CHARACTERIZATION OF THE LETHAL HOST-PATHOGEN INTERACTION BETWEEN TOBACCO MILD GREEN MOSAIC VIRUS AND TROPICAL SODA APPLE By JONATHAN ROBERT HORRELL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007 1 © 2007 Jonathan R. Horrell 2 To my Father, who has always inspired the best in me 3 ACKNOWLEDGMENTS I thank all the people in the Plant Pathology Department at the University of Florida for giving me the chance to further my education. I thank Dr. Francis Zettler, without whose encouragement I would have never endeavored to undertake graduate studies. I especially thank my advisor, Dr. Raghavan Charudattan, for his positive outlook, infectious enthusiasm, and support. Thanks, also, to the other members of my graduate advisory committee: Dr. Jane Polston, Dr. Ernest Hiebert, and Dr. Dennis Lewandowski. I additionally thank: Mark Elliott and Jim DeValerio for their guidance; Kris Beckham and Rob England for their invaluable practical expertise and guidance; Dr. Carlye Baker, Dr. Jeff Jones, Dr. Harry J. Klee, Dr. Wen- Yuan Song, and Dr. Jeff Rollins, for technical expertise and advice; Eldon Philman and Herman Brown for greenhouse maintenance, insect control, and general camaraderie; and Gene Crawford for always having a good story to tell. Furthermore, I express gratitude and thanks to Dr. Deborah M. Mathews (U.C., Riverside) and Dr. Lynn Bohs (University of Utah, Salt Lake City) for technical and material assistance. Finally, I give my sincerest thanks to the taxpayers of the State of Florida, without whose contribution my graduate school education would not have been possible. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 LIST OF ABBREVIATIONS........................................................................................................11 ABSTRACT...................................................................................................................................17 CHAPTER 1 LITERATURE REVIEW .......................................................................................................19 Host Biology...........................................................................................................................19 Pathogen Biology....................................................................................................................24 Host-Pathogen Interaction ......................................................................................................38 Cases of virus-induced lethal systemic hypersensitive response (LSHR).......................42 Cases of gene-for-gene interactions between tobamoviruses and Solanaceae................45 2 IDENTIFICATION OF COLUMNEA ISOLATE PV-0113 .................................................55 Introduction.............................................................................................................................55 Materials and Methods ...........................................................................................................59 Results.....................................................................................................................................64 Discussion...............................................................................................................................66 3 COLUMNEA ISOLATE PV-0113: SUBTYPE, SATELLITE, AND DISEASE PROGRESS ............................................................................................................................76 Introduction.............................................................................................................................76 Materials and Methods ...........................................................................................................78 Results.....................................................................................................................................81 Discussion...............................................................................................................................84 4 DISEASE PHENOTYPE AND COMPARISON OF PV-0113 WITH OTHER CULTURES............................................................................................................................93 Introduction.............................................................................................................................93 Materials and Methods ...........................................................................................................95 Results.....................................................................................................................................98 Discussion.............................................................................................................................101 5 5 CROSS-PROTECTION EFFECTS......................................................................................110 Introduction...........................................................................................................................110 Materials and Methods .........................................................................................................111 Results...................................................................................................................................113 Discussion.............................................................................................................................113 6 TMGMV PV-0113: PURIFICATION, LYOPHILIZATION, AND RNA EXTRACTION.....................................................................................................................116 Introduction...........................................................................................................................116 Materials and Methods .........................................................................................................117 Results...................................................................................................................................120 Discussion.............................................................................................................................121 7 DILUTION ENDPOINT AND BIOLOGICAL ACTIVITY OF PV-0113..........................123 Introduction...........................................................................................................................123 Materials and Methods .........................................................................................................127 Results...................................................................................................................................127 Discussion.............................................................................................................................128 8 CLONING OF PV-0113 COAT AND MOVEMENT PROTEIN GENES .........................138 Introduction...........................................................................................................................138 Materials and Methods .........................................................................................................141 Results...................................................................................................................................147 Discussion.............................................................................................................................148 9 TRANSIENT EXPRESSION OF PV-0113 COAT PROTEIN............................................158 Introduction...........................................................................................................................158 Materials and Methods .........................................................................................................161 Results...................................................................................................................................169 Discussion.............................................................................................................................172 10 GENERAL DISCUSSION ...................................................................................................189 11 SUMMARY AND CONCLUSIONS...................................................................................196 LIST OF REFERENCES.............................................................................................................198 BIOGRAPHICAL SKETCH .......................................................................................................214 6 LIST OF TABLES Table page 1-1 Strains/isolates of Tobacco mild green mosaic virus (TMGMV)......................................51 1-2 Size of TMGMV genes and gene products........................................................................52 1-3 Relationship between genotype and disease phenotype in Capsicum sp. peppers ............52 2-1 Species-specific primers used in reverse transcriptase polymerase chain reaction ...........68 2-2 Experimental host range and symptoms