RNase III-deficient Bacillus thuringiensis enables stable dsRNA production and enhances HD1 activity against Spodoptera frugiperda

BACKGROUND: Spodoptera frugiperda is one of the most important invasive agricultural pests in China. Integrating RNA interference (RNAi) with Bacillus thuringiensis (Bt)-based biocontrol represents a promising strategy for pest control. However, efficient dsRNA production in Bt remains challenging as endogenous RNase activity limits dsRNA stability and accumulation. RESULTS: In this study, an RNase III-deficient Bt strain, DBΔrnc, was generated using an optimized CRISPR-Cas9 system. Deficiency of RNase III markedly enhanced intracellular dsRNA accumulation. A hairpin dsRNA construct exhibited higher transcript levels than the dual-promoter construct, especially in the DBΔrnc background. Oral delivery of Bt expressed hairpin dsRNA targeting the endochitinase gene SfCHI of S. frugiperda induced effective gene silencing and caused pupation defects. Moreover, co-application with Bt HD1 strain significantly increased larval mortality in both neonate and 2^nd-instar larvae. CONCLUSION: Collectively, our findings highlight that the RNase III-deficient strain DBΔrnc is an efficient chassis for dsRNA expression. Furthermore, the enhanced insecticidal activity from co-application of DBΔrnc(hpdsCHI) and HD1 demonstrates the feasibility of integrating RNAi with Bt-based pest control.