The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.

The western corn rootworm remains one of the most important pests of corn in the United States despite the use of many pest management tools. Cry3A, the first coleopteran-active Bacillus thuringiensis toxin isolated, has not been useful for control of the corn rootworm pest complex. Modification of Cry3A so that it contained a chymotrypsin/cathepsin G protease recognition site in the loop between ␣-helix 3 and ␣-helix 4 of domain I, however, resulted in consistent activity of the toxin ("mCry3A") against neonate western corn rootworm. In vitro chymotrypsin digests showed that there was a substantial difference between the enzyme sensitivity of mCry3A and the enzyme sensitivity of Cry3A, with mCry3A rapidly converted from a 67-kDa form to a ϳ55-kDa form. The introduced protease site was also recognized in vivo, where the ϳ55-kDa form of mCry3A toxin was rapidly generated and associated with the membrane fraction. After a point mutation in mcry3A that resulted in the elimination of the native domain I chymotrypsin site (C terminal to the introduced chymotrypsin/ cathepsin G protease site of mCry3A), the in vitro and in vivo digestion patterns remained the same, demonstrating that the introduced site was required for the enhanced activity. Also, 55-kDa mCry3A generated by cleavage with chymotrypsin exhibited specific binding to western corn rootworm brush border membrane, whereas untreated 67-kDa mCry3A did not. These data indicate that the mCry3A toxicity for corn rootworm larvae was due to the introduction of a chymotrypsin/cathepsin G site, which enhanced cleavage and subsequent binding of the activated toxin to midgut cells.