ABSTRACT BROWN, JESSICA ASHLEY. Genetic Changes in TSWV Associated with Virus Accumulation in and Transmission Efficiency of Thrips tabaci. (Under the direction of Drs. George Kennedy and Tim Sit). Plant viruses rely on insect vectors for transmission to new plant hosts, but many of the specifics of virus-vector interactions are not fully understood. Tomato spotted wilt virus (TSWV) is transmitted by Thrips tabaci, which has been shown to vary greatly in its ability to transmit different virus isolates. A previous study examining transmission of 89 distinct pairings between TSWV isolates and T. tabaci isolines showed a significant effect of virus isolate, thrips isoline, and their interaction on transmission efficiency. The ability of a single isoline to transmit multiple virus isolates varied up to 18-fold, and the transmissibility of each isolate by multiple T. tabaci isolines varied up to 45-fold. In addition, significantly higher transmission rates were observed among sympatric TSWV isolate-isoline pairings than allopatric pairings, suggesting local adaptation. This study uses a subset of the 89 TSWV isolate and T. tabaci pairings to identify determinants of transmission by first examining the relationships between virus titer in source leaves, titer in individual transmitting and non-transmitting thrips, and transmission efficiency. Quantitative real-time PCR results of the TSWV L RNA segment show that virus titer in individual thrips was unrelated to the virus titers in the source leaves from which they acquired virus, and was not a significant variable underlying differences in transmission efficiency among TSWV isolates. We further investigated these virus isolate-thrips isoline pairings by sequencing the entire TSWV genome using next-generation sequencing from T. tabaci isolines, the leaf discs they fed from, and post-feeding leaves from F. occidentalis, F. fusca, and T. tabaci. Phylogenetic analysis revealed panmixia occurring in a number of the viral genes but slight geographic structuring in the GnGc and RdRp. There was lower genetic diversity and recombination in virus from plants compared to virus from T. tabaci. The analysis of variants, deviation from neutral equilibrium, and population statistics showed evidence for mutation and purifying selection being strong factors in the evolution of TSWV isolates during the transmission cycle. To test polymorphisms found in the next-generation sequencing and their effect on transmission from T. tabaci, we attempted to develop a T7 RNA polymerase-based partial reverse genetics system for TSWV. This system involves the co-infiltration of Nicotiana benthamiana leaves with Agrobacterium tumefaciens strains containing plasmids encoding the T7 RNA polymerase, TSWV nucleocapsid, S RNA, and viral suppressors of RNA silencing as well as the S RNA encoding the green fluorescent protein in place of the nucleocapsid as a reporter gene. We successfully developed expression plasmids containing the nucleocapsid, S RNA, and S RNA encoding GFP. Even though there was no success in planta, the plasmids developed here are large steps for future research to continue attempting a partial reverse genetics system for TSWV and identifying determinants of transmission. © Copyright 2019 by Jessica Ashley Brown All Rights Reserved Investigating the Genetic Changes in TSWV Associated with Virus Accumulation in and Transmission Efficiency of Thrips tabaci. by Jessica Ashley Brown A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Plant Pathology Raleigh, North Carolina 2019 APPROVED BY: _______________________________ _______________________________ George Kennedy Tim Sit Committee Co-Chair Committee Co-Chair _______________________________ _______________________________ Alana Jacobson Steven Lommel _______________________________ Ignazio Carbone BIOGRAPHY Jessica was born in Raleigh, North Carolina on March 1, 1990. She went to Needham B. Broughton high school, where she first became interested in chemistry during her junior year. She attended Meredith College for her undergraduate degree and switched from chemistry to biology sophomore year. That year was also when she first experienced research, studying the effects of secondary metabolites present in sewage water on fresh water organisms. After that she also isolated a putative protease inhibitor from a Kenyan plant that could potentially be developed as a treatment for HIV/AIDs as another undergraduate research project. Senior year she interned at The Hamner Institutes in the drug safety sciences institute, helping associate rare adverse drug reactions with genetic variants in humans. Her love of research, especially in molecular and microbiology led her to plant pathology under the guidance of Tim Sit and George Kennedy studying vector-virus interactions. ii ACKNOWLEDGMENTS I would like to thank my committee members for guiding me throughout all these years I especially want to thank Tim Sit for his constant support, snacks, and most importantly, coffee. I am glad George Kennedy and Alana Jacobson allowed me to be a part of this research. I would like to thank the USDA for funding my research. I would like to thank Thomas Chappelle for helping me with statistics. I also want to thank everyone that has given me advice for methods and analysis including David Rasmussen, Dorith Rotenberg, Andy Baltzegar, and many others. I am grateful for the friendships and support system I had with other graduate students in plant pathology especially, Alyssa Koehler, Katie Neufeld and my lab mate Casey Ruark. I wouldn’t be here if it weren’t for my mom and my brother supporting my career decisions even though they didn’t understand it (and still don’t). Most importantly I want to thank Scott Linak for being the most supportive, patient, and loving person in the world. iii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ..................................................................................................................... viii Chapter 1: Literature Review ......................................................................................................1 Plant-Virus Transmission .............................................................................................................1 Orthotospoviruses ........................................................................................................................3 TSWV Genome Organization ......................................................................................................5 Thrips ............................................................................................................................................6 Thrips tabaci .................................................................................................................................7 Viral Replication ..........................................................................................................................9 Determinants of Transmission ....................................................................................................11 TSWV Genetic Diversity ............................................................................................................12 Summary .....................................................................................................................................13 Literature Cited ...........................................................................................................................15 Chapter 2: Transmission of a persistently-propagative plant virus by its insect vector is not solely influenced by virus titer ............................................................................................34 Abstract .......................................................................................................................................35 Introduction ................................................................................................................................36 Materials and Methods ................................................................................................................38 Results ........................................................................................................................................44 Discussion ...................................................................................................................................47 Acknowledgements .....................................................................................................................51 Literature Cited ...........................................................................................................................52 Chapter 3: Population genetic analysis of Tomato spotted wilt virus within its vector T. tabaci and plant host E. sonchifolia ............................................................................................69 Abstract .......................................................................................................................................70 Introduction ................................................................................................................................71 Materials and Methods ...............................................................................................................74