GENOMICS AND EFFECTOROMICS OF XANTHOMONADS By NEHA POTNIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1 © 2011 Neha Potnis 2 To my husband, Deepak, and my parents for their unconditional love and support 3 ACKNOWLEDGMENTS I would like to express my gratitude to Dr. Jeffrey B. Jones, my committee chair for his constant support and encouragement. I am thankful to him for sharing his expertise and his ideas at every step during this project. I would also like to thank my co-chair, Dr. David Norman for his guidance and financial support during my graduate studies. I would also like to extend my gratitude to my committee members, Dr. Boris Vinatzer, Dr. Jim Preston, and Dr. Jeffrey Rollins for their valuable suggestions in my project and support. I really appreciate valuable guidance from Dr. Robert Stall. I would like to thank Jerry Minsavage for technical help during the experiments, helpful suggestions and constructive criticism. Virginia Chow contributed to the identification of genes encoding glycohydrolases involved in cell wall deconstruction and their respective genome organizations. During research work, I collaborated with Dr. Frank White, Dr. Ralf Koebnik, Dr. Brian Staskawicz, and Dr. Joao Setubal to write research articles and reviews. I would like to thank them all for giving me the opportunity. I thank my labmates Jose Figueiredo, Franklin Behlau, Jason Hong, Mine Hantal, and Hu Yang for co-operation and assistance and for making the lab, a pleasant place, to work. I would also like to thank faculty and staff of the Plant Pathology department. I am grateful to my Indian friends here in Gainesville for their support and lively company during my stay here. I warmly thank my loving husband, Deepak, who has been supportive throughout my PhD, with all his love and encouragement. My heartfelt thanks go to my parents for supporting my decision to fly here away from them, who have been so caring and loving. They helped me to shape my career and always guided me at every step in my life. Thank you all for making this possible. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 XANTHOMONAS-PLANT INTERACTIONS AND GENOMICS .............................. 14 Background ............................................................................................................. 14 Type III Secreted Effectors and Their Role in Plant-Pathogen Interactions ............ 14 Avirulence Genes ................................................................................................... 17 Contributions of Comparative Genomics Era .......................................................... 19 Project Goal and Objectives ................................................................................... 20 2 COMPARATIVE GENOMICS REVEALS DIVERSITY AMONG XANTHOMONADS INFECTING TOMATO AND PEPPER ..................................... 21 Background ............................................................................................................. 21 Materials and Methods ............................................................................................ 24 Genome Sequencing ........................................................................................ 24 Assembly and Annotation ................................................................................. 25 Whole Genome Comparisons .......................................................................... 25 Phylogenetic Analysis ...................................................................................... 26 Phylogeny Reconstruction ................................................................................ 26 Prediction of Effector Repertoires, Cloning of Candidate Effectors and Confirmation Using AvrBs2 Reporter Gene Assay ........................................ 27 Cloning of Pepper Specificity Genes in Xp. ...................................................... 28 Results .................................................................................................................... 28 Draft Genome Sequences of Xv Strain 1111, Xp Strain 91-118 and Xg Strain 101 were Obtained by Combining Roche-454 (Pyrosequencing) and Illumina GA2 (Solexa) Sequencing Data. ............................................... 28 Relationships of the Strains to Other Xanthomonads using Whole Genome Comparisons ................................................................................................. 29 Four Xanthomonads Show Variation in the Organization of the Type III Secretion Gene Clusters ............................................................................... 30 A Reporter Gene Assay Confirms Translocation of Novel Type III Effectors .... 30 Core Effectors among Four Xanthomonads Give Insights into Infection Strategies of the Pathogen ............................................................................ 31 5 Effectors Unique to Xp Might be Responsible for Restricting Growth on Pepper........................................................................................................... 32 Species-Specific Effectors ................................................................................ 33 Few Effectors are Shared among Phylogenetically Related Group Strains ...... 35 Xg Shows Evidence of Effector Acquisition by Horizontal Gene Transfer. ....... 35 All Four Xanthomonads Contain Ax21 Coding Gene but Only Xcv Contains a Functional Sulfation Gene. ......................................................................... 36 Two Type II Secretion Systems are Conserved in All Four Xanthomonas Genomes. ...................................................................................................... 37 Xanthomonads Possess Diverse Repertoires of Cell-Wall Degrading Enzymes, which are Present in Diverse Genomic Arrangement Patterns. .... 38 Genes Involved in Several Type IV Secretion Systems are Present in Genomes and Plasmids ................................................................................ 40 Type V Secreted Adhesins Function in Synergism During Pathogenesis ........ 41 Type VI Secretion System is Present in Xcv, Xv and Xp .................................. 42 LPS Locus Displays Remarkable Variation In Sequence and Number of Coding Genes and Shows Host Specific Variation ........................................ 43 Analysis of DSF Cell-Cell Signaling System ..................................................... 44 Cyclic Di-GMP Signaling .................................................................................. 45 Copper Resistance (cop) Genes are Present in Xv and Copper Homeostasis (coh) Genes are Present in All Strains .................................... 46 Genes Unique to Xp as Compared to Pepper Pathogens Give Clues to its Predominance over Xcv in the Field and Host Specificity ............................. 47 Pepper Pathogenicity/Aggressiveness Factors Increased In Planta Growth of Xp .............................................................................................................. 48 Genes Specific to Xg as Compared to Other Tomato/Pepper Pathogens may Explain its Aggressive Nature on Tomato and Pepper .......................... 48 Genes Common to All Tomato Pathogens but Absent from Other Sequenced Xanthomonads ........................................................................... 49 The Evolution of Pathogenicity Clusters Corresponds to the MLST-Based Phylogeny ..................................................................................................... 50 Concluding Remarks .............................................................................................. 50 3 AVIRULENCE PROTEINS AVRBS7 FROM XANTHOMONAS GARDNERI AND AVRBS1.1 FROM XANTHOMONAS EUVESICATORIA ELICIT HYPERSENSITIVE RESISTANCE RESPONSE IN PEPPER ................................ 78 Background ............................................................................................................. 78 Materials and Methods ............................................................................................ 79 Plant Material and Plant Inoculations ............................................................... 79 Bacterial Strains, Plasmids and Media ............................................................. 80 Library Preparation and Isolation of Clone with Avirulence Activity .................. 80 Deletion Mutant Construction ........................................................................... 81 Bacterial Population Dynamics in Infiltrated Leaf Tissue .................................. 81 Determination of Electrolyte Leakage from Infiltrated Leaf Tissue ................... 82 Site Directed Mutagenesis of avrBs7 ............................................................... 82 Sequence Analysis and Protein Homology Modeling ....................................... 83 6 Results ...................................................................................................................