estimate d in length at ~ 31 k b based on multiple restriction enzyme digestion analyses (data not shown). Digestion of pL450 clone with BamH I generated many different size fragments, which were cloned into pUFR034 and transformed by triparental mating into X. perforans strain 91 118 wild type and a hrpm utant The transconjugants were infiltrated into tomato leaflets to assess their ability to direct a nonhost HR. The 91 118 wild type and hrpmutant transconjugants with subclone pB38 containing a 4.5 kb insert elicit an HR in tomato plants but not in pepper plants (Figure 3 5 ). Additionally, pB38 clone was transformed by triparental mating into Xanthomonas strain INA42, which is an opportunistic bacterium isolated

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61 f rom orange and not pathogenic on tomato; furthermore, the strain does not induce a nonhost HR (Jones et al., 1993). The transconjugant of INA42::B38, which contained the clone pB38, induced an HR in tomato plant s but not in pepper ( Figure 3 5). A restricti on enzyme analysis with Bam H I and EcoRI of the pB38 subclone revealed two fragments of 1.5 Kb and 3.0 Kb, respectively The fragments were further subcloned into pUFR034 resulting in pB38 1 (1.5 Kb) and pB38 3 (3.0 Kb). The clones were introduced by into t he X. perforans hrp mutant and the transconjugants were tested for the nonhost HR phenotype. The results showed that only the 91 hrp ::B38 3, carrying the pB383 clone, triggered a n HR on tomato. The ends of the pB383 plasmid were sequenced, and a compar ison to the genome of Xcc 306 revealed that 5 end was 93% identical with ORF XAC3855 and another end showed 94% identity with XAC3859. Due to the fact that the homology in the XAC3855 started in the middle of the gene, we chose to work with the following ORFs, XAC3857, XAC3858 and XAC3859. A sequence homology search of all three HR -elicitor predicted proteins was carried out with the NCBI Blastp program ( http://blast.ncbi.nlm.nih.gov/Blast.cgi). The out put of the search indicated that XAC3857 showed high homology to many other Xanthomonas spp. conserved hypothetical proteins (CHP), including XCV3975 (96% identity and 98% similarity) from Xcv strain 85 10; CHP (90% identity and 95% similarity) from X. o pv. oryzicola strain BLS256, CHP (89% identity and 93% similarity) from Xoo strain POX99A; and CHP (84% identity and 94% similarity) from X. campestris pv. campestris strain ATCC33913. To better visualize the homology, we used the Clustal W alignment progr am to demonstrate similarity. In addition, the protein sequence translated from the nucleotide sequence of pCR3857 clone was used to search for homology in other Xanthomonas spp. ( Figure 3 6). XAC3858 showed very similar high homology with different Xanthomonas spp. conserved hypothetical proteins ( Figure 3 7). ORF

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62 XAC3859 exihibited high homology to D alanyl D alanine dipeptidase precursor, including to D alanyl D alanine dipeptidase precursor (92% identity and 95% similarity) from Xcv strain 8510; D alan yl D alanine dipeptidase (82% identity and 89% similarity) from Xoo strain POX99A; D alanyl D alanine dipeptidase (84% identity and 91% similarity) from X. o. pv. oryzicola strain BLS256; and vanX (72% identity and 79% similarity) from X. c. pv. campestris strain B100. Additionally, this gene is part of VanY superfamily, which is related to vancomycin resistance (Evers and Courvalin, 1996; Bussiere et al ,. 1998; Lee et al ., 2000) W e also search for the presence plant inducible promoter box (PIP box) sequen ce upstream of the XAC3859, which was not found. Moreover, the translated protein sequence showed certain level of homology with other Xanthomonas spp. (Figure 3 8) Based o n the DNA sequence of B38 3 subclone, where the tree genes described above are pres ented and the transcriptional organization ( Figure 3 9), we can speculate that these genes could be part of an operon due the fact that all the genes are the same direction. Discussion Besides the fact that the hrp regulators share a degree of homology among some important plant pathogenic bacteria, most hrp regulatory mutants are totally impaired with disease symptoms, bacterial growth and HR elicitation (Wei et al 1992; Xiao et al. 1994; Wengelnik et al 1995; Wengelnik and Bonas 1996; Brito et al 1999; Sneath et al. 1990). In this study, the ability of X cc to induce symptoms on highly susceptible grapefruit wa s dependent on a functional T3SS which corroborate s with the previous studies mentioned above However, the ability of 306:: hrpG and 30 6:: hrpX mutants to induce an HR on no n -host plants is unusual with respect to hrp mutants The infiltration of 306:: hrpG and 306:: hrpX mutants into non host tomato plants induced strong hypersensitive r esponse s This phenotype was reported with a

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63 hrcC m utant on P. syringae pv. tabaci which completely lost pathogenicity on tobacco leaves but still induce a strong HR in non -host tomato plants. The HR -elicitor for the hrcC mutant was determined to be flagellin protein (Marutani et al ., 2004). A p arallel s tud y in this laboratory demonstrated that a clone, pXfa450, from X. fuscans pv. aurantifolli (Xfa) genomic library elicited an HR in tomato plants in the T3SS independent manner. Speculating that the genome sequence between X. citri subsp. citri and X. f pv. aurantifolli are similar, we used the 450 clone to identify the unknown T3SS independent HR elicitor from X. f. pv. aurantolli In the search for the Xcc T3SS independent HR clone a series of subclone s from the 450 cosmid were isolated into X. perfora ns strain 91-118 hrp mutant (91 hrp ). A 3.0 Kb subclone was identified and called B38 3 which was able to elicit the nonhost HR phenotype. The sequence analysis of the B383 demonstrated that two ORFs, XAC3857 and XAC3858, are homologous to conserved hypothetical proteins in other Xanthomonas spp. but have no association with known HR -elicitor was observed. The third gene, XAC3859 contains a conserved domain, which is part of the VanY superfamily and involved in resistance to antibiotic vancomycin (Evers and Courvalin, 1996; Bussiere et al,. 1998; Lee et al., 2000) ; and it is highly conserved in other Xanthomonas spp This implies that this phenotype, probably, is not connected with flagella, or flagella related proteins. Moreover, the transcriptional reg ion where the genes are located showed that all 10 genes possess the same orientation. This may indicate that these genes are part of a n operon and the phenotype is linked to two or more genes, instead of a single gene. However, in order to confirm this hypothesis mutational analysis, creating a polar affect, must be performed.

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64 Table 3 1. Bacterial strains and plasmids used in the study Strain or plasmid Relevant characteristics Source Strains Xanthomonas citri subsp. citri 306 Wild type, As iatic strain, isolated in Brazil, Rif R DPI a 306:: hrpG hrpG single recombinant of pCR hrpG KnR, AmpR This study 306:: hrpX hrpX single recombinant of pCR hrpX Kn R Amp R This study hrpG ::XV9 hrpG complemented with pXV9, Kn R AmpR, TcR This stud y hrpX ::XV9 hrpX complemented with pLH rpG /X, Kn R Amp R Tc R This study Xanthomonas perforans 91 118 Wild type, pathogenic to tomato, Rif R Jones et al (2004) 91::450 91 118 carrying the 450 cosmid This study 91 hrp ::450 91 118 defective T3SS car rying the 450 cosmid This study 91 hrp ::Bs3 91 118 defective T3SS carrying a AvrBs3 homolog This study 91::B38 91 118 carrying the B38 plasmid This study 91:: hrp 91 118 defective T3SS This study 91 hrp ::B38 91 118 defective T3SS carrying the B38 pla smid This study 91::B38 3 91 118 carrying the B38 3 plasmid This study X. fuscans pv. aurantifolli Xfa C 5979 Xc 70, isolated in Brazil DPI Opportunistic xanthomononad INA42 Isolated in orange R. E. Stall b INA42::B38 4.5 Kb Xaa fragment cloned in to BamH I p UF034 R. E. Stall Escherichia coli DH5 FrecA 80dlacZ M15 Invitrogen Plasmids pCR2.1 TOPO Phagemid, Cb R Kn R Invitrogen pBluescript KS+/ Phagemid, pUC derivative; Amp R Stratagene pRK2013 Kn R tra + mob + Daniels et al (1984) pLAFR3 Tc R rlx + RK2 replicon BJS c pUFR034 Kn R Tn903, IncW, Mob + De Feyter et al (1990) pCR hrpG hrpG partial fragment in pCR2.1 TOPO This study pCR hrpX hrpX partial fragment in pCR2.1 TOPO This study pCR3857 3857 ORF and endogenous promoter in This study

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65 Table 3 1. Continued pCR2.1 TOPO pCR3859 3859 ORF and endogenous promoter in pCR2.1 TOPO This study pXV9 ~25kb of hrp cluster of X. c. pv. vesicatoria 75 3 cloned in pLAFR3 Bonas et al ( 1991 ) pXfa450 32 kb Xaa fragment cloned into pLAFR3 G. Minsavage d B38 4.5 Kb Xaa fragment cloned into BamH I p UF034 Thi s study B38 .3 3.0 Kb Xaa fragment cloned into BamH I and EcoR I p UF034 This study B38 .1 1 .5 Kb Xaa fragment cloned into BamH I and EcoR I p UF034 This study a DPI, Division of Plant Industry of the Florida Department of Agriculture and Consumer Services, Gainesville, FL, USA. b R.E. Stall, Robert E. Stall, University of Florida, Gainesville, FL. c BJS, B. J. Staskawicz, University of California, Berkley, CA. d G. Minsavage, Gerald Minsavage, University of Florida, Gainesville, FL.

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66 Figure 3 1. Phenotype of grapefruit leaves were used to distinguish among the strains. Strains were infiltrated into the leaves at concentrations of 5 x 108 CFU/mL. Labeling: (1) hrpG ::XV9; (2) 306:: hrpG ; (3) Xcc strain 306 wild type; (4) 306:: hrpX ; (5) hrpX ::XV9. The grapefruit leaves were photographed 7 days after infiltration.

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67 Figure 3 2 Phenotype of 306 wild type, 306:: hrpG and 306:: hrpX in leaflet of tomato cv Bonny Best The strains were hand -infiltrated into the leaflet s at concentrations of 5 x 108 CFU/ mL. The tomato w as photographed 24 h after infiltration

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68 F igure 3 3 Hypersensitive response assay in tomato cv. Bonny Best and pepper cv. ECW 20R (A ) left: 91 hrp ::450; right : 91::450. ( B) left : 91 hrp ::450; right -down : 91 -118 wild -type; right -up: 9 1 hrp The strains were infiltrated into the leaves at concentrations of 5 x 108 CFU/mL. The tomato and pepper leaves were photographed 24 h after infiltration.

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69 A B Figure 3 4 Electrolyte leakage in tomato plant is changed in WT::pLAFR3, compati ble interaction, and WT:: 450, incompatible interaction. ( A ) Comparisson of ion leakage among compatible interaction WT::pLAFR3; WT::pAvrBs3 (carrying a AvrBs3 homolog) and WT:: 450. ( B) Comparisson of ion leakage among incompatible interaction hrp -Mut::pL AFR3; hrp -Mut::pAvrBs3and hrp-Mut::450. The bacterial suspension was infiltrated at concentration of 3 x 108 CFU/ml. 0 50 100 150 200 250 300 350 400 450 0 24 48MicromhosHours after infiltration Electrolyte Leakage Wild -Type WT + pLAFR3 WT + pAvrBs3 WT + pL450 0 50 100 150 200 250 0 24 48MicromhosHours after infiltration Electrolyte Leakage Hrp Mutant hrp Mut + pLAFR3 hrp Mut + pAvrBs3 hrp Mut + pL450

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70 F igure 3 5 S ubclone, B38, triggers hypersensitive response (HR) independent of T3SS in X. perforans in non -host plants, tomato cv. B onny Best and pepper cv. ECW20R. ( A ) left: 91::38 (open arrow) ; right : 91 118 wildtype (open arrow); left: 91 hrp ::B38 (close arrow); right : 91:: hrp (close arrow). ( B) left: 91 hrp ::B38 (open arrow) ; right : 91:: hrp (open arrow); left: OPP ::B38 (close ar row) ; right : OPP INA42 (close arrow). (C ) 1. 91 118 wild-type; 2. 91:: B38; 3. 91:: hrp ; 4. 91 hrp ::B38; 5. OPP INA42; 6. OPP ::B38. The strains were infiltrated into the leaves at concentrations of 5 x 108 CFU/mL. The tomato and pepper leaves were photogra phed 24 h after infiltration.

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71 XAC3857 (A) MNLPLHFGLLGSLEAGLIALILGVVVFAAVERGGRRLQFNHGHTLGIACLLAVAIGAGYDIWHLVYTSIV RLESPLYARLALARIHDPNELGSRVVLEVAGALAGVVLGWKLFSSGGWENDPPSA ( B ) XOOBLS MNLPLHFGLLGSLEAGLIALVLGVLVFAAVERIGRRLQFNHGQTLGIACLLTVAIGAGYD 60 XOO99 MNLPLHFGLLGSLEAGLIALILGVLVFAAVGRIGRRLQFNHGQMLGIACLLTVAIGAGYD 60 XCC306 MNLPLHFGLLGSLEAGLIALILGVVVFAAVERGGRRLQFNHGHTLGIACLLAVAIGAGYD 60 3857 MNLPLHFGLLGSLEAGLIALILGVVVFAAVERGGRRLQFNHGHTLGIACLLAVAIGAGYD 60 XCV85-10 MNLPLHFGLLGSLEAGLIALVLGVVVFAAVERGGRRLQFNHGHTLGIACLLAVAIGAGYD 60 XCC33913 MNLPLHFGLLGSLEAGLIALVIGVLLFAGVEHLGRRLQFTHGHTLGIACLLAVAIGAGYD 60 ********************::**::**.* : ******.**: *******:******** XOOBLS IWNLVYTSIVRLESPLYARLALAKIHDPNELGSRVVLDVAGALAGVVLGWKLFSSGSWDK 120 XOO99 IWNLVYTSIVRLESPLYARLALAKIHDPNELGSRVVLDVAGALAGVVLGWKLFSSGSWDK 120 XCC306 IWHLVYTSIVRLESPLYARLALARIHDPNELGSRVVLEVAGALAGVVLGWKLFSSGGWEN 120 3857 IWHLVYTSIVRLESPLYARLALARIHDPN ELGSRVVLEVAGALAGVVLGWKLFSSGGWEN 120 XCV85-10 IWHLVYTSIVRLESPLYAHLALAKIHDPNELGSRVVLEVAGALAGVVLGWKLFSSGSWEN 120 XCC33913 MWHLVYTSVVRLESPLYARVALAKIHDPNELGSRVVLEVAGAIAGVVLGWRLFSSATWDD 120 :*:*****:*********::***:*************:****:*******:****. *:. XOOBLS DAPSA 125 XOO99 DAPSA 125 XCC306 DPPSA 125 3857 DPPSA 125 XCV85-10 DPPSA 125 XCC33913 DAPAA 125 *.*:* Figure 3 6 Protein alignment of XAC3857 gene and Xanthomonas spp. ( A ) Original protein sequence of the ORF XAC3857 from X. citri subsp. citri strain 306; ( B) Protein alignment among X. citri subsp. citri strain 306 (XCC306), XAC3857 clone (3857), X. campestris pv. vesicatoria strain 8510 (XCV85 10), X. oryzae s ubsp. oryzicola strain BLS256 (XOOBLS), X. oryzae subsp. oryzae strain POX99A (XOO99), and X. campestris subsp. campestris strain 33913 (XCC333913). Asterisks indicate that the amino acids in that column are identical in all sequences. Colon shows that con served amino acids substitutions have been observed. Dot means that semi -conserved substitutions are observed.

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72 XAC3858 (A) MSDPERFVDIACPYCGEWITLALDLTGGDQHYIEDCQVCCKPIAVSVRWDEEGEAQVSARGQDDA ( B ) XCC306 M S D P E R FV D IA C P YCG E WI T LAL D L TGG D QHY I ED CQ V CC K PIAVS V R W DEEG E A Q V S A R 60 XCV8510 M S D P E R FV D IA C P YCG E WI T LAL D L TGG D QHY I ED CQ V CC K PIAVS V R W DEEG E A Q V T A R 60 XOO99 M S D P E R FV D IA C P YCG E WI T LAL D L TGG D QHY I ED CQ V CC K PI S V R V R W DEEG E A Q V S A R 60 XOOBLS M S D P E R FV D IA C P YCG E WI T LAL D L TGG D QHY I ED CQ V CC K PI S V R V R W DEEG E A Q V S A R 60 XCC8004 M S D P E R FV D IA C P YCG E WI T LAL D L SGG D QHY I ED CQ V CC K PI S V S V Q W DEEG E A Q V S A R 60 XCC33913 M S D P E R FV D IA C P YCG E WI T LVL D L SGG D QHY I ED CQ V CC K PI S V S V Q W DEEG E A Q V S A R 60 *********************.***:*****************:* *:*********:** XCC306 GQ DD A 65 XCV8510 GQ DD A 65 XOO99 GQ DD A 65 XOOBLS GQ DD A 65 XCC8004 GQ DD A 65 XCC33913 GQ DD A 65 ***** Figure 3 7 Protein alignment of XAC3858 gene and Xanthomonas spp. ( A ) Original protein sequence of the ORF XAC3858 from X. citri subsp. citri strain 306; (B) Protein alignment among X. citri subsp. citri strain 306 (XCC306), X. campestris pv. vesicatoria strain 85 10 (XCV85 10), X. oryzae subsp. oryzae strain POX99A (XOO99), X. oryzae subsp. oryzicola strain BLS256 (XOOBLS), X. campestris subsp. campestris strain 8004 (XCC8004), and X. campestris subsp. campestris strain 33913 (XCC333913). Asterisks indicate that the amino acids in that column are identical in all sequences. Colon shows that conserved amino acids substitutions have been observed. Dot means that semi -conserved substitutions are observed.

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73 XAC3859 (A) MTDNLLFRNGRCWRQCVVQRVLLMSAVAPFALVAGEVHVSSARTAAEAGLVDVHALAPDIALDMRYASSN NFTGRVVPGYDAPRCYLLRPAADALARVAQTLKADGYRLQVFDCYRPVRSVNAFVTWAADLQDQATKPQY YPRVAKRALLGDYIAPTSGHSRGATLDLGLLECRPAGCRAVDMGTGFDF FDARAHTDAPDISAAQRAHRQ RLLRAMAAEGFANYPMEWWHFTFRPEPTPETAYDVPVD ( B ) XOOBLS -------------------------------------MIAMAP S ALVAS D V H V SSA K T A 22 XOO99 ---------------M K E T LLF RK G R C W R Q LFL QG ALLMIAMAP S ALVAS D V H V SSA K T A 45 XCC306 ---------------M T D N LLF R NGR C W R QCVV Q R VLLMS AVAPFALVAG E V H V SSA R T A 45 3859 -----------------------------------------------------V SSA R T A 7 XCV8510 ---------------M T D N LLF R NGR C W R QCFV QG ALLMS AVAPFALVAGQ V H M SSA R T A 45 XCC33913 ------------------------M R VWR -----W S LALAVLC A E F N VAA T P T I S PA TTA 31 XCCB100 M SY AW R MP Y P C F S A D NTSQT V CTS M R VWR -----W S LALAVLC A E F N VAA T P T I S PA TTA 55 :*.* ** XOOBLS A E A G LV D V H ALAPD IAL D M R H A SSNN F TGQ VVP GCN AP R CY LLQ PAA E ALA R LA HTL E A E 82 XOO99 A E A G LV D V H ALAPD IAL D M R H A SSNN F TGQ VVP GCN AP R CY LLQ PAA E ALA R VA HTL E A E 105 XCC306 A E A G LV D V H ALAPD IAL D M R Y A SSNN F TG R VVP GY D AP R CY LLR PAA D ALA R VA QTL K A D 105 3859 A E A G LV D V H ALAPD IAL D M R Y A SSNN F TG R VVP GY D AP R CY LLR PAA D ALA R VA QTL K A D 67 XCV8510 A E A G LV D V H ALAPD IAL D M R Y A SSNN F TG R MVP GY D AP R CY LLR PAA D ALA R VA QTL K A E 105 XCC33913 A E A G LI D V R T LAP N I D V D M R Y A G R D N F TG R VVP GY AAP TCY LLR PAA E ALA R VA R AV E A D 91 XCCB100 A E A G LI D V R T LAP N I D V D M R Y A G R D N F TG R VVP GY AAP TCY LLR PAA E ALA R VA R AV E A D 115 *****:**::***:* :***:*. :****::*** ** ****:***:****:*::::*: XOOBLS G D R S H V S D SYR PVR S V K AFVAWAAD L QNQTTR A QYY P R V D KR ALL G D Y IAP TSGHRR G A T 142 XOO99 G D R L Q V S D CYR PVR S V K AFVAWAAD L QNQTTR A QYY P R V D KR ALL G D Y I S P TSGHRR G A T 165 XCC306 GY R L Q VFD CYR PVR S V N AFV T WAA D L Q D Q A T K P QYY P R VAKR ALL G D Y IAP TSGHSR G A T 165 3859 GY R L Q VFD CYR PVR S V N AFV T WAA D L Q D Q A T K P QYY P R VAKR ALL G D Y IAP TSGHSR G A T 127 XCV8510 GY R L Q VFD CYR PVR S V K AFV T WAA D L Q D QST K A QYY P R V D KR ALL G D Y IAP TSGHSR G A T 165 XCC33913 G E R L Q VFD CYR PVR AVQ AFVAWA R D L Q A QST K A QYY P R V D KR ALL G D Y IA E TSGHSR G A T 151 XCCB100 G E R L Q VFD CYR PVR AVQ AFVAWA R D L Q A QST K A QYY P R V D KR ALL G D Y IA E TSGHSR G A T 175 :* *.*****:*:***:** *** *:*:.****** *********: **** **** XOOBLS L D L G LL QC R FAA CQ AVD M GTG F D FFD A R A HTD APD I S AA Q R A H R Q R LL R AMAAE G FA NYP 202 XOO99 L D L G LL QC R S AA CQ AVD M GTG F D VFD A R A HTD APD I S A GQR A H R Q R LL R AMAAE G FA NYP 225 XCC306 L D L G LL E C R PA GCR A V D M GTG F D FFD A R A HTD APD I S AA Q R A H R Q R LL R AMAAE G FA NYP 225 3859 L D L G LL E C R PA GCR AVD M GTG F D FFD A R A HTD APD I S AA Q R A H R Q R LL R AMAAE G FA NYP 187 XCV8510 L D L G LL E C LPGGC R AVD M GTG F D FFD A R A HTNTP D I S AA Q R A H R Q R LL Q AMAAE G F SNY P 225 XCC33913 L D L G LL TC KRGTC APV D M GT PFD FFD A R A HTD APD I S A TQR A H R Q R LL R AMAAQG FV NYP 211 XCCB100 L D L G LL TC KRGTC APV D M GT PFD FFD A R A HTD APD I S A TQR A H R Q R LL R AMAAQG FV NYP 235 ****** .***** **.*******::***** *********:****:** *** XOOBLS M E WWH F T F R P E P T P D T A Y AV S V D 225 XOO99 M E WWH F T F R P E P T P D T A Y AV S V D 248 XCC306 M E WWH F T F R P E P T P E T A Y D VPV D 248 3859 M E WWH F T F R P E P T P E T A Y D VPV D 210 XCV8510 M E WWH F T L R P E P T P E T A Y D VPV D 248 XCC33913 M E WWH F T F R P E P T P D T A Y D VPV N 234 XCCB100 M E WWH F T F R P E P T P D T A Y D VPV N 258 *******:******:*** *.*: Figure 3 8 Protein alignment of XAC3859 gene and Xanthomonas spp. ( A ) Original protein sequence of the ORF XAC3859 from X. citri subsp. citri strain 306; ( B) Protein alignment among X. citri subsp citri strain 306 (XCC306), X. campestris pv.

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74 Figure 3 8. Continued vesicatoria stra in 85 10 (XCV8510), X. oryzae subsp. oryzae strain POX99A (XOO99), X. oryzae subsp. oryzicola strain BLS256 (XOOBLS), X. campestris subsp. campestris strain B100 (XCCB100), and X. campestris subsp. campestris strain 33913 (XCC333913). Asterisks indicate t hat the amino acids in that column are identical in all sequences. Colon shows that conserved amino acids substitutions have been observed. Dot means that semi -conserved substitutions are observed.

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75 Figure 3 9 Transcriptional organization of the ORF s XAC3857 to XAC3859 region in Xanthomonas citri subsp. citri. The region contains11 genes. The arrowheads indicate direction of transcription.

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76 CHAPTER 4 GENETIC CHARACTERIZA TION OF HRPW OF X anthomonas citri subsp. citri Introduction The Gram -negative ba cterium Xanthomonas citri pv. citri (Xcc) is a plant pathogen that causes citrus canker diseases in most citrus cultivars (Stall and Civerolo, 1991; Schaad et al., 2006). Many pathogenic proteobacteria rely on the type III secretion system (T3SS), encode d by hypersensitive reaction and pathogenicity ( hrp ) genes, to secrete bacterial effector proteins into the extrac ellular milieu and translocate them into the e ukaryotic host cell cytosol and consequently modulate host physiology (Chang et al ., 2004; Tang et al., 2006). To deliver these T3 effector proteins into host cells three biological barriers must be crossed, the membranes in the host and the inner and outer bacterial T his process involve s the T3SS pore -forming translocon complex (Cornelis, 2006). In X anthomonas spp. the hrpF is a typical translocon component involved in pore -formation in the planar lipid bilayers but it is not required for secretion (Rossier et al ., 2000; Buttner et al ., 2002). Moreover, pathogens carrying a defective translocon are impaired in T3 -effectors translocation, thus, inhibiting disease progress (Rossier et al ., 2000; Sugio et al ., 2005; Cornelis, 2006) Lorenz et al. (2008) classified the T3S substrates in two groups: (I) extracellular components of the secretion apparatus, and (II) effector proteins that are translocated into the host cell modifying cells metabolism (He et al ., 2004; Mudgett, 2005). One class of proteins that crosses the Hrp type III secretion apparatus is composed of the harpin proteins, which are T3SS depe ndent and secreted in the extracellular milieu during interaction (Perino et al., 1999; Tampakaki and Panopoulos, 2000). Harpin proteins are unique to phytopathogens and do not show homology to any other known protein, heat stable, glycine rich and lack of cysteine residues. Importantly, harpins trigger h ypersensitive r esponse (HR) cell death in non -host plants

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77 and may contribute to effector translocation (Wei et al ., 1992; He et al ., 1993; Arlat et al ., 1994; Charkowski et al ., 1998; Kim et al., 2004; Kvit ko et al ., 2007). Additionally, in recent studies the harpin from Erwinia amylovora (HrpN) is weakly translocated into tobacco cells (Bocsanczy et al., 2008); purified harpin modulates the accumulation of salicylic acid (Clarke et al ., 2005); induces activ ation of mitogenactivated protein kinases (MAPKs) (Desikan et al., 2001), elevation of cytosolic Ca2+ (Cessna et al ., 2001), activation of active oxygen species (AOS) and ion flux modulation (Reboutier et al ., 2007). Since the first harpin, HrpN, was isolated from E. amylovora (Wei et al ., 1992), many harpin -like proteins or genes have been identified from diverse plant pathogenic bacteria, including Hpa1 from X. oryzae pv. oryzae (Zhu et al., 2000), HpaG from X. a pv. glycines and X. c pv. vesicatoria (Kim et al., 2003; Thieme et al ., 2005), and PopA1 from R. solanacearum (Arlat et al ., 1994), One of the most widely distributed members of the harpinlike protein group is HrpW. HrpW was first described in E. amylovora, and subsequently identified in P. s. pv. tomato and Xcc (Charkowski et al., 1998; Kim and Beer 1998; da Silva et al ., 2002). HrpW contains a C terminal domain homologous to class III pectate lyase (PEL) and an N terminal harpin -like domain. While located within the respective hrp gene clus ters of E. amylovora and P. syringae the hrpW gene of Xcc strain 306 is located downstream of the hrp cluster (da Silva et al ., 2002). The role of harpinlike proteins, including HrpW, play in bacterial pathogenisis or whether or not HrpW is even required for pathogenesis in Xcc is unknown. Therefore, a genetic and biochemical analysis of the hrpW gene of Xcc was undertaken. Additionally, because it was thought that hrpW was important, was hypothesized that hrpW is conserved among diverse X. citri strains. To address this hypothesis hrpW sequence s of X. citri subsp. citri were produced and compared along with similar sequences derived from different X. citri strains.

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78 Material and Methods Bacterial Strains and Plasmids Bacterial strains and plasmids used i n this study are listed in Table 4 1 Media and Growth Conditions Escherichia coli cells were grown in at 37oC in Luria Bertani (LB) medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) sodium chloride, pH 7.5). For solid media BD Bacto agar was added at 15 g l1. Xanthomonas strains were cultivated at 28oC on nutrient agar (NA) medium and on tryptone sucrose agar (Zhu et al., 2000). All bacterial strains used in this study were stored in 20% glycerol in sterile tap water and maintained at 80oC. Triparental matings were performed on nutritent yeast extract -glycerol (NYG) agar (Daniels et al ., 1984). Antibiotics were used in the following concentrations: ampicillin (Amp) 100 g ml1; kanamycin (Kn) 50 g ml1; rifampicin (Rif) 100 g ml1; spectino mycin (Spc) 100 g ml1; and tetracycline (Tc) 12.5 g ml1. Recombinant DNA Techniques Standard methods were used for DNA cloning, restriction mapping and gel electrophoresis as described by Sambrook et al (1989) Restriction enzymes, T4 DNA ligase, and G oTaq Flexi DNA Polymerase were used following the manufacturers recommendations (Promega Madison, WI, USA). Chemicals were purchased from Sigma -Aldrich (St. Louis, MO, USA), and Fisher Scientific (Hampton, NH, USA). PCR w as performed in a PCR Express T hermal Cycler (ThermoHybaid, Ashford, UK). Topo TA cloning kit was purchased from Invitrogen (Carlsbad, CA, USA). Plasmids were introduced into Escherichia coli DH5 competent cells as described by Sambrook et al (1989) and into Xanthomonas spp. by electr oporation and conjugation. The conjugation was performed using pRK2013 as helper plasmid in triparental matings (Daniels et al 1984), and the electroporation using aliquots of Xcc competent cells were mixed with

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79 plasmid DNA and electroporated using a Bio Rad electroporation instrument. The conditions applied were 200 OHMs (resistance), 25 FD (capacitance) and 2.5 Volts. After the pulse, the cells were immediately diluted by adding 250 L of NA medium. The cells were incubate for 1 h at 28oC with constant shaking, and plated on NA containing kanamycin, then incubated at 28oC for 2 days (Sun et al ., 2003). Insertion mutants in X. c. subsp. citri were generated using single homologous recombination methodology as described by Sugio et al (2005) and the dele tion mutants were generated using the s uicide vector pOK1 as described by Huguet et al (1998). The oligonucleotides used in this study are listed in Table 4 2. All oligonucleotides were designed by using the Primer3 (v. 0.4.0) program ( http://frodo.wi.mit.edu ) to minimize secondary structure and dimer formation and were chemically synthesized by Genomechanix (Gainesville, FL USA). DNA sequence data were analyzed by DNA Sequencing Core Laboratory of the Interdisciplinar y Center for Biotechnology Research, University of Florida Gainesville, FL (UF ICBR). DNA Sequence Alignment and Phylogenetic Analysis of hrpW Gene hrpW gene from X. c subsp. citri and different Xcc strains were sequenced using forward and reverse, UpH rpWF1 and DHrpWR1, respectively, primers based on the sequence of hrpW from Xcc genome sequence (da Silva et al ., 2002), and designed to cover the entire gene. The amplified fragments were purified using QIAquick PCR purification Kit (Qiagen). The purified PCR products were sequenced in both directions at the University of Florida ICBR DNA Sequencing Core. Nucleotides sequence alignments were done using the Clustal W program. For phylogenetic studies, we performed a homology search using NCBI BLAST to ident ify hrpW homologs in other bacterium strains besides Xcc. Based o n protein level similarity, the DNA sequence of X. c pv. campestris strain 33913, R. solanacearum strain UW551, Acidovorax avenuae subsp. citrulli strain AAC00 1, Pseudomonas viridiflava, Er wina amylovora, and Erwinia tasmaniensis strain Et1/99 were taken from GenBank accession

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80 numbers NP_636593.2, ZP_00943832.1, YP_969269.1, CAA74158.1, and YP_001906489.1, respectively. The phylogenetic trees were built by first aligning the nucleotide seque nce automatically using Clustal W program and then performing manually alignment using MacClade (version 4.0, Sinauer Associates). PAUP* was used for analysis and creating parsimony consensus trees, parsimony bootstrap trees, maximum likelihood (ML) trees, ML bootstrap trees, and Bayesian consensus trees. Parsimonious trees were created using the default settings and performing a heuristic search that resulted in one tree. Bootstrap analysis for the parsimony tree was conducted with 1,000 bootstrapping repl icates. For ML analysis, PAUP* performed a heuristic search under the default ML settings, which resulted in initial tree and the base likelihood score. Next, the data were analyzed by assessing models for nucleotide substitution using the ML approach as i mplemented in the program Modeltest 3.7 (Posada and Crandall, 1998). A best -fit nucleotide substitution model selection was selected for our set of aligned sequences, and model selection was conducted on the basis of hierarchal likelihood ratios tests (hLR T). The best -fit moder from the likelihood settings was the (GTR+G) selected by Akaikes information criterion (AIC). ML bootstrap analysis was performed with 1,000 bootstrapping replicates. Bayesian analysis was performed by using Mr. Bayes 3.1 (Huelsenbe ck et al., 2001). Settings for the analysis were set at 1,030,000 generations, sampled at every 1000 generations. The burnin was set at sample 250 and the consensus trees were created by PAUP*. Generation of hrpW Deletion Mutants In -frame deletion deriva tive of hrpW 306 hrpW was generated by using pOK1 suicide vector. To generate a full length deletion of hrpW divergent primers POWF1 and POWR1 were used to amplify upand downstream regions of DNA flanking hrpW from pCR2.1TOPO clone pCRHrpW -EP. Each 25 l PCR contained 5x buffer, 0.7 mM MgCl2, 0.2 mM each

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81 deoxynucleotide triphosphate (Promega), 2 pmol of each primer, and 0.04 U of Taq polymerase. The reaction mixtures were heated for 4 min at 95oC and amplified over 1 cycle of 1 min at 95oC, 1 min at 53oC, and 7 min at 72oC, followed by 30 cycles of 45 sec at 95oC, 30 sec at 60oC, and 5 min at 72oC. Through the amplification process were added a unique Sal I restriction site, used to recircularize the construct. The PCR product was purified using QIAquick PCR Purification Kit system (QIAGEN), and then, directly cloned into pCR2.1 TOPO containing kanamycin and ampicillin resistance genes following manufacturers protocol (Invitrogen). To construct the pO hrpW suicide vector, the deleted region was excised w ith BamH I and Xba I, which flanking the pCR2.1 polylinker. The deleted region, a 1.0 Kb fragment, was cloned into the suicide vector pOK1 linearized with the same two enzymes. The mutated sequence was then introduced into the Xcc genome by homologous re combination in two steps described by Huguet et al. (1998). The recombination events were analyzed by PCR using UpHrpWF1 and DHrpWR1, which cover the deleted region. In addition, the PCR fragment generated here was sequenced and the sequence generated was analyzed a multiple alignment tool Clustal W (http://www.ebi.ac.uk/Tools/clustalw2/index.html) to confirm the in -frame deletion in hrpW gene. Construction of hrpW -Harpin Domain For the generat ion of hrpW /harpin domainHrpW harpin mutant a 1.2kb region flanking hrpW gene was PCR amplified from Xcc 306 genomic DNA. The fragment was TOPO -cloned into pCR2.1 and transformed in E. coli following the manufacturers procedure (Invitrogen). D erivativ e plasmid s carrying the insert were digested with Aat II restriction enzyme, which deleted 87 -bp from the harpin domain, creating the pCR harpin. To create the pO harpin suicide vector, the pCR harpin was double digested with BamH I and Xba I, which flank the

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82 pCR2.1 polylinker. The deleted fragment was cloned into the suicide vector pOK1 linearized with the same two enzymes. The mutated sequence was introduced into the Xcc genome by homologous recombination in two steps described by Huguet et al. (1998), cr eating HrpW harpin mutant. To confirm the recombination event and the harpin mutation, we PCR amplified the deleted region using UpHrpWF1 and DHrpWR1 primers and sequenced the product. The nucleotide sequences were analyzed by Clustal -W program (http://www.ebi.ac.uk/Tools/clustalw2/index.html) to identify the deleted region. Blast program was used for homology searches (Altshul et al ., 1997). Complementation Tests The hrpW gene carrying the nativ e promoter was PCR amplified using UpHprWF1 and DoHprWR1 primers. Cloning into pCR2.1 TOPO, creating pCRHrpW -EP, and DH5 transformation were performed was described above. The digested HrpW EP fragment was subcloned from pCR2.1TOPO by restriction with Ba mH I and Xba I, and ligated into the BamH I and Xba I linearized pOK1 suicide vector to create pOHrpW -EP. E. coli PIR was transformed with pOHrpW EP. The transfomants were introduced in a 306:: harpin mutant by triparental mating. Mixing 306:: harpin muta nt cells as recipient with pOHrpW EP as donor and with pRK2073 as the conjugational helper on NYG agar. The ratio of recipient to donor to helper was 2:1:1 (vol/vol/vol). After 24 h of incubation at 28oC, the mating mixtures were resuspended in sterile tap water, and aliquots were spread onto NA plates containing rifamycin and spectinomycin (100 g ml1 and 100 g ml1, respectively) for selection of transconjugants. Transconjugant colonies were subsequently inoculated in grapefruit leaves to screen for com plementation of the pathogenicity.

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83 Plant Material Grapefruit cv. Duncan ( Citrus paradisi ) plants were grown from seed in 15 cm plastic pots in Terra -Lite agricultural mix (Scott Sierra Horticultural Products Co. Marysville, OH). The plants were kept in the glasshouse of the University of Florida in Gainesville, Florida at temperature ranging from 2530oC. Pepper plants ( Capsicum annuum ) cv. ECW and the near isogenic line ECW20R containing the Bs2 resistance gene were planted from seeds in Plugmix (W. R. Grace & Co., Cambridge, MA, USA). After two weeks, the seedlings were transferred to Metromix 300 (W. R. Grace & Co) in 10 cm plastics pots. The plants were kept in the glasshouse of the University of Florida in Gainesville, Florida at temperature ranging from 2530oC. Plant Inoculations All the Xanthomonas spp. and mutants were cultured on nutrient agar plates for 18 h at 28oC. For preparation of bacterial suspensions, 18 h cultures were harvested from the NA plates and suspended in sterile tap water, a nd standardized to an optical density at 600 nm (OD600) = 0.3 (5 x 108 CFU/ml) with a Spectronic 20 Genesys spectrophotometer (Spectronic -UNICAM, Rochester, NY, USA). For pathogenicity tests, bacterial suspensions of the wild -type, mutant and complemented Xcc were infiltrated at 5 x 108 CFU/ml into abaxial surface of citrus leaves by using hypodermic syringe and needle. Plant responses were evaluated after 3 4 days for water -soaking and 6 7 days for citrus canker symptoms. The inoculated plants were kept in a growth room at 2830oC up to 10 days after inoculation. The sensitivity of the Xcc wild type and its hrpW mutants w as evaluated with pin -prick inoculation in grapefruit leaves. Bacteria were suspended in sterile tap water at a concentration of 5 x 108 CFU/ml. A drop of the suspension was placed on the adaxial surface of young

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84 grapefruit leaf. Immediately after a needle was pierced through the bacterial suspension and through the leaves, the inoculum drops were wiped off with sterile cotton. The inocul ated plants were then grown in a glasshouse up to 30 days. The diameter of circular lesions, including cork tissue and water -soaked margin, was measured after inoculation. E licitation of HR was tested by infiltrating bacterial suspensions, which w ere pre pared with sterile tap water at concentration of 5 x 108 CFU/ml, into leaflets of tomato cv. Bonny Best and pepper plants cv. ECW and ECW20R with a hypodermic syringe and needle. The inoculated plants were incubated in a growth room at 24 28oC and assessed 24 h after infiltration for HR induction. Score are the means of those for three leaves. All the experiments were repeated at least three times. Bacterial Populations Bacterial suspensions with 5 x 108 CFU/mL were infiltrated in grapefruit leaves with a syringe and 27 -gauge needle (Klement, 1963). A 0.5 cm2 of inoculated area from each leaf, each sample was infiltrate d in three leaves, was excised and macerated in 1 mL of sterile tap water, 50 L were spread onto NA plates. The plates were incubated at 28oC for 2 3 days, thus, the colonies were counted. Translocation Activity in 306:: harpin Mutant A pL799 clone, carrying avrGf1 gene from Xcc strain Aw that triggers HR in grapefruit (Rybak et al., 2009), was conjugated by triparental mating into Xcc wil d type and HrpW harpin m utant to investigate if the mutation on the harpin domain has any affect in the T3SS functioning. The transconjugant 306::799 and harpin::799 were inoculated at concentration of 5 x 108 CFU/mL in grapefruit and kept at growth room at 28oC. The plants were assessed for HR induction up to 5 days after infiltration.

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85 Construction of hrpW ::avBs2 Fusion For translocation studies, pepper plants cv. ECW and ECW 20R were infiltrated with X. campestris pv. vesicatoria strain XV1922 (wild type), 1922::WBs (pUWBs) leading to the expression of the first 109 aminoacids from hrpW gene fused with avrBs2 C-terminal with 62 574 amino acids. The strains were resuspended to an OD600 of 0.3 and infiltrated into the mesophyll of pepper leaves. 24 hr a fter in filtra tion, the inoculated pepper plants were assessed for HR elicitation. The plasmid was constructed as follows. First, 327 bp fragment was amplified using the primers NTW -F1 and NTW R1 introducing a Bgl II site in both ends. The PCR fragment was excised from the agarose gel 1% and purified with QIAquick Gel Purification Kit system (QIAGEN). The digested PCR fragment was cloned into pBS1 ( Bgl II:: avrBs262574::HA) plasmid resulting in pBWBs. For construction of pUWBs plasmid, pBWBs was digested usi ng BamH I and Kpn I and ligated into the expression vector pUFR034 digested with the same restriction enzymes. The resulting plasmid pUWBs was used to transform X. c. pv. vesicatoria strain XV1922 by triparental matings. Furthermore, the selected transconj ugants were analyzed for induction of HR in pepper leaves. Results Xanthomonas citri subsp. citri hrpW Phylogeny Comparison of phylogenies from sequences of conserved chromosomal genes is an effective method to evaluate the evolution among different pathovars in the same genus. In this study, we analyzed the full sequence of hrpW gene from Xcc strain 306 with strains collected world -wide and Florida State, and plant pathogenic bacteria that carry hrpW homologous sequence s The neighbor joining (NJ) tree obt ained via analysis of the hrpW sequence (Figure 4 1) shows that the genomospecies of X. citri, in general, cluster together very well, with the

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86 exception of two X. citri strains XS20030004 and XS199700018 (represented in the tree as XC04 and XC018, respe ctively). However, sequence alignment analysis revealed that these two strains, XS20030004 and XS199700018, show a certain level of nucleotide similarity (Figure 4 2). The close similarity between hrpW from Xcc strain 306 and X. c. pv. campestris str ain 33913 and E. amylovora has already been demonstrated by da Silva et al. (2002). Meanwhile, the phylogenies of R. solanacearum strain UW551 (UW551), Acidovorax avenae subsp. citrulli strain AAC00 1 (AAC00), Pseudomonas viridiflava (PV), and Erwinia tasmanie nsis strain Et1/99 indicate that hrpW sequence is also conserved in these strains (Figure 41). The maximum parsimony (MP) analysis of the hrpW sequence set yielded one parsimonious tree, which was virtually identical to NJ tree, except for the positions o f taxa within two strains, R. solanacearum strain UW551/ Acidovorax avenae subsp. citrulli strain AAC00 1 (AAC00). The maximum likelihood (ML) tree was identical to the NJ tree, with the exception that the positions of Acidovorax avenae subsp. citrulli str ain AAC00 1 (AAC00) and Pseudomonas viridiflava (PV) to E. amylovora and Erwinia tasmaniensis strain Et1/99 The hrpW phylogeny data in the NJ tree were supported by the bootstrap values of 100 in both the ML analysis and the MP analysis. Total Deletion o f hrp W Gene Is Irrelevant for Xcc Pathogenicity and HR in Non -Host Plants To determine the relevance of hrpW gene for the pathogenicity for Xcc strain 306 in a compatible and incompatible reaction, deletion in the 0.9 kb coding sequence of hrpW was generat ed using Aat II restriction enzyme. One clone, 306:: hrpW representing a hrpW full length deletion was selected for further characterization. Using sequence alignment we showed the precise location of the mutations in the HrpW ( Figure 4 3). Grapefruit lea ves were infiltrated with bacterial suspensions of 5 x 108 CFU/ml and the generation of symptoms was assessed 7

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87 days after inoculation. 306:: hrpW mutant induced typical canker symptoms on grapefruit at 7 days postinoculation, additionally, the symptoms incited by the mutant are macroscopically identical with the Xcc wild type (Figure 4 4). Xcc strain 306 induced h ypersensitive r esponse (HR) in tomato plants In order determine if the mutation in hrpW affect Xcc HR activity, we inoculated tomato (Bonny Best ) and pepper (ECW and ECW 20R) leaves with bacterial suspensions of 5 x 108 CFU/ml and observations of macroscopic tissue collapse typical of HR were made 24 h postinoculation. At this concentration, both strains Xcc wild type and 306:: hrpW induced an HR. Moreover, no difference was noted in the bacterial population among the strains tested in this experiment (Figure 4 5). Harpin Domain in hrpW Gene is Required for Pathogenicity In order t o explore the effect of a deletion i n the harpin domain of hrpW g ene may have on pathogenicity and HR elicitor activity of Xcc, we used d eletion mutagenesis to generate harpin mutant, W harpin, which 87 bp were deleted (Figure 4 3). The HR activity of the W harpin mutant was the same as that of wild type hrpW Surprisin gly, the W :: harpin mutant showed a severe reduction on the ability to induce symptoms compared with the Xcc -306 (Figure 4 4). T he W :: harpin mutant phenotype was restored when we replaced the deleted harpin portion, using pOHrpW EP, by the harpin wild typ e region in cis Considering the fact that 306:: hrpW mutant did not affect the pathogenicity and W :: harpin blocked the progress of symptom development in grapefruit w e may propose that this phenotype is given by the expression of a defective HrpW protei n, which may destabilize some protein interaction necessary for the disease progress. Although the W :: harpin mutant is affecting the phenotype in a compatible reaction, no change in the W:: harpin mutation population was observed compared with the Xcc wil d type or the 306:: hrpW mutant ( Figure 4 5).

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88 Complementation of HrpW:: harpin Mutant In -Cis The first attempts in recovery the W:: harpin mutant in trans using pUHrpW EP, which carry the hrpW gene drive by it native promoter, were unsuccessful. In order to demonstrate a non -polar mutation e ffect in W harpin mutant, we complemented the W harpin mutant using a 0.9 kb PCR fragment from Xcc containing the full length hrpW gene, and cloned into the pOK1 suicide vector, pOHrpW EP. Inoculations performed in gra pefruit leaf showed very similar symptoms with harpin::harpin complemented and Xcc wild type (Figure 4 -4). The results reported lead us to speculate that this incapability to restore the pathogenicity in trans is due to a dominant -negative effect of the d efective protein, in cis exerted in the wild type protein expressed in trans To determine if our theory is correct, we conjugated the pU harpin plasmid in Xcc strain 306, creating 306:: harpin(T) transconjugant, that express es the defective harpin domain in trans Therefore, if the expressed defective harpin protein is negatively dominant in cis this dominance should be maintained when the defective protein is expressed in trans Grapefruit leaves were inoculated by pin -prick method with Xcc wild type, W:: harpin, 306:: harpin(T), harpin::harpin and 306:: hrpW All strains test ed showed typical canker lesion fo rmation 28 days postinoculation (Figure 4 6 ). The mutations and complemented mutant were confirmed by size PCR -fragment ( Figure 4 7 ). The results demonstrated that the lo ss of pathogenicity by the W harpin mutant it is not caused by negative dominance effect. Therefore, more experiments need to be performed to explain the role of harpin domain for Xcc virulence. Translocon Machinery, T 3 SS, Is Not Affected by harpin Mutant in Xcc The effect delete of W :: harpin mutant may ha ve on the ability of Xcc strain 306 to deliver protein effectors through the T3SS was investigated using pL799 clone, which contain the avirulence gene avrGf1 that triggers HR in grapefruit and it is T3SS -dependent (Rybak et al .,

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89 2009). The W :: harpin transconjugant, W harpin::799, was infiltrated in grapefruit at 5 x 108 CFU/mL and compared with Xcc wild type strain with avrGf1 clone (pL799) The HR elicitation was observed in both strains with the same intensity five days after inoculation. HprW is not Translocated into the Mesophyll of Plant Cell To determine the targeting of HrpW protein to host cells a fused protein was constructed with HrpW N -terminal and C -terminal from AvrBs2 of X. c pv. vesicatoria The fused protein was expressed in X. c pv. vesicatoria strain XV1922, a avrBs2 defective strain, to test HrpW translocation into pepper leaves. As a positive control we used the XV1922-1 clone, which contains the avrGf11106::avrBs262574 and gives HR in pepper cv ECW 20R. The inoculation assay shows no HR induction after 24 hours, which means that HrpW protein is not translocated into the plant cells Discussion HrpW was first identified in E. amylovora (Kim and Beer, 1998). Thereafter, hrpW was reported in many phytopathogenic bacteria (Gaudriault et al ., 1998; Charkowski et al., 1998; da Silva et al., 2002; Shrestha et al., 2004) including xanthomonads. Therefore, we used the hrpW sequence from Xcc strain 306 (da Silva e t al ., 2002), to establish the diversity among several Xcc strains collected world -wide (Table 4 1). The phylogram of the Xcc strains indicate s very high level of sequence conservation among all Xcc strains analyzed in this study and that it is somewhat, less conserved among the other phytopathogenic bacteria (Figure 46). With the exception of Xcc strains XS20030004 and XS199700018, which diverge from the group showing high similarity to hrpW sequence from Xcc strain 306. Although the dendrogram demonst rated that these two strains branched differently from the other strains, the sequence alignment showed a considerable level of similarity between these two specific strains (Figure 4 2 ). The slight diversity reported in the XS2003 0004 and XS199700018 st rains may be

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90 explained due to a flexible element that could affect DNA similarity among pathovars in the same species, e. g. transposable elements, which are often variable within strains of species (Parkinson et al., 2007). Taken together that hrpW is hi ghly conserved among Xanthomonas citri strains and it downstream location outside of hrp cluster reported by da Silva et al (2002), we hypothesized that hrpW might be involved in the Xcc strain 306 pathogenicity. Here, we show that the full sequence of Hr pW is not required for the Xcc pathogenicity and virulence, and neither, for HR induction in non -host tomato plants. Similar results were reported with hrpW mutants from E. amylovora and P. s. pv. tomato (Kim and Beer, 1998; Charkowski et al ., 1998). Moreo ver, previous reports demonstrated that HrpW of E. amylovora inhibits HR induced by the HrpN (Reboutier et al., 2007), and the expression of hrpW in trans also blocked the HR induction by P. fluorescens (Charkowski et al., 1998). Based on these reports, we can speculate that the lack of HrpW in Xcc eliminates an unknown antagonistic inte raction wi th other pathogenic factors. Studies comparing HrpW protein from Xcc and X. c pv. campestris with Hrp W harpins of Pseudomonas and Erwinia species, showed that the HrpW protein from both Xanthomonas did not elicit HR in non -host plants. Surprisingly, the deletion of a few amino acids in the N terminal, which encode s the putative harpin domain, totally shutdown the Xcc virulence in grapefruit plants but does not affe ct the bacterial population in planta and the ability to trigger HR in non -host tomato plants. Although it is well known that harpins are HR -elicitor s their role in bacteria pathogenicity is still unclear Some of the harpins identified are directly linke d with pathogenicity such as, HrpN in E. amylovora and E. chrysanthemi (Wei et al ., 1992), HrpN also is involved in the translocation of T3 effector DspA/E (Bocsanczy et al ., 2008 ) while HrpZ from

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91 P. s. pv. phaseolicola is associated with pore -forming acti vity that might be involved in nutrient release and/or entry for bacterial effectors (Lee et al., 2001). Intrigu ing ly, the absence of the entire hrpW gene did not compromise the Xcc pathogenicity in a compatible reaction in contrast what was observed with the deletion on the harpin domain. We therefore suggest that the protein -protein interaction complex that HrpW form with other hrp genes is disrupted due to change in the protein conformation expressed by the harpin mutant. Alegria et al (2004) demonstr ated that HrpW forms a protein complex with three other Xcc proteins, HrpD6 and HrpB1 which are cytoplasmic proteins that shown homology with proteins in X. c. pv. vesicatoria that are essential for pathogenicity and conserved hypothetical protein s respe ctively (Rossier et al ., 2000) and HrpB4, although the interaction of this third proteins is not physically demonstrated. Xcv HrpB4 protein is essential for pathogenicity and T3-effectors translocation and not secreted or translocated, and shows associatio n with signal sensors (Rossier et al., 2002; Alegria et al., 2004). These findings support our suggestion that a defective hrpW protein encoded by ha r pin mutant destabilizes this complex, consequently, reducing the HrpB4 interaction or the sense of a speci fic signal. However, the translocation of T3effector, AvrGf1, into the host cell was not terminated in the harpin mutant. Considering this fact, we may speculate that this phenotype was caused by the interruption or reduction of the translocation of a spe cific crucial factor for pathogenicity. Our hypothesis is also supported by recently studies showing that phytopathogenic bacteria carrying a defective harpin protein impaired the translocation of several type III secretion system effectors (Kvitko et al ., 2007; Bocsanczy et al ., 2008). In addition, we have characterized the stability of the T3SS in the harpin mutant and translocation of HrpW from Xcv strain XV1922 to pepper plants. We show that the deletion on

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92 the harpin domain does not affect the T3 effe ctor translocation inside of plant cells. Furthermore, the use of AvrBs2 C -terminal, a Xcv T3 -effector protein, as a reporter demonstrated that the HrpW is not translocated into the mesophyll of host cells. However, using CyaA reporter system demonstrated that HrpN, a harpin protein from E. amylovora, is slightly translocated into tobacco plant cells (Bocsanczy et al ., 2008) The lack of translocation reported here may be due the fact that harpin domain in Xcc is smaller and has highly homology with the Pel domains of the Pseudomonas and Erwinia than with harpin domains (Kim et al ., 2004).

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93 Table 4 1. Bacterial strains and plasmids used in the study Strain or plasmid Relevant characteristics Source Strains X. citri subsp. citri 290 Isolated fr om Saudi Arabia R. E. Stall a 112 Isolated from China R. E. Stall 131 Isolated from Maldive Islands R. E. Stall 106 Isolated from Australia R. E. Stall 46 Isolated from India R. E. Stall 257 2 Isolated from Thailans R. E. Stall 62 Isolated from Japan R. E. Stall 126 Isolated from Korea R. E. Stall 101 Isolated from Guam R. E. Stall XI2000 00194 Isolated from Florida DPI b XS1999 0038 Isolated from Florida DPI X1999 12815 Isolated from Florida DPI XN03 2912 Isolated from Florida DPI XI3001 00098 I solated from Florida DPI X2000 12878 Isolated from Florida DPI XI1999 00112 Isolated from Florida DPI XN03 11#3 Isolated from Florida DPI XS2003 0004 Isolated from Florida DPI XS1997 00018 Isolated from Florida DPI 306 Wild type, Asiatic strain, iso lated in Brazil, Rif R DPI 306:: hrpW hrpW 855 bp deleted, Rif R This study W:: harpin Harpin 87 bp deleted, Rif R This study 306:: hrpG hrpG single recombinant of pCR hrpG Kn R Amp R Rif R This study harpin::Harpin Harpin complemented with p OHrpW EP, Rif R This study 306:: harpin(T) 306 carrying pU Harpin, Kn R Rif R This study 306:: 799 306 carrying pL799, Tc R Rif R This study X. c. pv. campestris XV1922 Pepper race 6, Rif R Jones, J. B. c 1922::WBs strain XV1922 carrying pU WBs Kn R Rif R This study harpin::799 Harpin carrying pL799, Tc R Rif R This study Escherichia coli DH5 FrecA 80dlacZ M15 Invitrogen PIR Host for pOK1; Sp R oriR6K replicon UB d Plasmids pCR2.1 TOPO Phagemid, Cb R Kn R Invitrogen pBluescript KS+/ Phagemid, pUC derivative; Amp R Stratagene pRK2013 Kn R tra + mob + Sm R Daniels et al (1984) pOK1 Suicide vecto r, pKNG101 derivative, Sm R /Suc S Huguet et al (1998)

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94 Table 4 1. Continued pLAFR3 Tc R rlx + RK2 replicon BJS e pUFR034 Tn903, IncW, Mob + Kn R De Feyter et al (1990) pCR hrpW hrpW partial fragment in pCR2.1 TOPO This study pCR harpin Harpin 87 bp del eted with Aat II Kn R AmpR pCRHrpW EP pCR2.1 with1.8 kb hrpW plus native promoter, Kn R Amp R This study pOHrpW EP pOK1 clone containing hpW plus native promoter, Sm R This study pO Harpin/PEL pOK1 with HrpW Sm R This study pO Harpin pOK1 with Harpin Sm R This study pL22 X cc strain A w pLAFR3 cosmid that contains hrpX hrpG and hsp90 Xo homologues, Tc R Rybak et al (2009) pL799 pLAFR3 with DNA fragment from Xcc A w that contains avrGf1 TcR Rybak et al (2009) pBS1 Bgl II:: avrBs2 62574 ::HA of p Bluescri pt KS+/ Amp R Mudgett, M. B.f pBWBs hrpW 1 109 cloned in pBS1 This study p U WBs hrpW 1 109 fused to avrBs2 62574 cloned p UFR034 This study pUHrpW EP Xcc pUFR034 clone containing hpW plus native promoter, Kn R This study pU Harpin Xcc pUFR034 clone contai ning harpin domain deleted, KnR This study a R.E. Stall, Robert E. Stall, University of Florida, Gainesville, FL. b DPI, Division of Plant Industry of the Florida Department of Agriculture and Consumer Services, Gainesville, FL, USA. c Jones, J. B., Unive rsity of Florida, Gainesville, FL, USA. d UB, Ulla Bonas, Martin Luther -Universit t, Halle, Germany. e BJS, B. J. Staskawicz, University of California, Berkley, CA f Mudgett, M. B., Stanford University, Stanford, CA.

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95 Table 4 2 Oligonucleotides sequence used in this study Gene Primer name Primer sequence length hrpW POWF1 5 ACGCGTCGACGTCAGCCGAAAGGAATATACAG 3 32 POW R1 5 CGCGTCGACCAACCGCTGTA GCAATTCCGAC 3 31 UpHprWF1 5 CCTATATCAGTGCTAACCCA CT 3 22 DoHprWR1 5 CCTATATCAGTGCTAACCCA CT 3 22 NTW F1 5 GGAAGATCTGATGTCTTTGGCCAACACACTGC 3 32 NTW R1 5 GGAAGATCTGCCGACGTAGAGATTGTTGTGTCC 3 33

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96 Figure 4 1. Neighbor joining analyses using full nucleotide sequence of hrpW gene from X. c subsp. citri strain 306. The numbers represent the parsimony t ree bootstrap and maximum likelihood values.

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97 XC04 -----------------------------------------------------------XC018 -----------------------------------------------------------XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 -----------------------------------------------------------AAC00 -----------------------------------------------------------EW -------------ATGTCAATTCTTACGCTTAACAACAATACCTC----GTCCTCGCCGG 43 ET1 -------------ATGTCAGTTCTTACGCTTAACATCACTATCCC----ATCCGTGCAGG 43 PV GGTCAGGGCGGACAAAGCGATCTGGACTCGCTGCTGCAGTCGCTCCAGGGCAACGGTCAG 300 XC04 -----------------------------------------------------------XC018 -----------------------------------------------------------XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 -----------------------------------------------------------AAC00 -----------------------------------------------------------EW GTCTGTTCCAGTCCGGG-GGGGACAACGGGCTTGGT---GGTCAT--AATGCAAATTCTG 97 ET1 GGCTGATGCAGCCCGGC-GCGGATAGCGGGTTTTCCACGGGAAAC--AACGCCAACGCCG 100 PV GGCGGTGACAAGCAGACCAAGGATTCCGGCGCTGCCGGCGGCGGCGAAAAGGAACTGCTG 360 Figure 4 2. Sequence al ignment of Xcc strains collected world -wide and sequences from X. c pv. campestris strain 33913, R. solanacearum strain UW551, Acidovorax avenuae subsp. citrulli strain AAC00 1, Pseudomonas viridiflava, Erwina amylovora and Erwinia tasmaniensis strain Et1/99.

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98 XC04 -----------------------------------------------------------XC018 -----------------------------------------------------------XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 -----------------------------------------------------------AAC00 -----------------------------------------------------------EW CGTTGGGGCAACAACCCATCGATCGGCAAACCATTGAGCAAAT-GGCTCAATTATTGGCG 156 ET1 GGTTGGGGCAACAGCCCTTCGACAAACAAACCATTGAGCAAAT-GGCGCAGTTATTGGGA 159 PV ACTCAGGTCTTGATGGCCCTGTTCGAGAAGATCCTTGGCGGCTCGGATTCATCATC--CG 418 XC04 -----------------------------------------------------------XC018 -----------------------------------------------------------XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 -----------------------------------------------------------AAC00 -----------------------------------------------------------EW GAACTGTTAAAGTCACTGCTATCGCCACAATCAGGTAATGCGGCAACCGGAGCCGGTGGC 216 ET1 GAGCTGTTAAAGCCGCTGCTATCGCCACAGGCAGGCAATGCGGCAACGGGCGCTAACGGC 219 PV GCGCGGGCAACGGCTCTGGCGGTGGTTCGGGCGGCGCCAGTGGCA-TTGGCGGTGGCGGA 477 Figure 4 2.Continued

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99 XC04 -----------------------------------------------------------XC018 -----------------------------------------------------------XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 ---ATGCTACGCGCCTCATCCGAGGGGCGCGGGCTCCCACAACCTTTCTTTGTCGAGACC 57 AAC00 -----------------------------------------------------------EW AATGACCAGACTACAGGAGTTGGTAACGCTGGCGGCCTGAACGGACGAAAAGGCACAGCA 276 ET1 GAGGGCCAGCTGGCCGGCGTGGGGAACGCCGGCGGTCCGTCAGAGCAGAATGGTGCCCTG 279 PV ACCGGCGCCAGCAAAGATGCCGGTGCTCTCGGTCAAGGGCAGGGACTCGGCGGCACTCAG 537 XC04 -----------------------------------------------------------XC018 -----------------------------------------------------------XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 GCCATGCCCATCCAGGTAGATCGCCCGAGTGACCATTTCCATGTGCCCTCCACGTGGAAT 117 AAC00 -----------------------------------------------------------EW GGAACCACT------CCGCAGTCTGACAGTCAG---AACATGCTGAGTGAGA---TGGGC 324 ET1 GGGGCAACG------CCACAGAATAACGGCCAG---AACGCGTTAAGTGAGA---TGGGC 327 PV GGCGCTACGGGCGGATCGAGCACTGAGGATCTGGTCAACACGCTGATGCAGAAACTGGGT 597 Figure 4 2.Continued

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100 XC04 -----------------------------------------------------------XC018 -----------------------------------------------------------XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 CAAGACACTGGTTCCAAGATCGATACCAGCCAACTTCAGCATGCCGTACAACTGCTCCAG 177 AAC00 -----ATGCGACGCCGTGCATCGGGCATGGCGTACCGGCGCTTCGGCGCCGCACTTTTCG 55 EW AACAACGGGCTGGATCAGGCCATCACGCCCGATGGCCAGGGCGGCGGGCAGATCGGCGAT 384 ET1 AACAACGGGCTGGATCAGGCCCTGACCCCCGATGGCCAGGGTGGAGGGCAAATCAGCGAT 387 PV GGTGGATCGCTGGATAACTCCATTCAGCCGACTGCCGATGGCGGCGGTGAGGTGTCCCAG 657 XC04 ------------------------GTGGCCTCCTACACACGGGCGGGTGTGGGGATATAC 36 XC018 -----------------------------------------------------------XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 ------------------------------------------------ATGCAACGCATG 12 UW551 CAGGTCTTGCAGCAGGTCCAAGCGAACAAGCTGTTCGGGAACGTGCTGAACCAGCCGGAA 237 AAC00 CGAGATCCATGAACCCCATCGCCTTTCTCTCGTCTTCCCTGACCGCACGGCATGCCGCGG 115 EW AATCCTTTACTGAAAGCCATGCTGAAGCTTATTGCACGCATGATGGACGGCCAAAGCGAT 444 ET1 AATCCGTTGCTGAAAGCCCTGCTGAAACTGATCGCCCGAATGATGGACGGGCAGAGCGAC 447 PV AACGGCAAGCTCAAAGAGCTGCTGGAAATGATCGCTCAGTTCATGGACAGCCATCCGGAG 717 Figure 4 2.Continued

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101 XC04 CATAGATCCTCCCACTATTGCCCAGACCTCTCTCCCAAAAGAGCACCG-TGCGCGCAATA 95 XC018 -----------------------------------------------------------XCC306 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XCC112 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC290 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC112 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC131 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC46 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC257-2 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC62 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC126 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC101 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC0194 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC0038 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC12815 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC2912 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC098 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC12878 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XCC11 ----------------------------ATGCTGATGTCGGAATTGCT-ACAGCGGTTGA 31 XC33913 CTCAGGGACATGCTGCAGCCACAGGATCAGCCATTCAGCGACGGTCAG-CCACCGACCAA 71 UW551 TTCGGCAACTCCGGTCAGGGGCATGGCGGCAGCCACGGTGGCGGCCATCACGGCGGCTCG 297 AAC00 CATCGGACCAGGCCGCCTCCGCGGCCCGGCACAACGGCAGCCAGGCACTGCAGCAATCCC 175 EW CAGTTTGGCCAACCT------------GGTACGGGCAACAACAGTGCCTCTTCCGGTACT 492 ET1 CCGCTCGGCCAGCAG------------GGCGCTGGCAGTCACAATGCGTCTTCCGGCACG 495 PV ACTTTCAATCAGCCGTCTGATGCTGCGGGCAAAGGTGGTGGCGGTGGCGGTGGTGGAACA 777 XC04 TAAGGGATTTGGGAAAGCG-CCTTTGCT-------GGCCTTTCCCCAATGGGGACCTGCC 147 XC018 -----------------------------------------------------------XCC306 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XCC112 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC290 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC112 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC131 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC46 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC257-2 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC62 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC126 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC101 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC0194 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC0038 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC12815 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC2912 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC098 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC12878 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XCC11 TACAGACCCAATTCCAACC-TCAAAACA-------CGTCGCAATACGATTGGAACCCTTC 83 XC33913 CCCGAATCCGACACCACCG-ATGCCGCC-------CTCGCGCCCGCCCTGTTCCACCAAC 123 UW551 ACCGGGTTCGGCGAAAACGGTCGATTCG-------GTTTGCCGTACGCCAAGCCGCCTGC 350 AAC00 TCGCCACCCTGCTCGAAGG-CCTCCTGA-------AGCGCATGCGCGGCGGAGCCGCCCC 227 EW TCTTCATCTGGCGGTTCCCCTTTTAACG-------ATCTATCA-----GGGGGGAAGGCC 540 ET1 TCCTCAACGGGGAGCTCCCCTTCCAACG-------ATCTATCA-----GGCAACGGCTTC 543 PV CCTTCCGTAGGCGGTGGCGGTGGCGGTGGCGGTACACCTTCCGTGGGCGGTGGCGGAGAT 837 Figure 4 2.Continued

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102 XC04 CACATTGGCAAAGGGGGA--GGGGCAAACCGAA-GCAAGTGCCGGGTCGGTAA------197 XC018 -----------------------------------------------------------XCC306 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XCC112 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC290 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC112 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC131 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC46 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC257-2 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC62 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC126 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC101 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC0194 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC0038 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC12815 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC2912 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC098 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC12878 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XCC11 GCCTTCCCATGGGACGTC--CAATGGCCAAGGG-GAGGGCAACGAGCATGGCG------133 XC33913 CCCGGCGGACAACACGGTAGCGGTACTGGCGAT-GGCAGCGGCGGCAATGGCG------175 UW551 GCAGCCGGACATTGAGCTGCCGGCCAACAAGCCCAACCCCGGCAAGCACAACAC---TTC 407 AAC00 GTCACCCCGCTCTGGCGG---AAGCGCCGCGGG-GCATGCCGCCGGGACGGCG------276 EW CCTTCCGGCAACTCCCCTTCCGGCAACTACTCTCCCGTCAGTACCTTCTCACCC-CCATC 599 ET1 CCTTCCGG---CCCTTCTTCCGGCGGCACAGCGCCAACCCATTCTGACTCACCG-CCGTC 599 PV GGTGGCGGAACACCTTCCGTAGGCGGCGGAGGCGGAGGTGGTGGCGGAACACCTTCCGTA 897 XC04 -GGAAAAGGAACGCGGGCGGGTGATAAAATTTTCTTTGGCCAACCACGTCTCGAG-GACG 255 XC018 -----------------------------------------------------------XCC306 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XCC112 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC290 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC112 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC131 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC46 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC257-2 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC62 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC126 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC101 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC0194 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC0038 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC12815 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC2912 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC098 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC12878 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XCC11 -GTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCG---CCACCACGCCC-----GAGA 184 XC33913 -GCAACCACAACAGAGGCAACGGCGGCACCTGCGGAACGGGAGCCGATCACCGC--GCAT 232 UW551 CGCGCCGACCACGGAGACGACCCCGCCTGCTGTTTCGCCCAAGCCCGATAC---G-TCTC 463 AAC00 -CCTCCGGCCTCGCACACCACGCAACGTGCCGTGGCGC--ACACCGATGCCGGCC-GCGC 332 EW CACGCCAACGTCCCCTACCTCACCGCTTGATTTCCCTTCTTCTCCCACCAAAGCA-GCCG 658 ET1 CACGCCAACCTCCCCCACCTCACCGCTTGATTTCCCGTCTTCCCCCACCGGCGGA-GCCG 658 PV GGCGGCGGTGGCGGGGGTGGTGGCGGTACACCTTCCATAGGCGGTGGCGGTGGTACGCCT 957 Figure 4 2.Continued

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103 XC04 TTCTTGATCATTTCGAAAAAA---TTCGGTTCAATTCGAACCGTT-ACCGGGGAAGCGGG 311 XC018 -----------------------------------------------------CAACGGC 7 XCC306 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XCC112 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC290 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC112 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC131 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC46 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC257-2 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC62 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC126 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC101 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC0194 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC0038 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC12815 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC2912 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC098 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC12878 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XCC11 CGTCCGACCATTCCGACAAA-----TCCGCTCACTC--GACCGTC-ACCGG--CAGCGGC 234 XC33913 CGCCCACCAACCCGGGTCAGGCGGTGAGGCCCTGCCCCGATGGTCCGCAGGCCCCACTTC 292 UW551 CGGCCTCCAAGCCTGACACGTC--CTCGACCTCCACATCCGCGACGGAAGGCAAGGTGAC 521 AAC00 CGGCACGGCGCACGCAGCGCGCGGCACGGCCACCACGGCCCATGCCGCGAAGCCTCCCGC 392 EW G----GGGCAGCACGCCGGTAACCGATCATCCTGACCCTGTTGGTAGCGCGGGCATCGGG 714 ET1 G----GGGCAGCACGCCGGTAACCGATCGTCCCGATCCTGTTGGCAGTTCAGGCGTAGGG 714 PV GCACCGACCGGCCCGACGGGAACACCTTCACCCACCGGTCCGACCGGCACAGGCACCAGC 1017 XC04 C-------CCCGTGGGGGAGCGGGTTTGCACGGAT---------CAACGTCCAA--CAGT 353 XC018 C-------CCGGGTGGGGAGCGG-TCGGCACCGATT--------CAACGTCCA---CAGT 48 XCC306 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XCC112 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC290 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC112 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC131 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC46 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC257-2 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC62 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC126 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC101 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC0194 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC0038 C-------CCG--TGGGGAGCGG --TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC12815 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC2912 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC098 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC12878 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XCC11 C-------CCG--TGGGGAGCGG--TCGCAC-GAT---------CAACGTCAA---CAGT 270 XC33913 C-------TCCCGGCAAGGTCGTCACCGCGC-CACC--------CGGCCTCCAGAACAAG 336 UW551 A-------TACGGCGTGAAGCCG-CCCGAGCCGACCGGCGTGGTCGACGTCAA---CAAG 570 AAC00 C-------AAC-ATGCGGGTCGG---CGCGCCGACGGGCACGGTGGAGGTGAA---CAAA 438 EW G--CCGGAAATTCGGTGGCCTTCACCAGCGCCGGCGC-TAATCAGACGGTGCTGCATGAC 771 ET1 G--CCGGGCAGGCCGTTGATTTCCCTGGCGCCAGCGC-CAATCCCACGGTGGTGCATGAC 771 PV GGCTCGGCCACTCCGGTCTCTTTCCCGACGGCCTCGGGCACGCCGACCGTCGTCAACGAG 1077 Figure 4 2.Continued

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104 XC04 CCGGATCGTGGTTTACAAAGGGGGAAGGTGTTTG-ACGGACACAACAAATCTTTAGTCGG 412 XC018 CCCGATCGTGGTC-ACAA---GGCGAGTTGTTCGTACGGACACAACATTCTCTACGTCGG 104 XCC306 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XCC112 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC290 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC112 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC131 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC46 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC257-2 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC62 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC126 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC101 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC0194 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC0038 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC12815 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC2912 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC098 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC12878 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XCC11 CC-GATCGTGGTTCACAA--GGGCGAGGTGTTCG-ACGGACACAACAATCTCTACGTCGG 326 XC33913 AC-CATCGTGGTGCATAA--GGGCGAGGTATTCG-ACGGCAAGGGTCAGACCTACATCGG 392 UW551 CC-GATCGTCGTCAAGGC--GGGTGAGACGTTCA-ATGGCGAGGGCAAGTATTACCGCC625 AAC00 CC-CATCGTCGTGAAGGC--CGGCCAGACCTTCG-ACGGCCAGGGCCGGCTCTTCAGCGC 494 EW AC-CATTACCGTGAAAGC--GGGTCAGGTGTTTG-ATGGCAAAGGACAAACCTTCACCG826 ET1 AC-CATTACCGTAAAAGC--GGGCCAGGTGTTTG-ATGGTAAAGGGCAAACCTTTACCG826 PV AC-CATCAAGGTGGGGCC--TGGCGAGACGTTCG-ACGGCGGCGGCAAGACCTTCACCG1132 ** ** ** ** ** ** XC04 CGGGTCGGGC-ATCGGCGATGG-TTGCAGTCCGAGCACCAGCAACCGATGTTTGTCGT-G 469 XC018 CGGGTTCGGC-ATCG-CGATGGATCGCAGTTCGAGCAACAGCAACCGATGTTCGTCGTCG 162 XCC306 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XCC112 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC290 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC112 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC131 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC46 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC257-2 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC62 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC126 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC101 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC0194 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC0038 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC12815 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC2912 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC098 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC12878 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XCC11 CGGGTCCGGC-ATCGGCGATGGTTCGCAGTCCGAGCACCAGCAACCGATGTTCGTCGTCG 385 XC33913 TGGCCCTGGT-CTGGGCGATGGCTCCCAGAACGAACACCAGCAGCCGCTGTTCGTCGTCG 451 UW551 CGACCAAGGCGATGGGCGACAACTCGCAGAACGAGCACCAGCAGGCCGTGTTCATCCTGG 685 AAC00 CGGGCCCGGG-CTCAACGGCGGCGGCACGGCGGAGACGCATCTGCCGGTCTTCATCCTGG 553 EW CCGGTTCAGAATTAGGCGATGGCGGCCAGTCTGAAAACCAGAAACCGCTGTTTATACTGG 886 ET1 CCGGTTCGGAATTAGGCGATGGTGGCCAGTCGGAAAGCCAGAAACCGCTGTTCAAACTGG 886 PV CAGGCAAGGCTCTGGGAGATGGCGGACAGGGCGAGGGACAGAAGCCGATGTTCGAACTGG 1192 * ** ** ** Figure 4 2.Continued

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105 XC04 AACAAGGG-GGCACC--T-CCAAAACGTGCGGATGAGCGGGGGCGGCGACGGCA-CCATC 524 XC018 GACAAAGGCGGCACCCTT-CCAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 221 XCC306 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XCC112 AACAA-GGTGTCGACGTCACCGAAAGGAATATACAGGTTGA------------------425 XC290 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC112 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC131 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC46 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC257-2 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC62 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC126 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC101 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC0194 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC0038 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC12815 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC2912 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC098 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC12878 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XCC11 AACAA-GGCGGCACCCTC--CAAAACGTGCGGATGAGCGGGGGCGGCGACGGCATCCATC 442 XC33913 AGGAC-GGCGGTAGCCTG--TGCAACGTCAACATGAAGGGCGGCGGCGACGGCGTGCATT 508 UW551 AGCCC-GGCGCGAAGCTG--GAGAACGTGCA---GTATTCCGGCGCCGATGGCATCCACC 739 AAC00 AGCCG-GGCGCGTCCGTG--AAGAACCTGCA---GTTCAAGGGCGGCGACGGCATCCACC 607 EW AAGAC-GGTGCCAGCCTG--AAAAACGTCACCATGGGCGACGACGGGGCGGATGGTATTC 943 ET1 AGGAC-GGGGCCAGCCTG--AAAAACGTCACTATCGGCAATAACGGCGCGGATGGCATTC 943 PV CTGAG-GGCGCCACCCTG--AAAAACGTGGTGTTGGGTGACAACGCTGCCGATGGTGTGC 1249 ** ** XC04 TGTTGGGGACGCC-ACGTTGAAGGAA-TCCGCAACC-GAACGTCACAGAGGATGCGATGA 581 XC018 TGCTCGGCGACGC-ACGCTGAAGGAAGTCAGCACCCTGAACGTCAGCGAGGATGCGATGA 280 XCC306 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XCC112 -----------------------------------------------------------XC290 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC112 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC131 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC46 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC257-2 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC62 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC126 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC101 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC0194 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC0038 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC12815 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC2912 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC098 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC12878 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XCC11 TGCTCGGCGACGCCACGCTGAAGAACGTCCACAACCTGAACGTCAGCGAGGATGCGATGA 502 XC33913 TCATGGGCAGCGGCAAGATGGTCAACTGCGTCAACGACGATGTCAGCGAGGATGCGGTCA 568 UW551 TGCTGGGCAGCGCCACGCTGGACCGCGTCGTCAACCGCCAGGTGGGCGAAGATGCTTTCA 799 AAC00 TGCTGGGCGACGCCAGGCTCGACAACGTGCACGGCCTGCAGACGGACGACGACTTCATCA 667 EW ATCTTTACGGTGAT--GCCAAAA-------------TAGAC---AATCTGCACGTCACCA 985 ET1 ATCTCTATGGCGAT--GCGAAAA-------------TAGAT---AACCTGCACGTCACTA 985 PV ATGTCCGTGCTGCCA-GCGAAAAAGC--CGTGAACGTGGAT---AACGTGCATTGGACCA 1303 Figure 4 2.Con tinued

PAGE 106

106 XC04 C-ATAGAGGGAGT---GCAAC-GGGAACAT-ACTCGCGC-TTGCCGGAAC--GCCTGCCA 632 XC018 CCATCGACTGGTCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 338 XCC306 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XCC112 -----------------------------------------------------------XC290 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC112 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC131 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC46 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC257-2 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC62 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC126 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC101 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC0194 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC0038 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC12815 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC2912 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC098 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC12878 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XCC11 CCATCGAC-GGCCCTGGCAACCGGGAACATGACTCGCGCATTGCCGGAAC--GCCTGCCA 559 XC33913 CCATCGAT-GGCCAGGGCAACCGCGAGCATGATGCGCGCATTGCCGGT-T--GTCGCCCG 624 UW551 CCATCGAC-GGTGTCAAGAACCGCCTGCACGATGCCAAGATCGCCGGGATCGACCCGGCA 858 AAC00 CCGTCGAT-GGCGCGGAGAACCGGGCCGTGGATGCGCAGCGCGCCGGCTATTCCGCCAAG 726 EW ACGTGGGT-------------------GAGGACGCGA---TTACCGTTAA--GCCAAACA 1021 ET1 ACGTGGGG-------------------GAGGACGCGA---TCACCGTGAA--GCCAAACG 1021 PV ACGTCGGC-------------------GAGGACGCGC---TGACCGTGAA--GGGTGAGG 1339 XC04 GCCGCC-GCCGGCGCGT-CCAAAGATCGAAGTGCTCGA--ATTCCTTCGACAATGCCTCC 688 XC018 GCGGCCTGCTCGCGCGT-CCAAAGATCGAAATCCTCGACAGTTTCTTCGACAATGCCTCC 397 XCC306 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XCC112 -----------------------------------------------------------XC290 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC112 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC131 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC46 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC257-2 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC62 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC126 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC101 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC0194 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC0038 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC12815 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC2912 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC098 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC12878 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XCC11 GCGGCCTGCCGGCGCGT-CCAAAGATCGAAATCCTCGACAGTTCCTTCGACAATGCCTCC 618 XC33913 GAGGTCAGC--GGCCGG-CCGAAGGTCGACATCATCAATTGCACGTTCAAGAATGCCGCA 681 UW551 TCGATCCCCGGGGGCACGCCCAAGGTGGAGATCATCAACAGCGCGTTCTACGGCGCCAAG 918 AAC00 AGCATCCCTCCGGGCCCGGCCCACGTGGAGATCACGAACTCGTCGTTCCAGAACAGCCAC 786 EW GCGCG--GGCAAAAAAT--CCCACGTTGAAATCACTAACAGTTCCTTCGAGCACGCCTCT 1077 ET1 GCGCG--GGCAAAAAAT--CTCACGTTGAAATCACCAACAGCACTTTCCAGAACGCCTCT 1077 PV GCGGT--GCCAAGGTCA--CCAACCTGAACATCACCAACAGCAGTGCCCAGGGTGCCAAT 1395 Figure 4 2.Continued

PAGE 107

107 XC04 CACAAGGTGATCCAGGACAT-CAGGCGGCAGATGTCT--GTAGGTA-CTTCATCGTGAAC 744 XC018 GACAAGGTGATCCAGGACAAGCACGCGGCAGATGTGCTGGTGCGTAACTTCAGCGTGAAC 457 XCC306 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XCC112 -----------------------------------------------------------XC290 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC112 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC131 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC46 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC257-2 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC62 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC126 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC101 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC0194 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC0038 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC12815 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC2912 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC098 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC12878 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XCC11 GACAAGGTGATCCAGGACAACCAGGCGGCAGATGTGCTGTTGCGTAACGTCAGCGTGAAC 678 XC33913 GACAAGGTCGTGCAGGACAATGGTGCTGCCGACGTGGTGATGTCGGGTTGCACCGTCAAC 741 UW551 GACAAAGTCGGTCAGATCAACGGCGACGTAGACCTGCAGGTGCGGGGCATGTACGTCAAT 978 AAC00 GACAAGGCGATCCAGATCAACGGCGACGTGGACCTGAAGCTGCGCGGCATCTATGCGAAC 846 EW GACAAGATCCTGCAGCTGAATGCCGATACTAACCTGAGCGTTGACAACGTGAAGGCCAAA 1137 ET1 GACAAGATCCTACAGCTGAATGCCGATACCAGCCTGACCGTTGATAACGTGAAGGCCAAA 1137 PV GACAAGGTGTTCCAGCTCAACGCCGATGCGAACGTGAACGTGGACAACTTCAAGGTCAAG 1455 XC04 GGTCCCGGCAAGGTGTTACGCAC-AACGGCGT-CACACCGACATGGGT-CACACGTCTCG 801 XC018 GGAGGCGGCAAGGTGTTCCGCGCCAACGGCGGACACACCGACATCGATTCACACGTCACG 517 XCC306 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XCC112 -----------------------------------------------------------XC290 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC112 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC131 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC46 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC257-2 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC62 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC126 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC101 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC0194 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC0038 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC12815 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC2912 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC098 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC12878 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XCC11 GGAGCCGGCAAGGTGTTCCGCACCAACGGCGGACACACCGACATCGATTCACACGTCACG 738 XC33913 GGCGCCGGCAAGGTCTTTCGTACCAACGGTGGACACGCTGATATCGACACGCATCTTCAG 801 UW551 GGTGCCGGCAAGGTCTTCCGTACCAACGGCGGCGACGAGCAGATCAAGGCGAATGTCGCG 1038 AAC00 CAGATCGGGCAACTGGCCGTCACGCGCGGCGGCTACCCGATCACCG---CGCATGTGGAC 903 EW GACTTTGGTACTTTTGTACGCACTAACGGCGGTCAACAGGGTAACTG---GGATCTGAAT 1194 ET1 GACTTTGGCACTTTCGTGCGCACCAACGGCGGCCAGCAGGGCAACTG---GGATCTGAAT 1194 PV GATTTCGGCACCTTCATGCGCACCAATGGCGGGCAGCAGGGTGACTG---GAACCTGGAC 1512 Figure 4 2.Continued

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108 XC04 G-CGAAGACTCCACCCTGAAAAGCATCAAGGAACCAGTATTCCGCCCCGATGCCCCCGGC 860 XC018 GTCGAAGACTCCACCCTTAAAGGCATCAAGGAAGCAGTATTCCGCAGCTATGCGCGCGGC 577 XCC306 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XCC112 -----------------------------------------------------------XC290 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC112 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC131 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC46 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC257-2 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC62 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC126 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC101 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC0194 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC0038 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC12815 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC2912 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC098 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC12878 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XCC11 GTCGAAGACTCCACCCTGAAAGGCATCAAGGAAGCAGTATTCCGCACCGATGCCCCCGGC 798 XC33913 GTCGAAAATTGCGATTTCCAACAGGTCAAGGAAGCGGTGTTCCGTACCGATGCGCCGGGC 861 UW551 GTCCGGGACTCGAATTTCCAGAACGTGGCGGAGGCGGTCTTCCGTACCGATTCCAAATAC 1098 AAC00 ATCGCCGACTCCACCGCGCAGAACCTGAAATCCTTCCTCTTCCGCTTCGACTCCCGGCAG 963 EW CTGAGCCATATCAGCGCAGAAGACGGTAAGTTCTCGTTCGTTAAAAGCGATAGCGAGGGG 1254 ET1 CTGAGTCATATCAGCGCGCAGAACGGTAAGTTCTCATTCGTGAAAAGCGACAGCGAGGGA 1254 PV CTGAAAAACATCAGCGCTGAAGACGGCAAGTTCTCCTTCGTGAAGAGCGACAGCGAAGGC 1572 XC04 GCACATG--TCAGGGT----GCA-TGTCCTCGCGAGGAT---GCTCCCCTTCAAGTGGAG 910 XC018 GCACATG--TCAGTCT----GCAGTGCGTCCGCGACGAT---GCTAGCCATCAAGTGGAG 628 XCC306 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XCC112 -----------------------------------------------------------XC290 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC112 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC131 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC46 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC257-2 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC62 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC126 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC101 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC0194 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC0038 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC12815 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC2912 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC098 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC12878 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XCC11 GCACATG--TCAGTCT----GCATGGCGTCCGCGACGAT---GCTCCCCATCAAGTGGAG 849 XC33913 GCGACGG--TGAAGCT----GGCAAACCTGAAGACCGAT---GCGCCCGACGAGGTGATT 912 UW551 TCGACTGCGTCGTTCTC---GGACGATGTGAAGTCGGAT---GCGCCCTTCGATGGGCTG 1152 AAC00 TCCACCGTGCGCATCGCCAACACCGACGTGGACGGAGGCCGCACGCCGGTGAACGTGATG 1023 EW CTAAACGTCAATACCAGTGATATCTCACTGGGTGATGTTGAAAACCACTACA---AAGTG 1311 ET1 TTAAACGTCAATACCCAGGATATCTCCCTGGATAATGTGCAAAACCACTACA---AAGTT 1311 PV CTGAACCTGACCACCAGCGGCATTGATCTGAAGAATGTCGAGAACGCCTACAGCAAGCTG 1632 Figure 4 2.Continued

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109 XC04 GCCCTGTACCCCAGTATGTCAGATG----CGCCGAGCTCAGCCGAAAGGAATA-GCAGGT 965 XC018 GCCATGTACACCAGTCAGGCAGATGGGGGCATCAGCGTCAGCCGAAAGGAATATACAGGT 688 XCC306 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XCC112 -----------------------------------------------------------XC290 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC112 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC131 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC46 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC257-2 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC62 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC126 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC101 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC0194 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC0038 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC12815 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC2912 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC098 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC12878 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XCC11 GCACTGTCCCCCAGTCAGGCAGATGGCGCACGCAGCGTCAGCCGAAAGGAATATACAGGT 909 XC33913 GCCCCCAGCGCTTTCCAGGCCACCGGAGCGACCCGAATCGGGCATCAGCCCTACAGCGGC 972 UW551 GCTCCCAACAAGAGCCAGGTGACGGGCACCAACAAGGTGAGCTACAAGGCCTACTCGGGC 1212 AAC00 GCCGGCAACCCCGCCAACGTGCACGGCACGCGCAGCGTGCGCCAGTCGATCCAGACGGTC 1083 EW CCGATGTCCGCCAACCTGAAGGTGGCTGAATGA--------------------------1344 ET1 CCCGCTTCCGCTAACCTGAAGGTGGCCGAATGA--------------------------1344 PV CCAGGCTCGACCAACCACAAGGAGGCTTGA-----------------------------1662 XC04 TTCAGGCTGTAGTATTGCAG-ATGACG-CTGAGTGGCCGCTCGTCCTTGCAGCG-AGCGG 1022 XC018 TTCAAACACTAGTCCTGCACCATGACGACTGGGTGGCCGCTCATCCTTGGAGCGGAGCGG 748 XCC306 TGA--------------------------------------------------------912 XCC112 -----------------------------------------------------------XC290 TGA--------------------------------------------------------912 XC112 TGA--------------------------------------------------------912 XC131 TGA--------------------------------------------------------912 XC46 TGA--------------------------------------------------------912 XC257-2 TGA--------------------------------------------------------912 XC62 TGA--------------------------------------------------------912 XC126 TGA--------------------------------------------------------912 XC101 TGA--------------------------------------------------------912 XC0194 TGA--------------------------------------------------------912 XC0038 TGA--------------------------------------------------------912 XC12815 TGA--------------------------------------------------------912 XC2912 TGA--------------------------------------------------------912 XC098 TGA--------------------------------------------------------912 XC12878 TGA--------------------------------------------------------912 XCC11 TGA--------------------------------------------------------912 XC33913 TGA--------------------------------------------------------975 UW551 TGA--------------------------------------------------------1215 AAC00 TGA--------------------------------------------------------1086 EW -----------------------------------------------------------ET1 -----------------------------------------------------------PV -----------------------------------------------------------Figure 4 2 Continued

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110 XC04 GCAACCGACTCACCGGCGCGGCCCCGCGCGACTCAACGCACCTCGGAAATTTCCACCCCG 1082 XC018 GCAACTGACTCACCGGCTCGACCGCGGGCGACTCAGCGCACTTCGGAAATTTCCACGCCG 808 XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 -----------------------------------------------------------AAC00 -----------------------------------------------------------EW -----------------------------------------------------------ET1 -----------------------------------------------------------PV -----------------------------------------------------------XC04 -CCAGCCCCATCGACAAGTTCTAGGCATCGCCGACCTGCCAGAGCTTGATGCGCAGCCGG 1141 XC018 TCCAGCCCCTTCACCAAGGTCTAGGCATTGCCGACCTGCCAGAGCTTGATGTCCATCCGG 868 XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 -----------------------------------------------------------AAC00 -----------------------------------------------------------EW -----------------------------------------------------------ET1 -----------------------------------------------------------PV -----------------------------------------------------------Figure 4 2 Continued

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111 XC04 ACCTCGTTCTGCGAGTCGGCGCAACGCAACCGCGTCTTCGTAACATGATTTCGTCCCCGC 1201 XC018 ACCTCGTTCTGCGAGTCGGTGCAACGCAAC-GCGTCTTCGTAACAGATTATCGTCCCCGC 927 XCC306 -----------------------------------------------------------XCC112 -----------------------------------------------------------XC290 -----------------------------------------------------------XC112 -----------------------------------------------------------XC131 -----------------------------------------------------------XC46 -----------------------------------------------------------XC257-2 -----------------------------------------------------------XC62 -----------------------------------------------------------XC126 -----------------------------------------------------------XC101 -----------------------------------------------------------XC0194 -----------------------------------------------------------XC0038 -----------------------------------------------------------XC12815 -----------------------------------------------------------XC2912 -----------------------------------------------------------XC098 -----------------------------------------------------------XC12878 -----------------------------------------------------------XCC11 -----------------------------------------------------------XC33913 -----------------------------------------------------------UW551 -----------------------------------------------------------AAC00 -----------------------------------------------------------EW -----------------------------------------------------------ET1 -----------------------------------------------------------PV -----------------------------------------------------------XC04 CGTGG-GTATG 1211 XC018 CGTTGTAAATA 938 XCC306 ----------XCC112 ----------XC290 ----------XC112 ----------XC131 ----------XC46 ----------XC257-2 ----------XC62 ----------XC126 ----------XC101 ----------XC0194 ----------XC0038 ----------XC12815 ----------XC2912 ----------XC098 ----------XC12878 ----------XCC11 ----------XC33913 ----------UW551 ----------AAC00 ----------EW ----------ET1 ----------PV ----------Figure 4 2 Continued

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112 W1 MSELLQRLIQTQFQPQNTSQYDWNPSPSHGTSNGQGEGNEHGGRRNGRGGDNFLATTPET 60 W 2 MSELLQRLIQTQFQPQNTSQYDWNPSPSHGTS ---------------------------32 W 3 MSE LLQRL ---------------------------------------------------8 ************************************************************ W1 SDHSDKSAHSTVTGSGPVGSGRTINVNSPIVVHKGEVFDGHNNLYVGGSGIGDGSQSEHQ 120 W 2 DHSDKSAHSTVTGSGPVGSGRTINVNSPIVVHKGEVFDGHNNLYVGGSGIGD GSQSEHQ 91 W 3 -----------------------------------------------------------************************************************************ W1 QPMFVVEQGGTLQNVRMSGGGDGIHLLGDATLKNVHNLNVSEDAMTIDGPGNREHDSRIA 180 W 2 QPMFVVEQGGTLQNVRMSGGGDGIHLLGDATLKNV HNLNVSEDAMTIDGPGNREHDSRIA 151 W 3 -----------------------------------------------------------************************************************************ W1 GTPASGLPARPKIEILDSSFDNASDKVIQDNQAADVLLRNVSVNGAGKVFRTNGGHTDID 240 W 2 GTPASGLPARPKIEILDSSFDNASDKVIQDNQAADVLLRNVSVNGAGKVFRTNGGHTDID 211 W 3 -----------------------------------------------------------************************************************************ W1 SHVTVEDSTLKGIKEAVFRTDAPGAHVSLHGVRDDAPHQVEALSPSQADGARSVSRKEYT 300 W 2 S HVTVEDSTLKGIKEAVFRTDAPGAHVSLHGVRDDAPHQVEALSPSQADGARSVSRKEYT 271 W 3 ---------------------------------------------------RS VSRKEYT 17 ************************************************************ W1 G 301 W 2 G 272 W 3 G 18 Figure 4 3. Sequence a lignment of Xcc strain 306 wild type (W1); W:: Harpin mutant (W2); and 306:: hrpW HrpW (W3). Dots show parts of hrpW gene that were deleted.

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113 Figure 4 4 Phenotype assay in grapefruit leaves. A Xcc strain 306 wild type (1), W:: harpin mutant (circ le) and harpin::Harpin complemented (2); B. Xcc strain 306 wild type (1) and 306:: hrpW mutant. The bacterial suspension at 5 x 108 CFU/mL were syringe and needle infiltrated and photographed 7 days later.

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114 Figure 4 5. Populations of Xcc 306 (Xcc306) 306:: hrpW mutant (hrpW Mut), W:: harpin mutant (harpin Mut), harpin ::harpin mutant complement (harp Comp), Xcc 306 wild type carrying the pU:: harpin plasmid (306.1) and 306:: hrpG mutant (hrpG Mut) in grapefruit leaves times after infiltration of 5 x 108 CFU/mL of each strain into mesophyll. 5 6 7 8 9 0 2 4 6 8 10 Log10 cfu/cm2Days after Inoculation Xcc 306 hrpW Mut harpin Mut harp Comp 306.1 hrpG Mut

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115 Figure 4 6. Canker symptoms on abaxial leaf surface of grapefruit. Symptoms developed 28 days after pin -prick inoculation with Xcc 306 (all circled), 306:: hrpW mutant mutant ( A ), W:: harpin mutant ( B), har pin ::harpin mutant complement ( C ), Xcc 306 wild type carrying the pU:: harpin plasmid ( D ), and 306:: hrpG mutant ( E).

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116 Figure 4 7. PCR amplification of genomic DNA of Xcc 306 (1/2), 306:: hrpW mutant mutant (3), W:: harpin mutant (4), harpin ::harpin mutant complement (5), Xcc 306 wild type carrying the pU:: harpin plasmid (6/7). : Lambda DNA/ EcoR I and Hind III markers (Promega).

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117 CHAPTER 5 CHARACTERIZATION OF AVRGF1 FROM XANTHOMONAS CITRI SUBSP. CITRI STRAIN AW Introduction Host resistance is oft en linked to the recognition of specific elicitors encoded by the pathogen avirulence ( avr ) genes by a single resistance ( R) gene in the host (Hammond-Kosack and Jones, 1997). This recognition is now commonly referred to as effector -triggered immunity (ETI ) (Flor, 1971; Jones and Dangl, 2006). Phytopathogenic proteobacteria, such as Xanthomonas employ a type III secretion system (T3SS) in the initial infection phase to inject T3 effector proteins through this apparatus (Bonas et al ., 1991; Fenselau et al., 1992). The activity of T3 -effectors is required collectively to modulate the plant cell environment to provide optimum conditions for bacterial colonization (Cornelis and van Gijsegem, 2000). While the general function of T3-effectors is to suppress plant defenses, some T3 -effectors trigger a rapid and localized programmed cell death known as the h ypersensitive r eaction (HR) (Lindgren et al ., 1986; Staskawicz et al., 1995;.Block et al ., 2008). The list of T3-effectors with dual activity include avrRpm1 fro m Pseudomonas syringae pv. maculicola (Ritter and Dangl, 1995), avrXa7 in X. oryzae pv. oryzae (Yang et al ., 2004); avrPto of P. s. pv. tomato (Chang et al ., 2000) and avrPtoB in P. s pv. tomato DC300 (Scofield et al., 1996; Tang et al ., 1999; Kim et al., 2002). Xanthomonas axonopodis pv. citri recently renamed to Xanthomonas citri subsp. citri Xcc (Schaad et al., 2005), is the causal agent of citrus canker, which is one of the most destructive citrus diseases world -wide. An Xcc strain that attacks Key lime ( Citrus aurantifolia ) and alemow ( C. macrophylla) plants but no t grapefruit ( C. paradis i ) and orange ( C. sinensis ) was isolated and named as Xcc Aw (Schubert et al ., 1996; Sun et al ., 2004). Rybak et al. (2009) investigated the factors involved in limiting the ability of XccAw to cause disease in grapefruit and isolat ed a single avirulence gene, termed avrGf1 that elicits a n HR in grapefruit. The

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118 predicted product of avrGf1 AvrGf1 has sequence similarity to HopG1 in P. s. pv. phaseolicola and P. s. pv. tomato and a putative T3 -effector protein in R. solanacearum (Ryb ak et al., 2009). A number of methods have been used to characterize avr proteins including reporter gene fusions, gene bombardment and Agrobacterium -mediated gene transfer (Duan et al ., 1999; Roden et al ., 2004). In order to understand the structural requirements for AvrGf1 function as an elicitor, transient expression and bacterial reporter gene fusion systems for AvrGf1 were devised. Materials and Methods Bacterial Strains and Plasmids The strains of Escherichia coli, X. citri subsp citri and Agrobacte rium strains, and plasmids used in this study are listed in Table 5 1. Media and Growth Conditions Escherichia coli DH5 was used as the cloning host throughout this study and cultured in Luria Bertani (LB) medium (Sambrook et al ., 1989) at 37oC with appropriate antibiotics. For solid medium, 1.5% BD Bacto agar was added. The strains of Xanthomonas were cultured on nutrien t agar (NA) medium and tryptone sucrose agar at 28oC (Zhu et al ., 2000). Agrobacterium tumefaciens strains were grown in YEP medium (An, 1987) and Murashige and Skoog medium without plant growth regulators (MSO) (Murashige and Skoog, 1962). Triparental mat ings were performed on nutrient -yeast extract -glycerol (NYG) agar (Daniels et al ., 1984). All bacterial strains were stored in 20% glycerol in sterile tap water and maintained at 80oC. Antibiotic selection used ampicillin (Amp) at 100 g ml1; kanamycin ( Kn), 50 g ml1; rifampicin (Rif), 100 g ml1; and spectinomycin (Spc), 100 g ml1. Recombinant DNA Techniques All DNA manipulations, including isolation of total DNA, the alkaline method of plasmid purification, ligation and gel electrophoresis were per formed as described previously (Sambrook

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119 et al., 1989). Restriction enzymes, T4 DNA ligase, and GoTaq Flexi DNA Polymerase were used following the manufacturers recommendations (Promega, Madison, WI, USA). Chemicals were purchased from Sigma -Aldrich (St. Louis, MO, USA), and Fisher Scientific (Hampton, NH, USA). The polymerase chain reaction (PCR) was performed in a PCR Express Thermal Cycler (ThermoHybaid, Ashford, UK). The Topo TA cloning kit was used for cloning PCR products (Invitrogen, Carlsbad, CA, USA). All the enzymes and kits were used according to specifications of the manufacturers. Constructs were transformed into competent Escherichia coli DH5 cells described by Sambrook et al (1989). The selected plasmids were introduced into Xcc 306 by triparental conjugations (Daniels et al ., 1984), and into A. tumefaciens GV3101 strains by electroporation (Van Larebeke et al ., 1974) Aliquots of competent cells were mixed with plasmid DNA and electroporated using a BioRad GenePulser II Electroporator instrument. The conditions applied were 200 OHMS (resistance), 25 FD (capacitance) and 2.5 Volts. After the pulse, the cells were immediately diluted by addi tion of 250 L of NB medium. The cells were incubated for 1 h at 28oC with constant shaking, and plated on NA and YEP containing kanamycin at 50 g ml1 and rifampicin at 100 g ml1, then incubated at 28oC for 2 days. Sequencing was completed at Interdisc iplinary Center for Biotechnology (ICBR), University of Florida Gainesville, FL, USA with an Applied Biosystem model 373 system (Foster City, CA, USA). Plant Material and Plant Inoculations Grapefruit cv. Duncan plants were grown from seed in 15 cm plas tic pots in Terra -Lite agricultural mix (Scott Sierra Horticultural Products Co., Marysville, OH). The plants were kept in the glasshouse of the University of Florida in Gainesville at temperatures ranging from 25 30oC. A scale to standardize the citrus l eaves was adopted in this study and consisted of young

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120 leaves, (three weeks -old leaves after pruning back the branches), intermediate aged leaves (three to five weeks old leaves after the pruning), and old leaves (five or more weeks old leaves after prunin g). Pepper plants ( Capsicum annuum ) cv. ECW and its NIL ECW20R containing the Bs2 resistance gene were planted from seeds in Plugmix (W. R. Grace & Co., Cambridge, MA, USA). After two weeks, the seedlings were transferred to Metromix 300 (W. R. Grace & Co ) in 10 cm plastics pots. The plants were kept in the glasshouse at temperatures ranging from 25 30oC. For preparation of bacterial suspensions, 18 h cultures were harvested from the NA plates, suspended in sterile tap water, and standardized to an optica l density at 600 nm (OD600) = 0.3 (5 x 108 CFU/ml) with a Spectronic 20 Genesys spectrophotometer (Spectronic -UNICAM, Rochester, NY, USA). Bacterial suspensions of the strains used in this study, except Agrobacterium strains, were infiltrated at 5 x 108 C FU/ml into the abaxial surface of citrus leaves by using a hypodermic syringe and needle, and the symptoms were assessed up to 10 days after inoculation. Plant responses were evaluated 3 4 days after inoculation for water -soaking or chlorosi and after 6 7 days for pathogenicity. Agrobacterium -mediated expression of avrGf1 The full -length avrGf1 coding sequence, 1.6 kb fragment, was amplified by PCR using a forward primer 01 -Fwd and reverse primer 02 Rev (Table 5 2 ). To construct the entry vector pEAvrGf1 the avrGf1 PCR product was cloned into Gateway entry vector pENTR/D TOPO following the manufacturers instructions (Invitrogen). The avrGf1 sequence was then inserted in -frame with the GFP sequence from the GATEWAY vector pGWB5 (Tsuyoshi Nakagawa, Resear ch Institute for Molecular Genetics, Shimane University, Japan) by performing an LR reaction between the entry clone and pGWB5, creating pGGf1B5. Additionally, the pEAvrGf1 entry clone was used to construct the binary plasmid pGGf1B2 with the GATEWAY LR Cl onase Mix (Invitrogen) and the destination vector pGWB2 (Tsuyoshi Nakagawa, Research Institute for

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121 Molecular Genetics, Shimane University, Japan). The pGWB2, which does not carry GFP protein, was used as a control to test the possibility that GFP is respon sible for the observed phenotypes. All pGWBs contain NPTII gene for kanamycin selection driven by the NOS promoter and terminator sequences. The green fluorescent protein (GFP) in the pGWB5 is driven by the CaMV 35S promoter and terminator. Ti plasmid car rying deletions on the N -term and C term of avrGf1 were constructed by PCR amplification, using custom primers ( Table 5 2 ) from the pL799 plasmid DNA. N -terminal deletions were constructed introducing an ATG before AvrGf1 codon 14 and 117, creating Gf1:: N 13 and Gf1:: N116 mutants. We also deleted 7 and 83 amino acids of the C -terminal region of AvrGf1, creating Gf1:: C7 and Gf1:: C83 mutants respectively to identify the region required for the AvrGf1 HR induction in Bs2 pepper plants. Thus, all avrGf1 PC R fragments were cloned into Gateway entry vector pENTR/D TOPO following the manufacturers instructions (Invitrogen). The selected entry vector carrying the avrGf1 was then LR recombined, where the cloning process involves the transfer of the avrGf1 into the expression vector (pGWB2 and 5) through a simple recombination reaction that maintain s protein reading frame using Invitrogen Gateway manipulations, with pGWB2 and pGWB5 binary vectors (Table 5 1). Each resulting construct in pGWB2 and pGWB5 binary vectors were conjugated into A. tumefaciens GV3101 by electroporation and the resulting transconjugants were selected on YEP medium containing Kn 50 g ml1 to use for plant transient expression in citrus. Young leaves of Grapefruit cv. Duncan were used a s a source of tissue for transient expression assay. The empty vectors pGWB2 and pGWB5 served as negative control plasmids. Bacteria were cultured overnight at 28oC in YEP liquid medium containing 50 g ml1 of kanamycin with 200rpm shaking. The optical de nsity (OD) of the bacterial cells was measured in

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1 22 the spectrophotometer and adjusted to an OD600 of 0.3 with YEP medium. Three milliters of cells were collected by centrifugation for 5 minutes at 3500 rpm, and gently resuspended with 0.9 mL of MSO medium. Acetosyringone was added to a final concentration of 0.01mM. The agrobacterium suspensions were infiltrated into the mesophyll of young grapefruit leaves. Bacterial Expression of avrGf1 The plasmids used in the expression avrGf1 in Xanthomonas included pCR799, a pCR2.1 TOPO -cloning vector. The avrGf1 gene and adjacent region were PCR amplified with HRF1 and HRR1 primers from clone pL799 (Table 5 2; Rybak et al., 2009). The parameters used for PCR were as follows: Step 1. 90oC for 5 min; step 2. 95oC for 1 min; step 3. 55oC for 1 min; step 4. 72oC for 1 min per kb, 30 cycles from step 2 to step 4; step 5. 72oC for 10 min. The PCR fragments were purified using QIAquick PCR Purification Kit system (QIAGEN). Purified fragments were directly cloned into pCR2.1 TOPO, containing kanamycin and ampicillin resistance genes following manufacturers protocol. Additionally, the LA plates were augmented with X D 1 thiogalactopyranoside (IPTG 0.5mM) for blue/white screening of the lacZ pro moter. To construct pHAvrGf1, the pCR799 and pHM1 were digested with Xho I and Sal I, respectively, linearizing both plasmids. The digested plasmids were ligated and transformed into E. coli DH5 Cloning was confirmed by PCR with primers flanking the avrGf1 regions, HRF1 and HRR1, as well as sequencing of the PCR fragment with the same set of primers. The search for nucleotide and amino acid sequence homology was conducted with the BLAST 2.0 algorithm ( h ttp://blast.ncbi.nlm.nih.gov/Blast.cgi ) to confirm the insertion (Altschul et al ., 1997). The resulting construct pHAvrGf1 was conjugated into Xanthomonas citri subsp. citri strain 306 by electroporation, and the resulting transconjugants were selected on NA medium containing Spc 100 g ml1. A construct encoding AvrGf11 -

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123 106::AvrBs262574 was generated for translocation assay in pepper cvs. ECW and ECW20R. For making pBSNGf1 plasmid, a PCR fragment encoding the N term for AvrGf1 was amplified from Xcc 306 genomic DNA using NGf1-F1 and NGf1-R1 primers, and cloned into the Bgl II restriction site in the pBS( BglII::avrBs262574::HA) plasmid. The BamH I Kpn I fragment containing AvrGf11106::AvrBs262574 region was excised from the resulting plasmid and ligat ed into the pUFR034 vector previously digested by the same enzymes, creating pUF12. The resulting plasmid was introduced into X. campestris pv vesicatoria strain XC1922 by triparental mating. Xcv carrying the construct was then tested for the ability to e licit an AvrBs2 HR in the two pepper genotypes. Results Induction of Hypersensitive Response by Transient Expression of avrGf1 Within Citrus To investigate whether the induced HR in grapefruit leaves is trigger ed by AvrGf1 alone or in combination with other proteins from Xanthomonas we expressed the full length of avrGf1 under control of the constitutive cauliflower mosaic virus 35S promoter in grapefruit leaves using Agrobacterium tumefaciens mediated gene delivery. The amplified avrGf1 fragment was clo ned into pGWB5 and pGWB2 binary vector (Figure 5 1) using Gateway cloning system and Agrobacterium transformed with pGGf1B5 and pGGf1B5 (both carrying the avrGf1 ), and pGWB5 and pGWB2 (empty vectors) were inoculated in grapefruit leaves. Transient expressi on of avrGf1 under the control of the 35S Cauliflower mosaic virus promoter in young grapefruit leaves resulted in a typical HR 4 5 days after inoculation ( Figure 5 2). The A. tumefaciens strain GV3101 by itself or carrying pGWB2 or pGWB5, empty vectors, did not show any visible sign of necrosis (Figure 5 2), implying that the HR is induced by the avrGf1 and not due to an Agrobacter ium protein, or the green fluorescent protein (GFP) or other protein expressed by T DNA genes delivered by the empty vectors.

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124 The N -terminal and C -terminal Are Required for HR Elicitation Deletion mutants were constructed affecting both ends of avrGf1 (Figure 5 -1a). Two different deletions were performed in each terminal The plasmids carrying the N and C terminal coding sequ ence deletions, p5 N13 and p5 N116, p5 C7 and p5 C83, respectively, were electroporated into the A. tumefaciens strain GV3101. The resulting strains were tested for their HR elicitation in grapefruit leaf tissue. The transient expression of the deletion mutants Gf1:: N13 and Gf1:: C7, with 13 deleted amino acids after the start codon and 7 deleted amino acids before the stop codon, produced a n HR comparable to that of A. tumefaciens carrying the full length avrGf1 31::Gf1B5 and 31::Gf1B2 (Figure 5 1). However, the HR induct ion of the Gf1:: N116 and Gf1:: C83, which carry 116 deleted amino acids after the start codon and 83 deleted amino acids before the stop codon, respectively, showed complete absence of a macroscopical HR induction (Figure 5 1).Thus, both N and C terminal are required for HR elicitation. Xanthomonas citri subsp. c itri Harboring avrGf1 Induces a Hypersensitive Reaction in Grapefruit To determine if the Xcc, which induce s a compatible reaction, was able to delivery AvrGf1 into the plant host cell of grapefru it leaf tissue, and then, trigger an incompatible reaction we transformed Xcc with pHArGf1, which carries the avrGf1 driver by its own native promoter. The transconjugants Xcc 306 expressing in -trans the avrGf1 gene (306:: avrGf1 ) were infiltrated into grap efruit and Valencia leaf tissue at 5 x 108 CFU/ml, and leaves were observed after 5 days for the development of visible HR ( Figure 5 3). The Xcc 306 by itself showed water -soaking in the edges of the infiltrated area, which is a classical symptom of diseas e development. The HR in planta of grapefruit elicited by 306:: avrGf1 did not differ among the three repeats.

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125 AvrGf1 is Translocated Inside the Host Plant Cell Rybak et al. (2009) reported that avrGf1 requires a functional T3SS and triggers a HR in grapefr uit plants. To further test if the AvrGf1 is translocated into host plant cells or only secreted into the apoplast, the N terminal coding region of AvrGf1 (codons 1 106) was fused to the C -terminal coding region of AvrBs2 (codons 62 574), a X. campestr is pv. vesicatoria type III effector (Kearney and Staskawicz, 1990; Bonas, 1991). We inoculated Xcv XV1922 expressing the AvrGf11106::AvrBs262574 fusion protein into pepper leaves. We chose Xcv XV1922 to express the fusion protein because it does not tri gger HR in pepper cv. ECW20R due a natural mutation at avrBs2 gene. The bacterial suspension was infiltrated at 5 x 108 CFU/ml into the leaf mesophyll. Resistant Bs2 pepper plants specifically recognize d AvrBs2 truncated protein leading to HR 24 h after inoculation ( Figure 5 4). Furthermore, no HR was observed in pepper plants without Bs2 resistant gene (Figure 5 4). This data showed that the firs t 106 amino acids were sufficient to translocate AvrGf11106::AvrBs262574 fused protein into Bs2 pepper plants. Discussion AvrGf1 was characterized by molecular analysis to understand the role of this protein in XccAw HR elicitation in grapefruit as originally identified by Rybak et al. (2009) They determined that AvrGf1 is a homolog of several T3 -effectors and it is T3SS -dependent. T3SS facilitate s the translocation of a collection of proteins into plant cells (Buttner and Bonas, 2002). The translocation signal in the avrBs2 gene of X. c. pv. vesicatoria is located between 1 58 amino acid and the region of 62 7 14 amino acids activate Bs2 disease resistance (Mudgett et al ., 2000). In this work, we used AvrBs2 as a sensitive reporter (Guttman et al., 2002; Roden et al ., 2004) to identify if the AvrGf1 is translocated inside the host cell in Bs2 resistance pepper p lants from Xcv strain XV1922 transfomed with the AvrGf11106::AvrBs262 574 fused protein. The

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126 AvrBs2/Bs2 report system has been used to identify new translocation effectors (Roden et al., 2004). The HR induced for the XV1922, expressing the fusion protein, showed that the translocation signal is present in the first 106 amino acids in the N terminus. The fact that the translocation signal is present in the N termin us is not surprising, since most T3effectors carry the translocation signal in the N terminal corroborating th e results reported by previous studies (Leach and White, 1996; Mudgett et al ., 2002; Roden et al., 2004). Additionally, Rybak et al (2009) demonstrated that AvrGf1 is translocated into grapefruit cells in a T3SS -dependent manner. The deletion mutagenesis analysis per the N termin us showed that deletion of 116 amino acids affect HR activity in pepper plant cv. ECW20. Gathering the data from the AvrGf1::AvrBs2 translocation study indicate that the first 116 amino acids on the N terminal de finitely play important roles in protein translocation and and HR induction. In contrast to the HopG1 in P. s. pv. tomato DC3000, which is a homolog, that does not elicit cell death in Nicotiana benthamiana (Wei et al ., 2007). Additionally, the deletion of only 83 amino acids in the C -terminal, directly upstream of the stop codon, was shown to be enough to block cell death elicitation. While, Xcv AvrBs2 protein required, at least, the deletion of 297 amino acids upstream of the stop codon to lose the abilit y to trigger HR in Bs2 pepper plants (Mudgett et al ., 2000). In this work, an Agrobacterium mediated transient expression protocol was established for grapefruit to analyze the gene -for -gene interaction between AvrGf1 and the putative R gene produc t contr olling the HR response in grapefruit. An Agrobacterium -mediated transient expression in citrus leaves has been reported, which followed the procedure described by Kapila et al. (1997) and uses vacuum to infiltrate citrus seedling (Duan et al ., 1999). However, the aim

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127 of th at experiment was to show that the expression of pthA pathogenicity gene from Xcc alone can induce citrus canker symptoms in the absence of the pathogen and no HR. The symptoms showed by the transient expression are much smaller than the l esions induced by Xcc, thus, this difference may be because of the relatively inefficient gene transfer (Duan et al., 1999). In contrast, in the procedure developed here we exclude the necessity of orange seedlings that are time -consuming and the vacuum ch amber. Furthermore, the procedure here demonstrated the same level of HR elicitation compared with the Xcc wild type strain. Infiltrated plants showed stable expression of avrGf1 through the HR in young tissue but the same results were not observed in inte rmediate and old leaves. Previous attempt s to perform Agrobacterium -mediated transient expression in mature leaves have failed. The inefficiency of this method to function in old leaves may be because, in general, plant cells have hard and thick cell wall s that reduce access to the bacterium. In addition, this procedure excludes the need to regenerate transformed cells that are used commonly for functional analysis of gene regulation. The circumvention of tissue culture is particularly valuable in the case of gene expression of woody trees such as citrus, which are difficult to regenerate (Ghorbel et al ., 1999; Chvez Brcenas et al., 2000; Tucker et al., 2002).

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128 Table 5 1. Bacterial strains and plasmids used in the study Desigantion Relevant characteristi cs Source Strains X citri subsp. citri 306 Wild type, Asiatic strain, isolated in Brazil, Rif R DPI a 306:: avrGf1 306 carrying pH avrGf1 Amp R Kn R Spc R This study X c ampestris pv c ampestris XV1922 Pepper race 6, Rif R This study XV192 2 1 1922 carrying pUF12, Kn R Rif R This study Agrobacterium tumefaciens GV3101 Rif R Lahaye, T. b 31 :: WB5 GV3101 carrying pGWB5, Amp R Rif R This study 31 :: WB2 GV3101 carrying pGWB2, Amp R Rif R This study 31 :: Gf1B5 GV3101 carrying pGGf1B5, Amp R Rif R This study 31 :: Gf1B2 GV3101 carrying pGGf1B2, Amp R Rif R This study Gf1 :: N13 GV3101 carrying p5 N13, Kn R Rif R This study Gf1 :: 5 N116 GV3101 carrying p5 N116, Kn R Rif R This study Gf1 :: 5 C7 GV3101 carrying p5 C7, Kn R Rif R This study Gf1 ::5 C83 GV3101 carrying p5 C83, Kn R Rif R This study Gf1 :: 2 N13 GV3101 carrying p2 N13, Kn R Rif R This study Gf1 :: 2 N116 GV3101 carrying p2 N116, Kn R Rif R This study Gf1 ::2 C7 GV3101 carrying p2 C7, Kn R Rif R This study Gf1 :: 2 C83 GV3101 carrying p2 C83, Kn R Rif R This study Escherichia coli DH5 FrecA 80dlacZ M15 Invitrogen Plasmids pCR2.1 TOPO Phagemid, Amp R Kn R Invitrogen pENTR D Entry vector Invitrogen pBluescript KS+/ Phagemid, pUC derivative; Amp R Stratagene pRK2013 Kn R tra + mob + Daniels et al ( 1984 ) pHM1 Broad host range vecto r with pUC19 polylinker, SpR Hopkins et al (1992) pLAFR3 Tc R rlx + RK2 replicon BJS c pUFR034 Kn R Tn903, IncW, Mob + De Feyter et al (1990) pGWB2 Binary gfp expression vector contains 35S promoter upstream of attR1 -CmR -ccdB -attR2 sgfp Lahaye, T. p GWB5 Binary vector Lahaye, T. pBS1 Bgl II :: avrBs2 62574 ::HA of Bluescript KS+/ AmpR Mudgett d pCR 799 avrGf1 total sequence in pCR2.1 TOPO Amp R Kn R This study pHAvrGf1 avrGf1 (pCR799) ligated with pHM1 This study

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129 Table 5 1. Continued Desigantion Rel evant characteristics Source pBSNGf1 avrGf1 1 106 fused to avrBs2 62 574 of pBS1 This study PUF12 avrGf1 1 106 :: avrBs2 62 574 fusion in pUFR034 This study pE avrGf1 avrGf1 total sequence in pENTR D Amp R Kn R This study pE N13 avrGf1 deleted 13 codon afte r ATG in pENTR D AmpR, KnR This study pE N116 avrGf1 deleted 116 codon after ATG in pENTR D Amp R Kn R This study pE C7 avrGf1 deleted 7 codon before stop codon in pENTR D AmpR, KnR This study pE C83 avrGf1 deleted 83 codon before stop codon in pENTR D Amp R Kn R This study pGGf1B5 avrGf1 total sequence in p GWB5, Rif R Kn R This study pGGf1B2 avrGf1 total sequence in p GWB2, Rif R Kn R This study p5 N13 avrGf1 deleted 13 codon after ATG in p GWB5, AmpR, KnR This study p5 N116 avrGf1 deleted 116 codon a fter ATG in p GWB5, Amp R Kn R This study p5 C7 avrGf1 deleted 7 codon before stop codon in p GWB5, Amp R Kn R This study p5 C83 avrGf1 deleted 83 codon before stop codon in p GWB5, Amp R Kn R This study p2 N13 avrGf1 deleted 13 codon after ATG in p GWB2, Amp R Kn R This study p2 N116 avrGf1 deleted 116 codon after ATG in p GWB2, AmpR, KnR This study p2 C7 avrGf1 deleted 7 codon before stop codon in p GWB2, Amp R Kn R This study p2 C83 avrGf1 deleted 83 codon before stop codon in p GWB2, AmpR, KnR This study a D PI, Division of Plant Industry of the Florida Department of Agriculture and Consumer Services, Gainesville, FL, USA. b T Lahaye Martin Luther -Universit t, Halle, Germany. c BJS, B. J. Staskawicz, University of California, Berkley, CA. d M. B. Mudgett, St anford University, Stanford, CA.

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130 Table 5 2. Oligonucleotides sequence used in this study Gene Primer Name Primer sequence Length avrGf1 HRF1 5 CGCCAGGAAGGGCCTGCCAT G 3 21 HRR1 5 CCTCAACCGGGCGCCAAGTT GCGT 3 24 01 F wd 5 CACCATGGCTCCGSGCSYGCATTCGG C 3 27 02 R ev 5 GTCGCTGCTGGTCATTGACTTTCCTGC 3 27 NGF1 F1 5 GGAAGATCTATTCGAGATGCAGACAGCTCAG 3 31 NGF1 R1 5 GGAAGATCTGCCTTCTTTTTCGCCGGACTTC 3 31 GF1:: N13 N13 F1 5 CACCATGCTGCACTTGAGAGATACATCCAT 3 30 HRR1 5 CCTCAACCGGGCGCCAAGTT GCGT 3 24 GF1:: N116 N116 F1 5 CACCATGTTCGTTCTGTCCACGACATCGC 3 29 HRR1 5 CCTCAACCGGGCGCCAAGTT GCGT 3 24 GF1:: C7 HRF1 5 CGCCAGGAAGGGCCTGCCAT G 3 21 C07 R1 5 TCCTGCATAGAATGCCCGTGCAGC 3 24 GF1:: C83 HRF1 5 CGCCAGGAAGGGCCTGCCAT G 3 21 C83 R1 5 TGCAACG GCATTTGCACTAGTAAACG 3 26

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131 Figure 5 1. Diagram of AvrGf1 and truncated versions used to test regions of the protein for elicitor activity. A ATG indicates the site at the N terminal coding portion was excluded, and the red triangle indicates the point of truncation of the C terminal coding region. B. Organization of the binary vectors, pGWB2 and pGWB5, used to clone the truncated proteins.

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132 Figure 5 2. Agrobacterium -mediated expression of avrGf1 in grapefruit leaf tissue. A Phenotype elicited by Agrobacterium harboring 31::Gf1B2 plasmid, grown overnight in YEP medium and directly infiltrated into grapefruit leaves (indicate by circle and arrow) and leaf infiltrated with Agrobacterium carrying the 31::Gf1B2 plasmid, grown under the conditions developed in the section 5 of materials and methods (indicate arrow), 31::Gf1B5 without cell treatment (indicate by circle) and 31::Gf1B5 treated cells; B. Lower panel: Agrobacterim carrying pGWB5 empty vector, Up panel: Agrobacterium tumefaciens strain G V3101; C Phenotype of the N -terminal mutants Gf1:: N13 (low) and Gf1:: N116 (up); D Upper panel: HR induced by strain containing 31::Gf1B5; lower panel: C -terminal mutant containing Gf1:: C7. Bacterial suspensions were prepared following the transient expression protocol developed in this study (see Methods and Materials). The plants were photographed 5 days after infiltration.

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133 Figure 5 3. Hypersensitive reaction induced by X citri subsp. citri 306 expressing avrGf1 Xcc wild type strain 306 (indicate by the arrow) and 306:: avrGf1 (circled). A G rapefruit leaf; B. Valencia orange leaf. The bacterial suspension was infiltrated at concentration of 5 x 108 CFU/ml. The plants were photographed 6 days after infiltration.

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134 Figure 5 4 Phenotypes elicited on pepper plants cv. ECW20R 24 h after inoc ulation. A. Xcv: X. campestris pv. vesicatoria wild type strain XV1922; Xcv::A, Xcv::B and Xcv::C: different X. c. pv. vesicatoria transconjugants carrying the pUF12. B. Close -up of the X. c. pv. vesicatoria transconjugants, Xcv::A and Xcv::C

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135 CHAPTE R 6 OVERALL SUMMARY AND DISCUSSI ON The most aggressive form of Asiatic citrus canker disease is caused by Xanthomonas citri subsp. citri (Xcc) and designated as an A -strain. Another strain, Xanthomonas fuscans pv. citri (Xfc) and known as a C -strain, is k nown also to incite citrus canker like symptoms in Key/Mexican lime ( Citrus aurantiifolia ) and h persensitive r esponse (HR) in grapefruit (C. paradisi ) (Stall and Civerolo, 1991; Rybak et al., 2009). The pathogenicity and virulence of Xcc rel y on the highly specialized type three secretion system (T3SS) delivery system, which transports a collection of effector proteins directly inside host cells and thereby amending the host cell environment for bacterial colonization (Bonas et al ., 1991; Fenselau et al., 1 992; Cornelis and van Gijsegem 2000). H ypersensitive r esponse and p athogenicity ( hrp ) genes encode the T3SS system, and they are also chaperones for some effectors in the translocation process ( Chang et al. 2004; Tang et al 2006) The aim of this study was to characterize the putative T3 -effectors involved in the pathogenicity of Xcc. To accomplish this aim and to further characterize some of the genes that were identified in the process, the study was separated into four objectives: I. Identification a nd characterization of candidate T3 -effector genes in Xcc based on genomic sequencing data by da Silva et al (2002) using mutagenesis analysis; II. Genetic characterization of a T3SS -independent HR elicitor activity in Xcc; III. Characterization of hrpW a nd the domains, harpin and pectate lyase, in the Xcc pathogenicity; and, IV. Molecular characterization of the Xfc avrGf1 gene and development of avrGf1 as an efficient transient expression system in citrus leaves. The candidate effector gene s in this stud y were selected from genes that were scanned for the presence of the plant inducible promoter (PIP) Box (TTCGC N15TTCGC), which is the transcriptional signal for HrpX regulator (Wengelnik and Bonas, 1996; Wengelnik et al., 1996a ;

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136 Wengelnik et al., 1996b ), another motif called the 10 boxlike motif (YANNNT; Y: C/T; N: A/T/C/G), which is localized 30 32 bases pair from the PIP box that has been proposed t o be necessary for HrpX regulon, and for genes with sequence similarity to known T3 -effectors (Cunnac et al 2004). Only two candidate T3 -effector genes with homology to known T3effectors were found without the PIP Box, avrPphE2, which was previously reported by da Silva et al. (2002) and xopX which was identified in another Xanthomonas on the basis of an analysis of T3SS -dependent extracellular protein secretion ( Koebnik et al 2006). Insertional mutagenesis was used as the primary research approach in this study to evaluate the contribution of candidate T3 -effector genes of Xcc in pathogenicity. Pat hogenicity assays in grapefruit showed that none of the mutants created in this study had visible effects on citrus canker phenotype, nor had visible effects on the HR elicitation activity on the non -host species tomato. At the same time, insertions into h rp genes previously known to control T3SS function ( hrpA hrpG and hrpX ) resulted in loss of pathogenicity. However, mutations in hrpA hrpG and hrpX also failed to result in the loss of the nonhost HR on tomato. Concluding, most of the genes studied here and show n to be conserved in other Xanthomonas were not found to contribute to the visible canker symptooms. The presence of the PIP -box also does not define the direct interaction with pathogencity or T3SS, such as avrPphE or xopX which may be pseudogene s and which may not be involved in pathogenicity. Therefore, the lack of PIP does not mean PIP -box does not define direct involvement. Further pathogenicity tests, possibly involving bacterial cell counts, will be conducted to determine what role, if any, the candidate genes play in citrus canker. The insertional and deletion mutagenesis performed in Xcc hrpW domains, harpin and pectate lyase (PEL), demonstrated that the deletion of harpin plays crucial function on Xcc

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137 compatible interaction in grapefruit. The hrpW null mutation is not impaired with the pathogenicity ability. Probably the phenotype observed with harpin mutant is caused by a change in protein conformation, thus altering the formation of a protein complex that HrpW and the other proteins (Aleg ria et al ., 2004). Additionally, attempts to complement the harpin mutant with full length hrpW genes driven by the native promoter in trans failed several times, although, when the mutation was replaced in the chromosome, the pathogenicity was recovered. This inability to complement the harpin mutant might be a dominant negative effect of the defective protein expressed by the mutant. The Xcc hrp regulator mutants, 306:: hrpX and 306:: hrpG preserved the ability to induce HR in non -host tomato plant. Thus we speculate that an unknown T3SS independent gene, or genes, controls the biosynthesis of the elicitor. Previously, a study by our group and involving the characterization of Xfc genes that are involved in incompatible host reactions revealed a clone de signated 450, which, when expressed in hrpmutant of the tomato pathogen X. perforans demonstrated same phenotype in non-host tomato plants as Xcc and Xfc. To identify the possible candidate(s) for the T3 independent HR phenotype, clone 450 was subclone d, and a screen was performed in tomato plants, revealing a subclone of 3.0 kb that triggered an HR when expressed in trans in the X. perforans hrpmutant. Sequence analysis of the flanking ends of this subclone revealed sequence similarity for the ends of a region containing three genes in Xcc, namely, XAC3857, XAC3858 and XAC3859. The search for homology with these t h ree ORFs did not show any relation with known HR elicitor. BLAST analysis of the NCBI Genbank indicated that none of the ORF had relatednes s to any knonw HR related proteins. Further characterization of the three genes will reveal whether one or more of the genes control the T3SS independent HR phenotype of Xcc and Xfa.

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138 Further experiments are in progess to develop an efficient Agrobacterium mediated transient expression procedure in citrus leaf tissue. This system will be used to characterize the critical domains of avrGf1 gene that are required for the HR in grapefruit and as a reporter system for candidate T3 -effector genes of Xcc and rela ted pathogens. In this study, a deletion mutagenesis analysis made in the N terminus and C -terminus revealed that the region between 13 and 116 amino acids (aa) in the N -terminus are required for HR elicitation, and also, the region 7to 83 aa before the st op codon was required. Moreover, a translocation assay using a AvrGf11106::AvrBs262574 fused protein in Bs2 pepper plants, revealed that the secretion signal resided within the first 106 aa of AvrGf1.

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139 APPENDIX A SEQUENCE AND ALIGNME NT 1 ATGTCGGAAT TGCTACAGCGGTTGATACAGACCCAATTCCAACCTCAAAACACGTCGCAA 61 TACGATTGGAACCCTTCGCCTTCCCATGGGACGTCCAATG GCCAAGGGGAGGGCAACGAG 121 CATGGCGGTCGCAGGAACGGACGCGGCGGTGACA ACTTCCTCGCCACCACGCCCGAGACG 181 TCCGACCATTCCGACAAATCCGCTCACTCGACCG TCACCGGCAGCGGCCCCGTGGGGAGC 241 GGTCGCACGATCAACGTCAACAGTCCGATCGTGGTTCACA AGGGCGAGGTGTTCGACGGA 301 CACAACAATCTCTACGTCGGCGGGTCCGGCATCG GCGATGGTTCGCAGTCCGAGCACCAG 361 CAACCGATGTTCGTCGTCGAACAAGGCGGCACCC TCCAAAACGTGCGGATGAGCGGGGGC 421 GGCGACGGCATCCATCTGCTCGGCGACGCCACGC TGAAGAACGTCCACAACCTGAACGTC 481 AGCGAGGATGCGATGACCATCGACGGCCCTGGCA ACCGGGAACATGACTCGCGCATTGCC 541 GGAACGCCTGCCAGCGGCCTGCCGGCGCGTCCAAAGATCG AAATCCTCGACAGTTCCTTC 601 GACAATGCCTCCGACAAGGTGATCCAGGACAACC AGGCGGCAGATGTGCTGTTGCGTAAC 661 GTCAGCGTGAACGGAGCCGGCAAGGTGTTCCGCA CCAACGGCGGACACACCGACATCGAT 721 TCACACGTCACGGTCGAAGACTCCACCCTGAAAG GCATCAAGGAAGCAGTATTCCGCACC 781 GATGCCCCCGGCGCACATGTCAGTCTGCATGGCG TCCGCGACGATGCTCCCCATCAAGTG 841 GAGGCACTGTCCCCCAGTCAGGCAGATGGCGCAC GCAGCGTCAGCCGAAAGGAATATACA 901 GGTTGA MSELLQRLIQTQFQPQNTSQYD WNPSPSHGTSNGQGEGNEHGGRRNGRGGDNFLATTPETSDHSDKSAHS TVTGSGPVGSGRTINVNSPIVVHKGEVFDGHNNLYVGGSGIGDGSQSEHQQPMFVVEQGGTLQNVRMSGG GDGIHLLGDATLKNVHNLNVSEDAMTIDGPGNREHDSRIAGTPASGLPARPKIEILDSSFDNASDKVIQD NQAADVLLRNVSVNGAGKVFRTNGGHTDIDSHVTVEDSTLKGIKEAVFRTDAPGAHVSLHGVRDD APHQV EALSPSQADGARSVSRKEYTG Figure A 1. hrpW nucleotide sequence and deduced amino acid sequence.

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140 B gl II AGATCTATTCGAGATGCAGACAGCTCAGGCCTTCAGGAAAAATAGGTCGTCAATCCGGTAGCAGTGAAATACACGGC GTTATATGCACATTTACGTCTTGTCATCCCGATGGCTTGTCGCTCCGGATCGCTGGTAACTCACCACGGTCGATGAG TGCCGCCAGCCCAGGCACGCACAGTCAACACGGCCTCGCCGCGGACTGATAGCAATCTTCCATGTGATACCCGCAGA GTGGATCGATATGTCGTCGGTAGCGCGCGGCCATGCTTTTCATAGTCCCTATTCCATTCGTCCATCAGCACTTCGCC TAGTCAACAAGCTCCG ATG GCTCCGAGCATGCATTCGGCGGCGTCGCCGGTTTCTGTCCTGCACTTGAGAGATACAT CCATGGCTCCGAGCATGCATTCGGCGGCGTCGCCGGTTTCTGTCCTGCACTTGAGAGATACATCCATGCGCACCAAA GCCCAACTCCCATTGACTGCCATTCAACGGTTTCTTGCCCATGATGCAGCGTCAACGCAGGCCCCCTCTGCATCGGC ATCCACATCGCTCCACAAAAATGAGACCGCAGGCTTGCTGGCAGCCTTGCCAGCGCGAAACGCCAGGCAAGGAGCGC B gl II AGAGGAAGTCCGGCGAAAAAGAAGGCAGATCTATG ACCGGCAAGCCGGCCCTGGT GGCGCTCGACAGCGAATTCTCC GAGCAGCGTCTGGCCGAAGTGCAGGCGCGCCAGATCACCG TGCAGACCCTGCAAGGCAAGCTTGCCACGCATCTTGC GCAGGCCGGCACGGCGCTCAAACCCGACAGCATTGCGGCA CGCTTTGCTGCCGGCACACTGGAGCCGGTGTATCTGG ATACCGCCGCCTTCAATGCCATGTCGCGCGGGCT GCCCGCACGCGCACGTGCGGCCGCAGGCCCGGTGCTGATC GAT GCACAACAAGGTCGCATCATCTTCAATCTGCAGCGCGCGT TTGCGCCTGGCGACACCTTCAGCGACGCGGCGCTTGC CGCGCTTGGCAAGCAGTTGAATCTTTCCGGCCACGGGCTG GCAACGCCGAACTGGCTGCAGCCTGCCGCAGGCACGC CGGGGCGGCGCAAGCTGCAGCAAGCCGCGCGCTATCACGG CCACGAGGTGCCGGCCCGCGACGGTGGCGCCGGGTTC TTCAAGGCCAACGACCATCGCCTGCTGGAAGGCAAGCAAG TGCTGTTGCGCAATCATCAAAAGTCGCTCGTGCACAA CCACTACTTCGAAGCACCCAGCACGCGTGCGTTCGGAAAG GACGTCATGGTGCATCGCGGGCTGTTCGATAATCACG CCGGCATTCCGGAAAACTCGCTGGCGTCCATCGATCATGC CTACGAGCAGGGCTACCGCAATCTGGAGCTGGACGTC GAAGTCA GTTCCGATGGCGTGCCGGTGTTGATGCACGATTTCAGCATCGGCCGCATGGCAGGCGACCCGCAGAACCG GTTGGTGTCGCAGGTGCCGTTTGCCGAGCTGCGTGAAATG CCGTTGGTGATTCGCAACCCGTCTGACGGAAACTACG TCAAGACCGACCAGACCATCGCCGGTGTGGAGCAGATGCT GGAGCACGTGCTCAAAAAGCCCGAGCCGATGTCGGTG GCGCTGGACTGCAAGGAAAACACCGGCGAAGCAGTGGCGATGCTGCTGATGC GCCGGCCGGACCTGCGCAAGGCTGC GGCGATCAAGGTCTATGCCAAGTACTACACGGGCGGCTTT GACCAATTCCTGTCCAATTTGTACAAGCACTACCAGA TCAACCCGTTGCACTCGCAGGATGCGCCGCGCCGCGCCGC GCTGGATCGCTTGCTGGCCAAGATCAACGTGGTGCCG GTCTTGAGCCAGGGCATGTTGAACGACGAGCGCTTGCGCG GCTTCTTTCGAAGCAATGAGCAGGGCGCCGCGGGGCT CGCAGACACCGCAATGCAGTGGCTGGACAGCTGGACCAAG ATGCGCCCGGTGATCGTGGAGGCGGTGGCCACCGACG ACAGCGATGCCGGCAAGGCCATGGAAATGGCTCGGACGCG GATGCGCCAGCCGGACTCGGCCTACGCGAAGGCCGCG TATTCGGTGAGCTACCGGTATGAGGACTTTTCCGTGCCGC GCGCCAATCACGACAAGGACTACTACGTTTACCGCAA CTTCGGTGAACTGCAAAAGCTCACC AACGAG Figure A 2. Nucleotide sequence of AvrGf11106::AvrBs262574 fused protein. The restriction sites and the start codon of each gene is underline.

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141 B gl II AGATCTGATGTCTTTGGCCAACACACTGCGCACTGGGGCGGACATA CCGCGCCAGACGCGCTCGT TTCGCTACA CGC AACCGCACTTCGG CTTGGCATCGATCAGCACAATCAGCAC GCACGATGATCGTCGAATAGCCGACCACGATGGGAGA GCCTCAGATGATTTCCTCAATCAACCCCTATATCAGTGCT AACCCACTCCTCAACCACACGCTTCAGACGAAGCAGC CCGCATTTTCCGACACGATCATT ATG CTGATGTCGGAATTGCTACA GCGGTTGATACAGACCCAATTCCAACCTCAA AACACGTCGCAATACGATTGGA ACCCTTCGCCTTCCCATGGGACGTCCAATGGCCAAGGGGAGGGCAACGAGCATGG CGGTCGCAGGAACGGACGCGGCGGTGACAACTTCCTCGCC ACCACGCCCGAGACGTCCGACCATTCCGACAAATCCG CTCACTCGACCGTCACCGGCAGCGGCCCCGTGGGGAGCGG TCGCACGATCAACGTCAACAGTCCGATCGTGGTTCAC B gl II AAGGGCGAGGTGTTCGACGGACACAACAATCTCTACGTCG GCAGATCTATG ACCGGCAAGCCGGCCCTGGT GGCGCT CGACAGCGAATTCTCCGAGCAGCGTCTGGCCGAAGTGCAG GCGCGCCAGATCACCGTGCAGACCCTGCAAGGCAAGC TTGCCACGCATCTTGCGCAGGCCGGCACGGCGCTCAAACCCGACAGCATTGCGGCACG CTTTGCTGCCGGCACACTG GAGCCGGTGTATCTGGATACCGCCGCCTTCAATGCCATGT CGCGCGGGCTGCCCGCACGCGCACGTGCGGCCGCAGG CCCGGTGCTGATCGATGCACAACAAGGTCGCATCATCTTC AATCTGCAGCGCGCGTTTGCGCCTGGCGACACCTTCA GCGACGCGGCGCTTGCCGCGCTTGGCAAGCAGTTGAATCT TTCCGGCCACGGGCTGGCAACGC CGAACTGGCTGCAG CCTGCCGCAGGCACGCCGGGGCGGCGCAAGCTGCAGCAAG CCGCGCGCTATCACGGCCACGAGGTGCCGGCCCGCGA CGGTGGCGCCGGGTTCTTCAAGGCCAACGACCATCGCCTG CTGGAAGGCAAGCAAGTGCTGTTGCGCAATCATCAAA AGTCGCTCGTGCACAACCACTACTTCGAAGCACCCAGCAC GCGTGCGTTCGGAAAGGACGTCATGGTGCATCGCGGG CTGTTCGATAATCACGCCGGCATTCCGGAAA ACTCGCTGGCGTCCATCGATCATGCCTACGAGCAGGGCTA CCGCAA TCTGGAGCTGGACGTCGAAGTCAGTTCCGATGGCGTGCCG GTGTTGATGCACGATTTCAGCATCGGCCGCATGGCAG GCGACCCGCAGAACCGGTTGGTGTCGCAGGTGCCGTTTGC CGAGCTGCGTGAAATGCCGTTGGTGATTCGCAACCCG TCTGACGGAAACTACGTCAAGACCGACCAGACCATCGCCGGTGTGGAGCAGATGCTGGAGCACGTGCTCAAAAAGCC CGAGCCGATGTCGGTGGCGCTGGACTGCAAGGAAAACACC GGCGAAGCAGTGGCGATGCTGCTGATGCGCCGGCCGG ACCTGCGCAAGGCTGCGGCGATCAAGGTCTATGCCAAGTA CTACACGGGCGGCTTTGACCAATTCCTGTCCAATTTG TACAAGCACTACCAGATCAACCCGTTGCACTCGCAGGATG CGCCGCGCCGCGCCGCGCTGG ATCGCTTGCTGGCCAA GATCAACGTGGTGCCGGTCTTGAGCCAGGGCATGTTGAAC GACGAGCGCTTGCGCGGCTTCTTTCGAAGCAATGAGC AGGGCGCCGCGGGGCTCGCAGACACCGCAATGCAGTGGCT GGACAGCTGGACCAAGATGCGCCCGGTGATCGTGGAG GCGGTGGCCACCGACGACAGCGATGCCGGCAAGGCCATGG AAATGGCTCGGACGCGGATGCGCCAGCCGGACTCGGC CTACGCGAAGGCCGCGTATTCGGTGAGCTACCGGTATGAGGACTTTTCC GTGCCGCGCGCCAATCACGACAAGGACT ACTACGTTTACCGCAACTTCGGTGAACTGCAAAAGCTCAC CAACGAG Figure A 3. Nucleotide sequence of HrpW1 109::AvrBs262574 fused protein. The restriction sites and the start codon of each gene is underlin e.

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163 BIOGRAP HICAL SKETCH Jos Francisco Lissoni Figueiredo was born in the city of Cosmorama, Brazil. During his undergraduate studies in biomedicine at the University of Baro de Mau, he started a trainee program in the sequencing genome project of the Xanthomonas c itri subsp. citri and Xanthomonas campestris pv. campestris. In 2001, he started the Master of Science program at University of State of So Paulo in the g enetics and p lant i mprovement program. After graduat ing with his m aster s he was invited by Dr. Frank F. White to work with Xanthomonas campestris pv. campestris in the Plant Pathology Department at Kansas State University. In 2004, he was admitted in the Plant Pathology Department Ph.D. program a t Kansas State University and transferred to the Plant Pat hology D epartment at University of Florida where he received his Ph.D. in 2009 conduct ing a research project on screen pathogenic factors involved in Xanthomonas citri subsp. citri disease progress