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Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen's RNA uptake efficiency.

Summary: Spray‐induced gene silencing (SIGS) is an innovative and eco‐friendly technology where topical application of pathogen gene‐targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross‐kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNA encapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi‐mediated protection against B. cinerea both on pre‐ and post‐harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection. [ABSTRACT FROM AUTHOR]