Purification of a trypsin inhibitor from Cocculus hirsutus and identification of its biological activity

Proteinase inhibitors play a significant role in plant defense against insect pests and phytopathogens by inhibiting their proteases. A thermotolerant monomeric trypsin inhibitor with molecular weight ∼18kD was purified from Cocculus hirsutus (ChTI) using trypsin sepharose affinity column. Western blot analysis using ChTI IgY revealed its presence in vegetative parts and seeds. The second and third instar larvae of H. armigera fed with ChTI (5000TIU/ml) resulted in 84.59 and 58.71% reduction in mean larval weight respectively. An increase in the larval growth period was observed in ChTI fed larvae at all instars and inhibitor fed larvae could not complete their life cycle. ChTI caused 74 and 59.53% inhibition of bovine trypsin and Helicoverpa gut proteases respectively. ChTI exhibited strain specificity and inhibited growth and development of plant fungal pathogens. Bioassay studies on yeast strains indicated that ΔYNK and MNN1 are more sensitive to ChTI. The results suggest that phosphodiester linkage in cell wall components is likely to be the key determinants for binding of ChTI. Taken together, these studies indicate that ChTI is a potential candidate for development of transgenic plants against foliar diseases and insect pests.