UC Davis

This dissertation contains the first comprehensive study using RNA-seq to reveal early responses (12, 24, and 72 hours post-inoculation (HPI)) to Sw-5b-mediated resistance against Tomato spotted wilt orthotospovirus (TSWV) infection. TSWV is a significant challenge to successful tomato production and is spread in nature by a relatively small number of thrips species, especially western flower thrips (WFT), Frankliniella occidentalis (Pergande). Investigations to identify TSWV resistance in tomato have been almost entirely conducted using mechanical inoculation with sap prepared from infected tissue. The Sw-5b gene encodes a nucleotide-binding leucine-rich repeat (NLR) protein, the most commonly used resistance gene against TSWV in commercial tomato. The mechanism by which the NLR recognizes the TSWV NSm elicitor has been elucidated; however, the downstream cascade of events leading to Sw-5b NLR-mediated resistance are not known, nor are differences in downstream defense signaling that could be associated with different methods of inoculation. In my dissertation, I investigated the early events in Sw-5b-mediated resistance, and the potential differences between thrips and mechanical inoculation, by examining the differential expression of genes at 12, 24, and 72 HPI with RNA-seq in the near isogenic lines, Solanum lycopersicum ‘Santa Clara’ (Sw-5b-, the TSWV-susceptible line), and S. lycopersicum ‘CNP-LAM 147’ (Sw-5b+, the TSWV- resistant line) following TSWV mechanical and thrips inoculation. I found that the Sw-5b NLR immune receptor induced earlier transcriptome responses to thrips inoculations (12 and 24 HPI), while transcriptome responses to mechanical inoculation were not detected until 72 HPI. Across the three-time points investigated, 80 and 95 differentially expressed genes (DEGs) were detected with thrips and mechanical inoculations, respectively, most of which were upregulated with both inoculation methods. The majority of DEGs differed depending on the inoculation method (66 uniquely expressed in mechanically inoculated treatments, and 49 uniquely expressed in thrips inoculated treatments). Only 26 DEGs were shared between the inoculation methods. Examination of the functional categories in which DEGs of interest were classified showed that while thrips and mechanical inoculation resulted in the detection of different DEGs, many DEGs of interest placed into similar functional categories, e.g., pathogenesis-related proteins and cytochrome P450s. At the same time, there were marked differences in the functional categories into which the detected DEGs were placed. Unlike mechanical inoculation, thrips inoculation resulted in DEGs in the NLR and receptor-like kinases and calmodulin-binding protein functional categories. These findings suggest that the inoculation method plays a role in the downstream signaling elicited by activation of Sw-5b. These data are the first to show that the TSWV inoculation method results in differences in Sw-5b-mediated immune signaling and that the Sw-5b NLR-immune receptor induces earlier transcriptome responses to thrips inoculations. Identification of transcriptomic responses at these early time points and with different inoculation methods provides new insight into the components of Sw-5b-mediated plant defense pathways. Also, in nature, TSWV is primarily spread by thrips; however, most resistance studies focus their attention on mechanical inoculation. My results support the use of thrips inoculations in studying Sw-5b resistance, and suggest that when mechanical inoculation is the sole method used in resistance investigations, genes most likely of greatest importance in nature may be missed. To examine the functional importance of some of the DEGs detected with RNA-seq, I established a preliminary functional analysis strategy using a Tobacco rattle virus based virus-induced gene silencing (VIGS) system, and a transgenic Sw-5b Nicotiana benthamiana (Nb::Sw-5b). I successfully used VIGS to silence Sw-5b and showed a loss of function resulting in a phenotype with no HR production and systemic infection. I established that VIGS could be used as a preliminary genomic tool to perform functional assessment of genes that may play an integral role in Sw-5b-mediated resistance against TSWV. Based on intensity of fold change, and functional classifications, 15 DEGs were selected for preliminary functional analysis using VIGS. While the outcome of these experiments was not entirely conclusive, results from TRV-based VIGS revealed two DEGs of interest; ERF1, and a receptor-like kinase family protein. The results suggest these two genes are possibly required for Sw-5b-mediated resistance against TSWV and are worthy of further investigation. The variability and difficulty in replicating results I encountered using the TRV-based VIGS system, suggests further investigations should consider other methods for screening DEGs of interest, such as CRISPR. Finally, I used TRV-based VIGS to explore the function of SGT1 in Sw-5b-mediated resistance by silencing of SGT1. In these experiments, I found that silencing of SGT1 resulted in loss of HR, and systemic infection of plants. These findings show that SGT1 is required for Sw-5b-mediated immune signaling. In contrast, silencing of EDS1 resulted in no effect on HR production or resistance to TSWV infection, indicating that like many CC-NLRs, Sw-5b does not require EDS1 for immune signaling.