RNA interference (RNAi) has been extensively used in crop protection platforms. Currently most approaches are based on the conventional use of transgenic plants expressing doublestranded RNAs (dsRNAs) against selected targets (endogenous plant genes, virus, fungi, bacteria). However, the use of Genetically Modified Organisms (GMOs) has raised scientific and public concerns. Approaches alternative to the production of Genetically Engineered material that could enable the direct exogenous application of RNA molecules to trigger RNAi has the potential to address public and industry concerns with regards to RNAi field-based technologyInterim_Report_Laurent_Deluc_February 2021 2 (Dalakouras et al., 2020). There are several methods to deliver RNA in plants. Our project proposes to develop dsRNAs application tools to mitigate the negative effects of Red Blotch Virus on grape berry composition. As a first step of a long-term project, we propose to identify through a genome wide study the genomic regions of the Red Blotch Virus that are targeted by grapevine during the early stages of viral infection. Since the acceptance of the project in June 2020, the PI has recruited a graduate student from Italy who will be working on the project. Through cooperation, the PL Deluc has been issued by the USDA-APHIS services a permit to import two bitmer infectious clones containing two variant strains (NY175 and NY358) of the Red Blotch virus (USDA -APHIS Permit #: 20-176-103m) These infectious clones will be essential to study the early phases of grapevine GRBV infection through two main methodologies that are describe below.
Layperson summary* RNA interference (RNAi) is a conserved biological response across living organisms (animal or plant cells) initiated by the presence of double-stranded RNA molecules from various pathogens, including viruses. The RNA interference mechanism initiated in the plants will lead to a cascade of molecular events that are meant to repress the activity of the virus and its propagation within the plant. Once infected, the plants will recognize and produce specific nucleic regions of the viral genome to activate the RNA silencing machinery. These regions are named “hot spots” regions. The main goal of this project is to identify these “hot spots” for the Red Blotch Virus. In the long-term, this knowledge could help developing innovative technology tools like ectopic RNA molecules application in vineyards to mimic the presence of the virus and to make the plants immune or “primed” to further infections like a vaccine will do. This might limit the propagation of the Red Blotch Virus from plants to plants and could potentially mitigate its negative effect on grape berry ripening of already infected plants. The current project will use Next Generation Sequencing technologies to identify these “hot spots” during the early stages of Red Blotch Virus infection in tissue culture material. Once the nucleic regions identified, the continuum of the project will involve trials for systemic application, through spray, of these RNA molecules to either infected plants to mitigate the negative effects of Red Blotch on ripening, or to non-infected plants, to trigger their immune responses to viral infection. If confirmed in a greenhouse setting, the next step will be to assess the ds-RNA formulation in field-trails.