Co-expression of Cocculus hirsutus trypsin inhibitor with Cry protein reduces resistant development in targeted insects along with complete mortality

Abstract

Crop losses due to various insects are a global problem. For decades, several types of research have been ongoing on this issue with limited success due to co-evolving insect pests. In this study, the modified Cocculus hirsutus trypsin inhibitor (ChTI), a serine proteinase inhibitor gene encoding a natural plant defense protein, inhibiting the digestive proteases in the midgut of phytophagous insects, was expressed alone and pyramided with the Bt-toxin Cry1Ab, to reduce resistance development against two herbivorous insects, Helicoverpa armigera and Spodoptera litura with complete insect mortality. In-silico analysis revealed the structural and conformational stability of ChTI protein. Furthermore, 80 % weight loss and reduced larval growth were observed in both insects challenged with 200 µg of the purified ChTI protein. The ChTI protein trypsin inhibitor activity was observed up to 5.5 U/mg expressed in E. coli, 3.0–3.5 U/mg in transgenic Arabidopsis plants, and up to 2.5–3.0 U/mg in transgenic tomato plants. Transgenic plants (Arabidopsis and tomato) were developed by pyramiding ChTI, and cry1Ab genes showed 100 % mortality against both insects while the transgenic plants expressing the ChTI gene alone showed up to 70 % mortality without yield penalty. Transcriptomic analysis of the transgenic plants further revealed that other pathogenesis-related defense proteins were also differently regulated.