Britt Eubanks,* Bahiya Zahl, Chance Lemon, Madesyn Samples, Satyanarayana Gouthu, and Laurent Deluc
*Oregon State University, 2750 SW Campus Way, Corvallis, OR 97331 (eubankbr@oregonstate.edu)

The emergence of fungicide resistance for grape powdery mildew (GPM; caused by Erysiphe necator) suggests the need to develop new strategies for GPM control. Recent research demonstrates that agricultural pests such as insects, nematodes, and fungi can be controlled through exogenous application of RNA molecules to trigger RNA interference (RNAi). This project’s primary goal is to develop a spray-induced gene silencing (SIGS) program to induce RNA interference mechanisms targeting both the grapevine and the fungus. The plant genes targeted for RNAi belong to a susceptibility-gene family to Erysiphe necator (Mildew Locus O: MLO genes). We also targeted three pathogen-regulated genes critical to the life cycle of the pathogen. Our first objective was to identify stretches of RNA molecules with maximum interference activity to repress MLO endogenous genes (VitviMLO 3,4,6,9,13,17) and fungal genes (Dicer-like protein 1 and 2 and CYP51). As a first step, we successfully cloned and validated by sequencing all candidate genes’ coding regions used to generate dsRNA molecules. Using these clones as a template, we have produced the dsRNA molecules using a commercial kit. We sprayed the MLO-based dsRNA solutions (20 ng/uL) of the MLO candidates combined with a wetting agent (SILWET-L77) on three-day acclimated tissue-cultured disk leaves of grapevine tissue-culture plants. RNA extraction is underway and will be followed by reverse transcription reactions and teal-time PCR experiments. Our second objective is to evaluate uptake and processing of dsRNA molecules from grapevine leaves using a fluorescent tag to assess how systemic the spray can be, and our third objective is to determine the efficacy of clay nanoparticles (layered double hydroxide [LDH]) to prolong dsRNA’s lifespan on the surface of grapevine leaves.

Funding Support: USDA