Improvement of host-induced gene silencing efficiency via polycistronic-tRNA-amiR expression for multiple target genes and characterization of RNAi mechanism in Mythimna separata

Host-induced gene silencing (HIGS) emerged as a new strategy for pest control. However, RNAi efficiency is reported to be low in Lepidoptera, which are composed of many important crop pests. To address this, we generated transgenic plants to develop HIGS effects in a maize pest, Mythimna separata (Lepidoptera, Noctuidae), by targeting chitinase encoding genes. More importantly, we developed an artificial microRNA (amiR) based PTA (polycistronic-tRNA-amiR) system for silencing multiple target genes. Compared with hpRNA (hairpin RNA), transgenic expression of a PTA cassette including an amiR for the gut-specific dsRNA nuclease gene MsREase, resulted in improved knockdown efficiency and caused more pronounced developmental abnormalities in recipient insects. When target gene siRNAs were analysed after HIGS and direct dsRNA/siRNA feeding, common features such as sense polarity and siRNA hotspot regions were observed, however, they differed in siRNA transitivity and major 20-24nt siRNA species. Core RNAi genes were identified in M. separata, and biochemical activities of MsAGO2, MsSID1 and MsDcr2 were confirmed by EMSA (electrophoretic mobility shift assay) and dsRNA cleavage assays, respectively. Taken together, we provide compelling evidence for the existence of the RNAi mechanism in M. separata by analysis of both siRNA signatures and RNAi machinery components, and the PTA system could potentially be useful for future RNAi control of lepidopteran pests.

Automated summary

The study introduces Host-induced gene silencing (HIGS) as a new strategy for pest control and demonstrates the successful application of transgenic plants targeting chitinase encoding genes in the maize pest Mythimna separata. The development of an artificial microRNA (amiR) based PTA system has significantly improved knockdown efficiency and caused more pronounced developmental abnormalities in recipient insects. Common features and differences in siRNA characteristics after HIGS and direct dsRNA/siRNA feeding are analyzed, along with the identification of core RNAi genes in M. separata. The study provides compelling evidence for the potential use of the PTA system in future RNAi control of lepidopteran pests.