BcMFSR Is Required for Efficient Uptake of Exogenous dsRNA and Spray-Induced Gene Silencing in Botrytis cinerea.

Spray-induced gene silencing (SIGS) uses exogenous double-stranded RNA (dsRNA) to suppress pathogen virulence or growth-related genes, offering a promising strategy for controlling grey mould caused by Botrytis cinerea at the gene level. In this study, we investigated the molecular mechanisms underlying the efficient uptake of dsRNAs by B. cinerea, making significant progress towards addressing key gaps in our understanding of SIGS. We identified four B. cinerea genes, BcBMP1, BcSOD1, BcGAR2, and BcVELB, as effective targets for grey mould control by employing an Arabidopsis thaliana SIGS screening system. Furthermore, by constructing a T-DNA insertion mutant library, we identified the ΔBcmfsR mutant, which exhibited significantly impaired dsRNA internalization. Functional assays revealed that the B. cinerea Major Facilitator Superfamily protein, BcMFSR, is critical for pH-dependent dsRNA uptake, facilitating SIGS efficacy. In the absence of functional BcMFSR, dsRNAs failed to reduce lesion size and B. cinerea virulence, regardless of the dsRNA sequence. BcMFSR is required for SIGS-mediated silencing of four genes. Moreover, fluorescent labelling confirmed the localization of both BcMFSR and dsRNA in vesicular structures within hyphae, suggesting its involvement in endocytosis. Our discoveries not only established the novel role of BcMFSR in RNA uptake but also provided a foundation for improving the efficiency of RNA pesticides and broadening their agricultural applications.