UC Davis

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.