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Two C-terminal deletion constructs were made to study the effect of such deletions on the biological activity of the CryV protein of Bacillus thuringiensis subsp. kurstaki. The results of feeding on neonatal larvae of Ostrinia nubilalis (European corn borer [ECB]) indicated that the 50% lethal dose of the full-length CryV protein was 3.34 g/g of diet (95% fiducial limits, 2.53 to 4.32 g/g of diet). Removal of 71 amino acids (aa) from the C terminus had little effect on toxicity, whereas deletion of 184 aa abolished the insecticidal activity of the CryV protein completely. Truncations of the full-length CryV protein were also generated with trypsin and the midgut protease of ECB. The proteolytically treated products were characterized by determining their Nterminal amino acid sequences. The CryV protein was found to be cleaved by both proteases through a two-step process. Initially an intermediary form was generated which contained aa 45 of full-length CryV as its N-terminal end. The C-terminal end of this peptide was not experimentally determined. However, analysis of the deduced amino acid sequence of CryV indicated that the C-terminal end of the intermediary form is likely either aa 655 or 659. Further N-terminal processing of the intermediary form resulted in a protease-resistant core form. The core included aa 156 to aa 655 or 659. While the intermediary form retained 100% of the ECB larval toxicity, the core form exhibited only ϳ22% of the toxicity of the full-length protein.

Genetic engineering may be used to introduce multiple insect resistance genes with different modes of action into crop plants. We explored the possible interactions of two differing gene products fed in the diet of cowpea weevil, Callosobruchus maculates (F.), a stored grain pest. The soybean cysteine protease inhibitor soyacystatin N (scN) and alpha-amylase inhibitor (alphaAI) from wheat have defensive function against this coleopteran. When artificial seeds containing both scN and alpha(AI) were infested with eggs of C. maculatus, the delays in larval development were longer than was predicted by summing the developmental delays seen when larvae were fed a diet containing the individual proteins, indicating that the effects of scN and alpha(AI) are synergistic. Alpha(AI) was readily hydrolyzed when incubated with insect gut extract. This proteolytic degradation was inhibited by scN, but not by Kunitz inhibitor (a serine protease inhibitor). Thus, degradation of alpha(AI) was due to proteolysis by insect digestive cysteine proteases. These data suggest that C. maculatus uses digestive enzymes not only to function in food protein digestion but also to defend the insects themselves by helping reduce the concentration of a toxic dietary protein.

Genetic engineering may be used to introduce multiple insect resistance genes with different modes of action into crop plants. We explored the possible interactions of two differing gene products fed in the diet of cowpea weevil, Callosobruchus maculates (F.), a stored grain pest. The soybean cysteine protease inhibitor soyacystatin N (scN) and alpha-amylase inhibitor (alphaAI) from wheat have defensive function against this coleopteran. When artificial seeds containing both scN and alpha(AI) were infested with eggs of C. maculatus, the delays in larval development were longer than was predicted by summing the developmental delays seen when larvae were fed a diet containing the individual proteins, indicating that the effects of scN and alpha(AI) are synergistic. Alpha(AI) was readily hydrolyzed when incubated with insect gut extract. This proteolytic degradation was inhibited by scN, but not by Kunitz inhibitor (a serine protease inhibitor). Thus, degradation of alpha(AI) was due to proteolysis by insect digestive cysteine proteases. These data suggest that C. maculatus uses digestive enzymes not only to function in food protein digestion but also to defend the insects themselves by helping reduce the concentration of a toxic dietary protein.

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