Identification of Bacillus thuringiensis delta-endotoxin Cry1C domain III amino acid residues involved in insect specificity

Cry1C domain III amino acid residues involved in specificity for beet armyw orm (Spodoptera exigua) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were ma de. Crossover points of these hybrids defined six sequence blocks containin g between 1 and 19 of the amino acid differences between Cry1E and G27. Blo cks B, C, D, and E of G27 were shown to be required for optimal activity ag ainst S. exigua. Block E was also required for optimal activity against the tobacco hornworm (Manduca sexta), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in blo ck B identified Trp-476 as the only amino acid in this block essential, alt hough not sufficient by itself, for full S. exigua activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in thi s insertion completely abolished activity against S. exigua but resulted in higher activity against M. sexta. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups o f amino acids as well as some individual residues in Cry1C domain III, whic h are strongly involved in S. exigua-specific activity as well as sometimes involved in M. sexta-specific activity.