Antimetabolic proteins from plants and their potential use in conferring resistance against corn rootworms

Abstract

The major digestive enzymes of larval Diabrotica undecimpunctata howardi, the southern corn rcotworm (SCR), have been investigated. A number of proteases have been identified, the majority cysteine proteases, some aspartic acid proteases are also present. In vitro assays of cysteine proteases showed that almost all activity could be arrested by E64 or chicken egg-white cystatin. This activity was also affected by TLCK, CpTI and thaumatin. Two inhibitory activities were demonstrated in protein extracts from rice seed. The first, oryzacystatin-I, caused marked inhibition of both the insect cysteine proteases and papain. The second produced strong inhibition of insect cysteine proteases but left papain virtually unaffected. Amino acid sequence data for this novel inhibitor was obtained, and significant homology demonstrated to the rice allergenic proteins (Izumi et a].,1992; Adachi et al., 1993). Oryzacystatin-I was expressed as a fully functional fusion protein (Rozc) in Escherchia coli, isolated, characterised and employed in feeding trials with larval SCR, a significant rise in mortality was demonstrated. Other protease inhibitors were also assayed in vivo, but none exhibited the efficacy of Rozc. A single iso-form of a-araylase was identified and characterised. In vitro assays with amylase inhibitors demonstrated the effectiveness of WAAI and the weak effect of BAAI. WAAI, employed in bioassay, produced a significant decrease in survival. Five lectins were tested by bioassay. WGA and GNA caused marked alterations in larval development, GNA was most effective. Saporin caused little effect when incorporated into artificial diet. WAAI, CpTI and GNA were employed in combination bioassays. An enhanced level of effectiveness was demonstrated with the double and triple combinations assayed. While further work is necessary, especially assaying protein efficacy in planta and establishing mechanisms of action, this project has successfully demonstrated the clear potential of plant antimetabolic proteins for use in the enhancement of inherent resistance of crop plants to insects, and of employing a number of proteins in a multi-mechanistic defence.