Co-expression of Cocculus hirsutus trypsin inhibitor with Cry1Ab toxin exhibits insect mortality and reduction in development of resistance in insects

ChTI (Cocculus hirsutus trypsin inhibitor), a plant serine protease inhibitor, is a natural plant defense protein effective against phytophagous insects by inhibiting digestive proteases in guts. We modified the ChTI gene by codon optimization and expressed it in E. coli, purified it, and performed insect bioassay, where significant weight loss with reduced larval growth was observed in both the insect pests; Helicoverpa armigera and Spodoptera litura. Further, we expressed the ChTI protein in Arabidopsis thaliana and Solanum lycopersicum, either individually or pyramided with Bt-Cry1Ab toxin and quantified the toxicity of the expressed proteins against both insects in the transgenic plants. We also performed transcriptomic analysis of transgenic Arabidopsis and tomato plants expressing ChTI protein, where the enhancement of different pathogenesis-related proteins was observed. Earlier reports say that insects gradually develop resistance against toxins, so we developed transgenic plants, co-expressing the two different toxins from diverse origin, achieving resistance against insects, and reducing the tendency of insects to develop resistance against the toxins. Comparative analysis of all the expressed gene constructs viz. pChTI, pcry1Ab, and pChAb was completed by molecular and insect biassays. Complete mortality against both insects was observed without compromising the yield.