The research in this thesis addressed aspects of the tomato-begomovirus interaction: molecular analyses of resistance and susceptible determinants, invasion biology in Costa Rica and the role of weeds as reservoirs. In Chapter II, I used an RNA-Seq approach to identify differentially expressed genes (DEGs) during infection by tomato yellow leaf curl virus (TYLCV) of the near isogenic lines LA3473-R and LA3474-S, which have the Ty-1 (resistant) and the ty-1 (susceptible) alleles, respectively. Differences in gene expression in the resistance and susceptible responses were observed. VIGS analyses of selected DEGs further revealed that genes encoding WRKY transcription factors, nucleotide-binding site and leucine-rich repeat (NBS-LRR) containing proteins, and receptor-like protein kinases (RLKs) may play a role in the resistance and susceptible responses to TYLCV infection. Sequence analyses of the cloned Ty-1 gene provided further insight into how some of the newly identified DEGs participate in the Ty-1 defense response. In Chapter III, I examined the invasion biology of three emergent/introduced begomoviruses in Costa Rica to assess their interactions in terms of disease severity and viral accumulation and genetics. Here, I first completed the molecular and biological characterization of tomato yellow mottle virus (ToYMoV) using full-length infectious DNA-A and DNA-B clones to fulfill Koch’s postulates for the ToYMoD and to show that the virus infects a subset of solanaceous species. Full-length infectious clones of an isolate of tomato leaf curl Sinaloa virus (ToLCSiV) from Costa Rica also were generated, and were used to assess genetic interaction (pseudorecombination) with ToYMoV. The infectious clones of ToYMoV, ToLCSiV and TYLCV were then used to inoculate tomato plants with each virus and all combinations, and symptoms induced and viral DNA accumulation were determined. Tomato plants infected with combinations of two or all three viruses developed more severe symptoms than plants inoculated with each virus alone. Symptoms of ToLCSiV and ToYMoV appeared earlier (~7 d post-inoculation [dpi]) than those of TYLCV (~10 dpi). However, by 14 dpi and beyond, TYLCV symptoms had become dominant in all mixed infections. Virus accumulation was determined by qPCR and revealed that, although symptoms of TYCLV became predominant in mixed infections with the NW bipartite begomoviruses, levels of ToYMoV and ToLCSiV were similar to those in plants infected with the individual viruses. ToYMoV and ToLCSiV DNA levels were negatively impacted early in infection (7 dpi), but later in infection (14 and 21 dpi) a neutral synergistic interaction was observed. These results are discussed in terms of begomovirus invasion biology and implications for begomovirus disease development and management in Costa Rica. In Chapter IV, I describe the molecular and biological properties of two bipartite begomoviruses associated with striking golden/yellow mosaic symptoms in Malachra sp. and Abutilon sp. plants on Hispaniola, and assess their potential to cause disease in crop plants. Sequence and phylogenetic analyses, together with infectivity studies with infectious clones, established that the symptoms in Malachra sp. were caused by the crop-infecting bipartite begomovirus tobacco leaf curl Cuba virus (TbLCuCV), whereas those in Abutilon sp. were caused by a new species of weed-infecting begomovirus, for which the name Abutilon golden yellow mosaic virus (AbGYMV) is proposed. Host range experiments showed that TbLCuCV also induced moderate to severe disease symptoms in Nicotiana benthamiana, tobacco (N. tabacum) and common bean plants (Phaseolus vulgaris) plants. In contrast, AbGYMV induced mild or no symptoms in these plants, indicating a high degree of adaptation to Abutilon sp. in the Dominican Republic, and low potential to cause crop diseases. Pseudorecombinants generated with the infectious clones of these viruses were highly infectious and induced severe symptoms in N. benthamiana and Malachra sp. Furthermore, both viruses co-infected Malachra sp., thereby increasing the probability of virus evolution via recombination and pseudorecombination. Together, these results suggest that TbLCuCV primarily infects Malachra sp. in the Caribbean Basin, and occasionally spills over to infect and cause disease in crops other than tomato, whereas AbGYMV is well-adapted to Abutilon sp. and has not been reported infecting crops.

Dissertation abstract In this dissertation I investigated aspects of two plant virus species, tomato necrotic dwarf virus (ToNDV) and Squash vein yellowing virus (SqVYV), which have emerged in different ways in California. In Chapter Two, I further characterized a new isolate of ToNDV (genus Torradovirus) associated with a necrosis disease outbreak in Kern County, CA in 2015. This involved obtaining a pure isolate (ToNDV-K15) and generating full-length infectious RNA 1 and RNA 2 clones. Using these clones, I determined the complete genome sequence of this isolate and used agroinoculation to fulfill Koch’s postulates for this disease and show that the host range of the virus was limited to solanaceous species, and that virus derived from the infectious clones was whitefly-transmissible. Finally, pairwise comparisons and phylogenetic analyses seemed to support species status for ToNDV. The infectious clones allowed me to investigate the role and function of movement-associated genes/proteins encoded by RNA 2. In Chapter Two, I used mutational analyses to investigate the role of ORF1 and MP in infection and identify important motifs in involved in cell-to-cell movement. Modeling was then used to infer how selected mutations may have impaired structure and function. Subcellular localization with GFP-tagged fusion proteins showed that ORF1 and MP were targeted to the cell membrane and plasmodesmata (PD); however, the interaction of ORF1GFP was transient and involved a network and motile vesicles, whereas MP accumulated in PD and co-localized with the TMV-MP PD marker. ORF1 binds, consistent with the hypothesis the vesicles are involved in trasport. Interestingly, phylogenetic analyses of the MP revealed evidence of recombination and an unexpected relationship with MPs of polerovirus (family Sobemoviridae). Together, these results provide further insight into the function these RNA-2 encoded proteins in torradovirus movement and evidence for a two-MP type strategy. In Chapter Three, I report the completion and analysis of the complete genome sequence of an isolate of SqVYV involved in a yellowing disease outbreak in cucurbits in Imperial Valley in 2014. The genome of this isolate (SqVYV-CA) has the typical organization of the SqVYV-type ipomoviruses, including lack of a HC-Pro gene/protein and a P1a and P1b genes/proteins at the 5’ end. Notably, my research revealed a recombination event in the P1a gene of SqVYV-CA, involving a portion of the P1 gene of an uncharacterized potyvirus and is predicted to generate a hybrid P1/P1a protein. Finally, using the divergent P1 sequence in the hybrid P1/P1a gene of SqVYV-CA, I developed a primer pair that allows for the specific RT-PCR detection of SqVYV-CA. I validated the specificity of the test, i.e., it did not detect SqVYV-FL or amplify non-specific fragments from the negative control. In 2021, this SqVYV-CA RT-PCR test allowed for the detection of the virus two of the four samples with yellowing symptoms from Fresno Co. This is the first report of SqVYV in the Fresno area and is a potential threat to cucurbit production, especially watermelon.

Genes involved in a viral resistance response in common bean (Phaseolus vulgaris cv. Othello) were identified by inoculating a geminivirus reporter (Bean dwarf mosaic virus expressing the green fluorescent protein), extracting RNA from tissue undergoing the defense response, and amplifying sequences with degenerate R gene primers. One such gene (a TIR-NBS-LRR gene, RT4-4) was selected for functional analysis in which transgenic Nicotiana benthamiana were generated and screened for resistance to a range of viruses. This analysis revealed that RT4-4 did not confer resistance to the reporter geminivirus; however, it did activate a resistance-related response (systemic necrosis) to seven strains of Cucumber mosaic virus (CMV) from pepper or tomato, but not to a CMV strain from common bean. Of these eight CMV strains, only the strain from common bean systemically infected common bean cv. Othello. Additional evidence that RT4-4 is a CMV R gene came from the detection of resistance response markers in CMV-challenged leaves of RT4-4 transgenic plants, and the identification of the CMV 2a gene product as the elicitor of the necrosis response. These findings indicate that RT4-4 functions across two plant families and is up-regulated in a non-virus-specific manner. This experimental approach holds promise for providing insights into the mechanisms by which plants activate resistance responses against pathogens.