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.

A unique, coleopteran-active protein, termed eCry3.1Ab, was generated following variable-region exchange of a Bacillus thuringiensis lepidopteran-active protein, Cry1Ab, with a Cry3A region. Our results support the hypothesis that this variable-region exchange is responsible for imparting strong bioactivity against the larvae of western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte), a pest species which is not susceptible to either parent protein sequence. This study demonstrates the potential of successfully engineering a portion(s) of a lepidopteranactive B. thuringiensis sequence so that it has activity against coleopterans. Further elucidation of the eCry3.1Ab activity indicated the importance of variable regions 4 to 6 that were derived from Cry1Ab instead of Cry1Ac. There was some flexibility in making domain III of engineered hybrid insecticidal proteins even more Cry1Ab-like and retaining activity, while there was less flexibility in making domain III more Cry3A-like and retaining activity. In vitro binding studies with brush border membrane vesicles demonstrated that there was specific binding of chymotrypsin-processed modified Cry3A (mCry3A), which was not diminished by addition of a 100-fold molar excess of chymotrypsin-processed eCry3.1Ab or unprocessed eCry3.1Ab. In addition, in the converse experiment, specific binding of chymotrypsin-processed eCry3.1Ab was not diminished by the presence of a 75-fold molar excess of chymotrypsin-processed mCry3A. These data support the hypothesis that eCry3.1Ab can interact with different binding sites than the activated form of mCry3A in the WCR brush border and may provide a different mode of action from the standpoint of resistance management.The three-dimensional structures of Bacillus thuringiensis ␦-endotoxins indicate that the Cry1, Cry2, Cry3, and Cry4 types of proteins have similar three-domain organizations (2, 10, 16, 18). The three structural domains are N-terminal domain I comprised of 7 ␣-helices, domain II containing three ␤-sheets in a so-called Greek key conformation, and C-terminal domain III, which is a ␤-sandwich in a so-called jellyroll conformation. The active portions of B. thuringiensis Cry proteins are also characterized by having five conserved blocks in their amino acid sequences, which are designated CB1 to CB5 from the N terminus to the C terminus (11). CB1 comprises approximately 29 amino acids, CB2 comprises approximately 67 amino acids, CB3 comprises approximately 48 amino acids, CB4 comprises approximately 10 amino acids, and CB5 comprises approximately 12 amino acids. The sequences before and after these five conserved blocks are highly variable and thus are designated the "variable regions," which are designated V1 toV6.

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Recent investigations indicated that Bacillus thuringiensis delta‐endotoxins (DET) possess aphidicidal activity in an artificial diet bioassay. Crystalline preparations of CryIIA, CryIIIA and CryIVD solubilized in a slightly alkaline sucrose/amino acid diet clearly imparted toxicity toward adults of potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera: Aphididae) after 4–5 days of continuous feeding. No obvious feeding deterrence was noted in these assays, as copious honeydew was produced and aphids often died in a feeding position. CryIIIA which was solubilized in aphid diet, but filtered to remove spores or crystalline toxin lacked aphidicidal activity. Spores from an acrystalliferous strain (EG2205) were not toxic by themselves at 7.75 × 105 spores/ml aphid diet, but did restore toxicity to the filtered CryIIIA solution. Therefore, low levels of spores may be very effective in concert with DET for aphicidal activity. Results also clearly demonstrated that a suspension of crystalline CryIIIA alone, without spores, exhibited toxicity. Therefore, DET may be more toxic to the aphids when imbibed as a fine suspension, perhaps indicating the need for slow solubilization into the aphid midgut.

Osmotic stress due to high sucrose concentration was imposed on the potato aphid, Macrosiphum euphorbiae (Thomas), in an artificial diet situation. Thin layer chromatography resolved melezitose and six additional honeydew oligosaccharides. Significantly higher levels of these oligosaccharides were recovered from the honeydew of this species in response to feeding on increased dietary sucrose concentrations, pointing to an osmoregulatory response. In addition, carbohydrase activities from the potato aphid were described. A degree of specificity toward hydrolysis of the sucrose substrate over other a-glucosyl sugars was demonstrated. Also, sucrose was optimal for the formation of the oligosaccharides. A strong a-galactosidase activity was found and transgalactosylation ability was indicated. These major trends in carbohydrase activity were also found in the foxglove aphid, Acyrthosiphon solani (Kaltenbach), and the oleander aphid, Aphis nerii Fonscolornbe.

The adenosine diphosphate (ADP)-ribosyltransferase, Vip2 (vegetative insecticidal protein), from Bacillus cereus in combination with another protein from the same organism, Vip1, has insecticidal activity against western corn rootworm larvae. The Vip2 protein exerts its intracellular poisoning effect by modifying actin and preventing actin polymerization. Due to the nature of this toxin, expression of Vip2 in planta is lethal. In this work, we attempted to build an enzyme precursor (proenzyme, zymogen) that would silently reside in one biological system (e.g. plants or yeast) and be activated in the other (insect larvae). Our approach involved engineering a random propeptide library at the C-terminal end of Vip2 and selecting for malfunctional enzyme variants in yeast. A selected proenzyme (proVip2) possesses reduced enzymatic activity as compared with the wild-type Vip2 protein, but remains a potent toxin toward rootworm larvae. In addition, upon analysis of the digestive fate of the engineered enzyme precursor in rootworm larvae, we demonstrated that 'zymogenized' Vip2 can be proteolytically activated by rootworm digestive enzyme machinery. This report represents an example of applying a protein engineering strategy for the creation of a plant-tolerated, zymogen-like form of an otherwise toxic protein. This approach may outline a novel path to address challenges associated with utilizing toxic proteins in certain biotechnological applications.

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