Plant-mediated RNAi via Panonychus citri-derived miRNAs disrupts adult emergence and confers mite resistance

MicroRNAs (miRNAs), small non-coding RNAs that trigger RNA interference (RNAi), are promising candidates for pest control, yet their efficacy against non-insect pests remains poorly explored. This study evaluates endogenous mite-derived miRNAs as transgenes for crop resistance against Panonychus citri, a major pest with rapid acaricide resistance that severely damages citrus, soybean, and other cash crops. Through developmental miRNA libraries of P. citri, we identify miR-750 and miR-200a-5p as key regulators during the deutonymph-to-adult transition, whose overexpression significantly inhibits adult eclosion. Both in vivo and in vitro assays confirm that miR-750 targets both NFYB and ADRM1, whereas miR-200a-5p specifically targets ADRM1; dsRNA-mediated silencing of these genes recapitulates eclosion defects induced by miRNA overexpression. We generate single-copy transgenic soybean lines with high expression of miR-750 (line 7#6) or miR-200a-5p (line 2#14). Detached-leaf and whole-plant bioassays using T1 generations reveal that both lines exhibit strong resistance against P. citri. Notably, neither transgenic line adversely affects the non-target predatory mite Neoseiulus barkeri, either via direct miRNA exposure or prey-mediated transfer. The resistance against P. citri and biosafety toward N. barkeri are retained in T2 generations. These findings highlight the potential of endogenous miRNAs as effective targets for developing eco-friendly and sustainable pest management strategies.