Nanomaterial-mediated delivery of tomato miR393 intrinsically targeting 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 increases the risk of resistance. Although RNA interference (RNAi) holds potential for disease management, its application is limited by RNA instability and ambiguous targeting. Here, we demonstrate that tomato microRNA393 (miR393) can move across kingdoms 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 suppressing its expression and disrupting fungal cell wall integrity. To protect miR393 from degradation, we developed a star polycation (SPc)-based nanocarrier delivery system. This nanomaterial can extend the degradation half-life of miR393 six-fold under RNase A and RNase III treatment (up to 2 h) and increase delivery efficiency by 35%-65%. SPc-loaded miR393 enhances tomato immunity by activating the jasmonic acid and salicylic acid pathways, suppressing auxin receptor genes, and inducing PR1 protein accumulation (6.8-fold). Inoculation assays further showed that SPc-loaded double-stranded miR393 can reduce gray mold lesion areas by 67% in tomato, 88% in strawberry, and 41% in grape. This study elucidates the dual mechanism by which miR393 controls gray mold-cross-kingdom regulation of pathogenic fungi and modulation of plant immunity-and provides a promising strategy for sustainable disease prevention and control.