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 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.

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

“…In the case of Cry1Ac, binding specificity governed by domain III is dependent on recognition of GalNAc residues in receptor proteins (Burton et al, 1999;JuratFuentes and Adang, 2004). A clear example of domain III determining specificity comes from introduction of rootworm specificity in Cry3Aa by replacing domain III with the same domain from Cry1Ab (Walters et al, 2010). This observation would be unexpected based on the specificity of Cry1Ab, which is inactive against coleopteran larvae, and suggests that significant similarity in domains II and/or III may not be an appropriate predictor of specificity for engineered toxins.…”

Genetic Improvement of Bt Strains and Development of Novel Biopesticides

“…A fusion toxin comprising a truncated version of Cry1Ba and domain II from Cry1Ia was also shown to be toxic to both coleopteran and lepidopteran pests (Naimov et al 2001;Naimov et al 2003). More recently, a hybrid Cry1Ab/3A toxin which was toxic to the western corn rootworm was also created (Walters et al 2010). Finally, optimization of Bt crystal proteins through DNA shuffling has also been reported (Lassner and Bedbrook 2001;Craveiro et al 2010).…”

Bt Crops: Past and Future

“…Genetically engineered Cry3A toxins (mCry3A and eCry3.1Ab) displaying increased activity against D. virgifera larvae (Walters et al 2008(Walters et al , 2010 are currently used in some Bt maize varieties (Table 15.1). Modified (Mod) Cry1A toxins targeting lepidopteran larvae and effective against larvae lacking the primary Cry1A toxin receptor (Soberón et al 2007) are a clear alternative to currently used lepidopteran-specific traits.…”