Development of Triangle RNA Nanostructure for Enhancing RNAi-Mediated Control of Botrytis cinerea Through Spray-Induced Gene Silencing Without Extra Nanocarrier.

Botrytis cinerea, a necrotrophic fungal pathogen responsible for gray mold, poses a severe threat to over 1400 plant species, causing significant pre- and postharvest losses worldwide. RNA interference (RNAi)-based strategies, particularly spray-induced gene silencing (SIGS), have emerged as environmentally friendly alternatives to chemical fungicides. However, the application of naked double-stranded RNA (dsRNA) suffers from poor stability and low cellular uptake. In this study, we engineered a self-assembling triangular RNA nanoparticle, termed Bc-triangle, targeting four virulence genes of B. cinerea-BcDCL1, BcPPI10, BcNMT1 and BcBAC. The nanostructure was designed using RNA origami principles and produced in Escherichia coli. Functional assays demonstrated that Bc-triangle significantly inhibited conidial germination and mycelial growth in vitro, and markedly reduced disease severity in plants. Compared with linear dsRNA, Bc-triangle showed superior persistence and efficacy, with lesion area reduction sustained up to 10 days post-spraying. qRT-PCR analysis revealed substantial downregulation of the target genes, especially BcNMT1, indicating enhanced RNAi activation. These findings establish RNA nanotechnology as a powerful platform for transgene-free, programmable, and sustainable control of fungal pathogens in crop production.