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Analysis of Variations in Susceptibility and Transcriptome Responses in Tomato to Two-Spotted Spider Mite Feeding

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Analysis of Variations in Susceptibility and Transcriptome Responses in Tomato to Two-Spotted Spider Mite Feeding Western University Scholarship@Western Digitized Theses Digitized Special Collections 2011 ANALYSIS OF VARIATIONS IN SUSCEPTIBILITY AND TRANSCRIPTOME RESPONSES IN TOMATO TO TWO-SPOTTED SPIDER MITE FEEDING Ingrid Fung Follow this and additional works at: https://ir.lib.uwo.ca/digitizedtheses Recommended Citation Fung, Ingrid, "ANALYSIS OF VARIATIONS IN SUSCEPTIBILITY AND TRANSCRIPTOME RESPONSES IN TOMATO TO TWO-SPOTTED SPIDER MITE FEEDING" (2011). Digitized Theses. 3258. https://ir.lib.uwo.ca/digitizedtheses/3258 This Thesis is brought to you for free and open access by the Digitized Special Collections at Scholarship@Western. It has been accepted for inclusion in Digitized Theses by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. ANALYSIS OF VARIATIONS IN SUSCEPTIBILITY AND TRANSCRIPTOME RESPONSES IN TOMATO TO TWO-SPOTTED SPIDER MITE FEEDING SPINE TITLE Analysis of Tomato-Spider mite Interactions THESIS FORMAT Monograph BY Ingrid Fung Graduate Program in Biology A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Ingrid Fung 2011 THE UNIVERSITY OF WESTERN ONTARIO SCHOOL OF GRADUATE AND POSTDOCTORAL STUDIES CERTIFICATE OF EXAMINATION Supervisor Examiners Dr. Vojislava Grbic Dr. Mark Gijzen Supervisory Committee Dr. Denis Maxwell Dr. Susanne Kohalmi Dr. Shiva Singh Dr. Priti Krishna The thesis by Ingrid Fung entitled: Analysis of Variation in Susceptibility and Transcriptome Responses of Tomato to Two-Spotted Spider Mite Feeding is accepted in partial fulfilment of the requirements for the degree of Master of Science Date Chair of the Thesis Examination Board ABSTRACT The two-spotted spider mite (Tetranychus urticae Koch) is a pest of tomatoes (Solarium lycopersicum) worldwide. The aim of this study was to generate data for comparison of spider mite resistant and susceptible tomato cultivar transcriptiome responses to mite feeding. Cultivars were screened for mite resistance using a platform developed and presented in this thesis. MicroTom (a brassinosteroid biosynthesis mutant) was found to be resistant to mite feeding, while MicroTom-D (a near isogenic line with increased brassinosteroid biosynthesis) was susceptible indicating that brassinosteroids may have a role in defences against spider miteTbeding. Jasmonic acid levels differed in these two lines endogenously and in response to spider mite feeding. Preliminary analysis of transcriptional response data indicates that MicroTom has a more dynamic early response to spider mite feeding than MicroTom-D. In addition, the basis of MicroTom resistance may involve changes in polyamine metabolism, and up regulation of cystiene protease inhibitors. Keywords: Solarium lycopersicum, tomato, Tetranychus urticae, two-spotted spider mite, cultivars, brassinosteroid, plant defence, transcriptome, MicroTom, herbivory ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Vojislava Grbic for giving me the opportunity to work in her laboratory, for her guidance throughout my studies, and for the resources she provided at every stage of my thesis. I am very grateful to have had the support of my advisory committee Dr. Priti Krishna and Dr. Susanne Kohalmi. The advice and input of Dr. Priti Krishna was always invaluable. I thank her for taking the time to give me guidance. My deepest gratitude goes to Dr. Susanne Kohalmi for always being so generous with her time, for editing my thesis, and for the countless other ways in which she supported me during my graduate studies. Without her this thesis would not have been possible. - — To the members of the Grbic lab thank you for all of the ways in which you have helped me complete my thesis. Specifically I would like to thank Leon Kurepin for conducting the hormone measurements on my Tomato samples. Special thanks go to Vladimir Zhurov and Stephane Rombauts without whom I would be lost in a sea of microarray data. Thank you both for working around the clock to ensure that I would have data for my thesis. Lastly I would like to send my gratitude to my mother Elaine and my boyfriend Julian who expect the world of me, support me regardless of my endeavors, and strive to give me the moon. I will continue to try and make you proud. IV TABLE OF CONTENTS CERTIFICATE OF APPROVAL..................................................................................II ABSTRACT.....................................................................................................................Ill ACKNOWLEDGEMENTS...............................................................................................IV LIST OF FIGURES..........................................................................................................VII LIST OF TABLES..........................................................................................................VIII LIST OF APPENDICIES...................................................................................................IX LIST OF ABBREVATIONS............................................................................................... X CHAPTER ONE - INTRODUCTION........................................................................... 1 1.2 Plant Defense Signaling......................................................................................... 1 1.3 Calcium and ROS Signaling..................................................................................2 1.4 Phytohormones.............................................................. 3 1.5 Tetranychus urticae...............................................................................................7 1.6 Solanum lycopersicum......................................................................................... 11 1.6 MicroTom.............................................................................................................14 1.7 Improvement of Tomato Crops............................................................................ 15 1.8 Tomato-Spider Mite Interactions......................................................................... 16 1.9 Goals and Objectives........................................................................................... 18 CHAPTER TWO - MATERIALS AND METHODS................................................. 19 2.1 Plant Material....................................................................................................... 19 2.2 Planting and Growth Conditions.......................................................................... 19 2.3 Herbivore Rearing................................................................................................ 19 2.4 Infestation Protocol..............................................................................................20 2.5 Time Course and Dosage Experiments................................................................23 2.6 Mite Distribution..................................................................................................23 2.7 Seaming Protocol................................................................................................24 2.8 Damage Quantification........................................................................................25 2.9 Cultivar Screen....................................................................................................26 V 2.10 Trichome Distribution Assay.............................................................................26 2.11 Mite Feeding Experiments.................................................................................28 2.12 Measurement of Hormone Concentrations........................................................28 2.13 RNA Isolation.................................................................................................... 31 2.14 Microarray Analysis.......................................................................................... 32 CHAPTER THREE - RESULTS.................................................................................. 36 3.2 Feeding Period and Number of Mites.................................................................. 36 3.3 Mite Distribution.................................................................................................. 39 3.4 Damage Quantification........................................................................................40 3.5 Screening Platform Protocol................................................................................43 3.6 Tomato Cultivar Screen.......................................................................................46 3.7 MicroTom Near-Isogenic Line Screen................................................................ 49 3.8 Analysis of Hormone Concentrations..................................... 54 3.9 Trichome Analysis............................................................................................... 57 3.10 Transcriptome Responses.................................................................................. 60 3.11 Expression of Jasmonic Acid Regulated Genes................................................60 3.12 Gene Ontology Analysis.................................................................................... 67 CHAPTER FOUR - DISCUSSION.............................................................................. 90 4.2 Preliminary Analysis of Transcriptome Responses of MT and MT-D Plants to
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