Nanomaterial-mediated delivery of tomato miR393 that intrinsically targets a cell wall-related gene of Botrytis cinerea confers efficient control of gray mold disease

Gray mold, caused by the fungal pathogen Botrytis cinerea, is a major threat to global agriculture. Conventional pesticide control exacerbates environmental pollution and the risk of resistance development. Although RNA interference (RNAi) holds potential for disease control, its application is limited by instability of RNA and ambiguous targeting. This study demonstrates that tomato microRNA393 (miR393) can cross-kingdom into B. cinerea and reduce its pathogenicity. Using RT-qPCR, GUS staining, and 5' RLM-RACE, we confirmed that miR393 specifically targets nucleotides 1777-1778 of the BcFKS1 gene in B. cinerea, thereby inhibiting its expression and disrupting the fungal cell wall. To protect against miRNA degradation, we constructed a nanocarrier delivery system based on Star Polycation (SPc) was constructed. This nanomaterial extended the degradation half-life of miR393's by 6-fold under RNase A and RNase III conditions (up to 2 h) and increased delivery efficiency by 35%-65%. SPc-loaded miR393 enhanced tomato immunity by activating the jasmonic acid/salicylic acid (JA/SA) pathways, suppressing auxin receptor genes, and inducing PR1 protein accumulation (6.8-fold). Furthermore, inoculation assays showed that dsmiR393 reduced gray mold lesion areas by 67% in tomato, 88% in strawberry, and 41% in grape. This study elucidates the dual mechanism of miR393 to control gray mold, including cross-kingdom regulation for pathogenic fungi and modulation of plant immunity. This work provides a new strategy for disease prevention and control in sustainable agriculture.