Characterization of insecticidal activity of black pepper seed protease inhibitor for deployment in transgenics to control Helicoverpa armigera

Helicoverpa armigera is one of the most important crop pests worldwide, and Bacillus thuringiensis (Bt) toxin proteins have been deployed in transgenic plants for controlling this pest in several crops. However, there is a possibility of development of insect resistance to Bt toxins deployed in transgenic plants. Therefore, we studied the biological activity of proteinase inhibitors (PIs) from seeds of black pepper as a candidate gene for deployment in transgenic crops. The PIs from black pepper seeds showed insecticidal activity against H. armigera gut proteinases (HGPs). Protease inhibitory activity of the pepper extract was confirmed by inhibition of gelatin hydrolysing activity of trypsin and HGPs on X-ray film by dot-blot method. Black pepper protease inhibitor (BPPI) inhibited more than 80% activity of bovine trypsin and trypsin-like activity of HGPs using chromogenic substrate benzoyl-arginyl p-nitroanalide (BAPNA). Electrophoretic analysis revealed the presence of a prominent protein band of 6 kDa molecular weight. Partially purified BPPI in artificial diet retarded the growth and development of H. armigera larvae in a dose-dependent manner. Trypsin inhibitory activity of the BPPI was observed in a pH range of 6.0 – 9.0, and temperature range of 30 – 50 ^oC. BPPI remained active and stable against the HGPs up to 4 h. BPPI inhibited the casein hydrolysing activity of bovine trypsin and trypsin-like activity in HGP extract, in vitro and in vivo, as determined by protease zymogram assay. The PIs with potent insecticidal activity against insect pests could be deployed in transgenic plants for sustainable crop protection.