Host‐delivered <scp>amiRNA</scp> ‐chimeric <scp> <i>Bt</i> </scp> protein, <scp>Cry1AcF</scp> , combinatorial mediated resistance against the polyphagous insect‐pest, <scp> <i>Helicoverpa armigera</i> </scp>

BACKGROUND Helicoverpa armigera is one of the most devastating polyphagous insect-pest affecting crop production worldwide. In the present study, we demonstrated the efficacy of the multiplexed artificial microRNA (amiRNA)-Bt combinatorial approach in combating H. armigera. Multiplexed amiRNA gene construct was developed for simultaneous targeting of genes encoding acetylcholinesterase-1, HaAce1, and 20-hydoxyecdysone receptor, HaEcR, of H. armigera. Arabidopsis pre-miRNA 164a and 164b precursor backbones were used for expression of amiR-Ace1 and amiR-EcR, respectively. Arabidopsis native miRNA sequence was replaced by the amiRNA sequence, and both the recombinant amiRNA precursors, intervened by potato GBSS intron, were placed under a single transcriptional unit under the control of NOS promoter and terminator. The Bacillus thuringiensis (Bt) gene construct was comprised of a chimeric Bt gene, cry1AcF, under CaMV promoter and NOS terminator. RESULTS Highest level of resistance against H. armigera was observed in combinatorial tobacco transgenic plants expressing both the Multi-amiR and Cry1AcF simultaneously compared to plants expressing either Cry1AcF only or Multi-amiR only as revealed by detached leaf insect bioassay. Combinatorial tobacco transgenic plants demonstrated 100% larval mortality in 4 days compared to 8-9 days in the case of Bt tobacco plants and 12-13 days in case of multi-amiR lines. The down-regulation of HaAce1 and HaEcR expression in larvae fed on leaves of combinatorial tobacco transgenic plants demonstrated the complementary role of RNA-interference (RNAi) in conferring robust H. armigera resistance. CONCLUSION The multiplexed amiRNA approach that alleviates off-target concerns of RNAi in combination with Bt could be a potent and durable management strategy against H. armigera menace. © 2025 Society of Chemical Industry.