Abram Smith, Satyanarayana Gouthu, Charles Dearing, and Laurent Deluc*
*Oregon State University, 2750 SW Campus Way , Corvallis, OR, 97331 (laurent.deluc@oregonstate.edu)

Grapevine red blotch virus (GRBV) poses a significant threat to winegrape production. Host factors critical for red blotch infection remain largely unknown in grapevines, impeding attempts to use CRISPR technology to generate red blotch-resistant grapevines. Our project seeks to identify these host factors. Our two main objectives are to identify the host factors using the yeast two-hybrid (Y2H) system and to validate their interaction with viral proteins in planta. For the Y2H experiment, we will conduct a comprehensive genome-wide screen using GRBV replication-associated proteins (C1, C2, C3, and C1-2) as baits against a comprehensive grapevine cDNA library from GRBV-infected plants as preys. Based on the strength of the interactions and their biological relevance from previous studies, we will select the top two most promising interactors for each bait (C1, C2, C3, C1+2) to conduct bimolecular fluorescence complementary assays from isolated grapevine protoplasts. All four baits have been successfully cloned into yeast, initiating the screening process. A prey library using coding-gene related RNA is currently being constructed. The next step will be to carry out mating of the bait and prey, as well as isolation of mated yeast. Further screenings on a series of selective media will identify high-confidence interactors, followed by Sanger sequencing to identify their biological function. To get ahead of the work on objective 2, we have optimized a protocol to isolate protoplasts from tissue cultured cells of microvines. If we reach our goals, we will lay the groundwork for further studies to produce CRISPR-engineered, GRBV-resistant grapevines.

Funding Support: California Department of Food and Agriculture