Structure-function studies on three virion ORFs of grapevine red blotch virus to elucidate disease etiology




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Grapevine red blotch virus (GRBV) is a monopartite single-stranded DNA Grablovirus belonging to the family Geminiviridae. It has been recognized as an emerging serious threat to the viticulture industries in the United States. Previous studies have demonstrated that the GRBV is transmissible in the greenhouse by arthropod sap-sucking vectors to cause Grapevine red blotch disease (GRBD). Host RNA silencing involving small interfering RNAs (siRNAs) has been characterized as a major defense mechanism against microbial pathogens and viruses in plants. Based on the known examples from other members of geminiviruses, we propose there is one or more RNA interference silencing suppressor proteins encoded in the GRBV genome (like other geminiviruses) to counter the host innate immunity mediated by RNA silencing. We hypothesize the GRBV silencing suppressor proteins interfere with a deeply conserved (in dicots) autoregulatory loop involving microRNA828/TAS4/MYBA5/6/7 that are effectors of anthocyanin biosynthesis, resulting in mis-regulation of miR828/TAS4 and hypothesized concordant effects on MYBA5/6/7 transcription factors targeted by TAS4 tasiRNA 3’D4(-) and potentially nine other MYB mRNAs that are direct targets of miR828 in grape. Increased levels of anthocyanin observed in GRBV-infected grapevine is hypothesized to be the symptom consequence of GRBV silencing suppressor proteins affecting the miR828/TAS4/MYBA5/6/7 autoregulatory loop. Anthocyanin accumulation that is the hallmark symptom may potentially serve as visual/olfactory cues for preferential feeding on infected vines by arthropod vectors, consistent with epidemiological evidence for rapid spread. The interactions between candidate GRBV silencing suppressors and the host microRNAs (miRNAs), trans-acting small interfering (tasi-) RNAs, phased-tasi-RNAs (phasi-RNAs) and host target mRNAs are the subject of this study. There are six GRBV ORF genes mapped to virion and complementary strands of the virus genome, but the gene functions are yet to be elucidated. I have characterized and tested the suppressor functions of the three ORFs from the virion strand: V1, V2, V3. I have identified V2 as a candidate silencing suppressor gene. I have optimized the conditions of V2 protein expression/purification in E. coli for future production of antibodies and characterization of V2 grapevine targets by in vitro and in vivo experiments. I have completed the sRNA profiling of GRBV infected plants, revealing other miRNA-mediated pathways which are also deranged more than hypothesized miR828/TAS4/MYBA5/6/7. A V2 hairpin binary vector was constructed and is being used to generate transgenic materials in tobacco and grapevine rootstock to test by GRBV challenge in the greenhouse whether hairpin silencing sequences directed to V2 can serve as a means to engineer GRBV-resistant transgenic grapevine.

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Grapevine Red Blotch Virus, Grapevine Red Blotch Disease, Silencing Suppressor Proteins, Anthocyanin, Small Interfering RNAs, MicroRNA828/TAS4/MYBA5/6/7 Autoregulatory Loop