Yan Qiu

Chitinase is a key enzyme involved in insect chitin metabolism, and RNA interference (RNAi) targeting the corresponding gene represents a promising strategy for eco-friendly pest management. However, the design parameters of double-stranded RNA (dsRNA), particularly fragment length and target position, remain insufficiently optimized. To investigate the effects of these parameters on the RNAi efficiency against the MsChi2 gene in Mythimna separata, we synthesized four dsRNAs of 60, 200, 500, and 1000 bp and administered them to larvae. The results indicated that 200 and 500 bp dsRNAs significantly inhibited larval growth, reduced survival, and silenced MsChi2 expression. Among these, the 200 bp dsRNA exhibited the most pronounced effects, including a 59% inhibition rate of larval body weight, 65.9% mortality, and 62% target gene silencing efficiency, accompanied by a significant reduction in chitin content and abnormalities in molting and pupation. Using 200 bp as the optimal length, five fragments targeting different coding regions of the gene were evaluated. All except one exhibited strong activity, and their RNAi efficiencies were positively correlated with the predicted numbers of effective small interfering RNAs. Collectively, these findings demonstrate that dsRNA length and target position jointly modulate RNAi efficiency, with 200 bp representing the optimal fragment length. This study provides a rational design framework for dsRNA development and establishes both theoretical and practical foundations for RNAi-based management of M. separata.

RNA interference (RNAi) is a crucial antiviral defense mechanism in plants, where Argonaute (AGO) proteins play a central role. However, the function of AGO proteins in the interaction between Soybean mosaic virus (SMV) and Nicotiana benthamiana remains unclear. In this study, SMV pathogenicity was confirmed using an SMV-GFP infectious clone, with typical symptoms and systemic GFP fluorescence observed 14 days post-inoculation. Real-time quantitative reverse transcription polymerase chain reaction analysis revealed dynamic regulation of multiple NbAGO genes upon infection. Notably, NbAGO1a, NbAGO1b, and NbAGO2 were significantly upregulated and positively correlated with viral accumulation, suggesting their critical roles in antiviral defense. Based on these findings, these three genes were selected as targets for artificial microRNA (amiRNA) silencing. Three amiRNAs were designed for each gene using the Arabidopsis miR1596 backbone, with the most effective sequences exhibiting silencing efficiencies ranging from 75.2% to 98.1%. A polycistronic tRNA-amiRNA (PTA) cassette was constructed using Golden Gate cloning technology to simultaneously target all three genes. Co-infection assays indicated that the PTA cassette enhanced SMV accumulation more effectively than single amiRNAs, as evidenced by increased GFP fluorescence (49.1–60.5%) and pronounced leaf necrosis. The PTA system downregulated the expression of NbAGO1a, NbAGO1b, and NbAGO2 by 18.4–26.7%. Furthermore, silencing NbAGO2 alone resulted in severe necrosis, underscoring its essential role in this antiviral defense mechanism. This study elucidates the importance of NbAGO1a, NbAGO1b, and NbAGO2 in antiviral immunity and demonstrates the utility of the PTA system for efficient multi-gene silencing, offering valuable insights for developing RNAi-based antiviral strategies.