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

Spray-induced gene silencing (SIGS) is a recent technology that enhances a plant’s immune system. It involves the ectopic application of double-stranded RNAs (dsRNAs) to trigger a plant’s defense mechanism, known as RNA-interference (RNAi). Unlike traditional methods relying on the generation of transgenic plants, SIGS offers a rapid and targeted disease management solution. By transiently enhancing RNAi, it eliminates the need for resources and time-consuming development of resistant plants. However, SIGS application in tree crops encounters significant challenges, including limited cellular uptake and environmental stability of dsRNAs. To address these limitations, we explored an innovative approach that combines SIGS with branched polyethyleneimine-functionalized carbon dots (CDs) as nanocarriers, which enhanced dsRNA stability and cellular uptake. We developed a cost-effective, microwave-assisted protocol to synthesize 10 nm monodispersed CDs, that achieved >50% protection of dsRNA against RNase III degradation at a 200:1 (w/w) ratio. These CDs effectively improved cellular uptake, demonstrated by the successful delivery of CD-complexed fluorolabeled-dsRNAs (Cy3-RNA) into intact plant tissue-cultured cells. To prove the applicability of this formulation in planta, we sprayed Cy3-RNA with CDs on the abaxial surface of greenhouse grapevine leaves via low-pressure spray. Fluorescence microscopy revealed penetration of Cy3-RNA into stomata and neighboring cells. Additionally, the efficacy of CD-complexed dsRNA in triggering RNAi was validated using a 21-mer dsRNA targeting eGFP in eGFP-expressing microvines. Notably, no cytotoxic effects were observed following CD application. Ongoing research quantitatively compares eGFP expression of microvine leaves sprayed with CD-complexed and naked dsRNA through qPCR, to demonstrate the improvement in nucleic acid delivery associated with CDs. This innovative approach aims to overcome critical barriers for dsRNA delivery, particularly those associated with cell wall-related constraints in plants. Through optimization of RNAi-based defense strategies in grapevine, this research contributes to advancement of sustainable viticulture practices.

Funding Support: Erath Family Foundation, Oregon Wine Board