Lepidopteran-active variable-region sequence imparts coleopteran activity in eCry3.1Ab, an engineered Bacillus thuringiensis hybrid insecticidal protein

Abstract

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 lepidopteran-active 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.

FIG. 1.

Sequence alignments of (A) the N-terminal region of eCry3.1Ab and variant engineered hybrid insecticidal proteins and (B) the C-terminal region of variant engineered hybrid insecticidal proteins. Solid underlining indicates “cap sequence” residues, gray shading indicates the engineered cathepsin G site, boxes indicate the conserved sequence blocks indicated beneath the boxes, the dotted vertical line indicates the junction between domain II (D II) and domain III (D III), and dashed underlining indicates the C-terminal Cry1Ab 38 amino acid “tail sequence.” Conserved blocks were defined as described by Höfte and Whiteley (11). Note that subscript designations of engineered hybrid insecticidal proteins are used to distinguish different proteins in the present work and do not correspond to any other sequence numbering convention.

FIG. 2.

Relationship of eCry3.1Ab to other engineered hybrid insecticidal protein variants. Identical sequence regions are indicated by the same pattern. The presence of bioactivity against first-instar WCR larvae is indicated on the right.

FIG. 3.

In vivo processing and binding of eCry3.1Ab in first-instar WCR larvae: Western blot of recovered whole-body homogenate supernatant or pellet fractions derived after larval feeding on a diet with (+) or without (−) biotinylated eCry3.1Ab. b-eCry3.1Ab, biotinylated eCry3.1Ab marker protein; b-c-mCry3A, biotinylated chymotrypsin-treated mCry3A marker protein. The filled and open arrowheads indicate the positions of processed and unprocessed forms of eCry3.1Ab, respectively.

FIG. 4.

Homologous and heterologous BBMV competition binding assay results obtained with (A) biotinylated chymotrypsin-treated mCry3A (c-mCry3A) and (B) biotinylated chymotrypsin-treated eCry3.1Ab (c-eCry3.1Ab) labeled proteins. The Western blots show the effects of using buffer alone (Ø) and a 100-fold molar excess of unlabeled proteins during BBMV incubation (A) or the effects of using buffer alone (Ø) and a 75-fold molar excess of unlabeled proteins (B).