Christian Mandelli* and Laurent Deluc
*Oregon State University, 2750 SW Campus Way, Corvallis, OR, 97331 
(mandellc@oregonstate.edu)

The lack of targeted approaches to control grapevine red blotch virus (GRBV) through vector management presents a significant challenge to the wine industry. Costly vine removal represents the only effective option, underscoring the urgent need for targeted alternatives. RNA interference (RNAi) is a conserved defense mechanism in plants, triggered by the virus’ presence during early infection stages. Currently, the RNAi-related molecular interactions between GRBV and grapevine are poorly documented. Establishing a solid understanding of the GRBV-host arms race is essential to develop effective control strategies that enhance the RNAi response. Our research aims to investigate these interactions, focusing on the plant’s RNAi response during early GRBV infection stages. After generating a population of GRBV-infected microvine plants through Agrobacterium-mediated infiltration, we observed a peak of viral replicase activity one week postinfection. Subsequent small RNA sequencing revealed the presence of 21, 22, and 24-nucleotide virus-derived small-interfering RNAs (vsiRNAs), indicating RNAi gene silencing activity. Using a custom bioinformatics pipeline, we identified nine GRBV genomic regions, or hotspots, targeted by the plant’s RNAi. Additionally, GRBV-targeted bisulfite sequencing revealed hypermethylation within these hotspots, peaking at 24 days postinfection (dpi), underlining a potential correlation between small RNA production and the methylation of the viral genome. Based on these findings, we evaluated hotspots-derived double-stranded RNAs (dsRNAs) to silence GRBV activity in infected tissue-cultured plants. Our preliminary data suggest a significant reduction of the viral replicase and V3 transcripts abundance at 12 and 24 dpi. Our results demonstrate the efficacy of enhancing RNAi through dsRNA application for GRBV silencing. Evaluation of other target genes is underway. To the best of our knowledge, this is the first report experimentally validating vsiRNAs produced during early GRBV-infection. Our findings underscore the potential of RNAi-based approaches, emphasizing the importance of foundational knowledge in optimizing such strategies for sustainable vineyard practices.

Funding Support: Oregon Wine Board