Administration of coexpressed artificial microRNA and bacteriophage MS2 virus‐like particles provides protection against Spodoptera litura

Dear Editor, Spodoptera litura, commonly known as the tobacco cutworm, is a polyphagous agricultural pest worldwide, causing significant economic losses to a wide range of crops. Over the past decades, S. litura has developed high resistance levels to multiple chemical insecticides (Li et al., 2024), and shown low susceptibility to transgenic Bacillus thuringiensis (Bt) cotton (Wan et al., 2008). This necessitates the exploration of alternative strategies for effective S. litura control. The RNA interference (RNAi)-based approach, employing double-stranded RNA (dsRNA) targeting essential genes in pests, has paved the way for a new generation of insect pest management (Zhu & Palli, 2020). However, RNAi efficiency in lepidopteran insects has been hindered by the rapid degradation of dsRNA by highly active nucleases in body fluids (Shukla et al., 2016; Guan et al., 2018). Therefore, enhancing the production of RNAi-inducing molecules and improving their stability are crucial for effective lepidopteran control. In a recent breakthrough, Jiang et al. (2024) demonstrated that MS2 virus-like particles (VLPs) could enhance the stability and efficacy of artificial microRNA (amiRNA) in the gut of lepidopterans (Jiang et al., 2024). To enhance RNAi efficiency in S. litura, the coexpression of MS VLPs and amiRNA in the pET28-BL21(DE3) RNase III– system has been proposed, enabling the production of higher quantities of dsRNA compared to the commonly used L4440-HT115(DE3) system (Ma et al., 2020). Chitin synthase (CHS) is a crucial enzyme for chitin formation, with the CHS1 gene playing a vital role in the pupation and molting processes of S. litura larvae (Yu et al., 2020). By using the Tribolium castaneum bantam scaffold as a backbone (Bally et al., 2020), we designed an amiRNA to target the CHS1 gene of S. litura (amiRCHS1). In our study, we expressed MS2 VLP and amiRCHS1 separately or in tandem in Escherichia coli BL21(DE3) RNase III– system (Ma et al., 2020). Upon isopropyl β-D-1-thiogalactopyranoside induction, northern blot analysis (File S1) revealed the expression of amiRCHS1 (138 nt) in E. coli that carried the constructs for either amiRCHS1 alone or VLP-amiRCHS1 (Fig. 1A). Simultaneously, sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis confirmed the expression of MS2 VLPs at 28 kDa in bacterial cultures expressing VLP alone and VLP-amiRCHS1 (Fig. 1B). These results indicate the capability of the pET28-BL21(DE3) RNase III– system to effectively coexpress both amiRNA and MS2 VLPs. To assess the RNAi efficacy of coexpressed amiRNA and MS2 VLPs, newly hatched S. litura larvae were provided with an artificial diet enriched with the engineered bacteria (File S1). Various parameters such as larval weight gain, mortality, pupal formation, and moth emergence were monitored. The results indicated that feeding on amiRCHS1 caused a significant reduction in larval weight gain and target gene expression (Table S1) on the 7th d after feeding (DAF) compared to control larvae (CK and VLP). This reduction was significantly more pronounced when larvae were fed with the coexpression of amiRCHS1 and MS2 VLPs (Fig. 1C–E). Corresponding to the weight reduction trend, larvae fed with VLP-amiRCHS1 exhibited the lowest pupation and molting rates (Fig. 1F–H). These data confirm that MS2 VLPs can effectively encapsulate amiRNA in the BL21(DE3) RNase III– strain, thereby enhancing RNAi efficiency in S. litura. To test whether the MS2 VLPs could mitigate the degradation of amiRNA by nucleases in the midgut of S. litura, crude extracts from the engineered bacteria were incubated with the intestinal fluid of S. litura (File S1). Subsequent analysis via northern blotting of total RNA extracted from the mixture revealed that naked amiRCHS1 was degraded within 10 min and completely degraded after 20 min. In contrast, amiRCHS1 encapsulated by MS2 VLPs remained detectable even after 20 min of incubation (Fig. 1I). These results indicate that MS2 VLPs have capacity to protect amiRNA from degradation in the intestinal fluid of S. litura. Taken together, our findings demonstrate the successful coexpression of amiRCHS1 and MS2 VLPs in the pET28-BL21(DE3) RNase III– system, leading to an efficient RNAi response in S. litura. Moreover, our study highlights the potential of MS2 VLPs in enhancing RNAi efficiency for the management of lepidopteran pests. This work was supported by the National Natural Science Foundation of China (32071477, 32272634). We are grateful to Professor Jie Shen from China Agricultural University for generously providing the E. coli BL21(DE3) RNase III– strain. The authors declare no conflict of interest. Table S1 Primers used in this work. File S1 Materials and methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.