Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte

Abstract

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is a significant pest of corn in the United States. The development of transgenic corn hybrids resistant to rootworm feeding damage depends on the identification of genes encoding insecticidal proteins toxic to rootworm larvae. In this study, a bioassay screen was used to identify several isolates of the bacterium Bacillus thuringiensis active against rootworm. These bacterial isolates each produce distinct crystal proteins with approximate molecular masses of 13 to 15 kDa and 44 kDa. Insect bioassays demonstrated that both protein classes are required for insecticidal activity against this rootworm species. The genes encoding these proteins are organized in apparent operons and are associated with other genes encoding crystal proteins of unknown function. The antirootworm proteins produced by B. thuringiensis strains EG5899 and EG9444 closely resemble previously described crystal proteins of the Cry34A and Cry35A classes. The antirootworm proteins produced by strain EG4851, designated Cry34Ba1 and Cry35Ba1, represent a new binary toxin. Genes encoding these proteins could become an important component of a sustainable resistance management strategy against this insect pest.

FIG. 1.

SDS-PAGE analysis of crystal proteins from various wild-type and recombinant strains of B. thuringiensis. Lanes: a, molecular mass standards (in kilodaltons); b, EG10650; c, EG4550; d, EG5899; e, EG11529; f, EG4851; g, EG11658; h, EG9444; i, EG11648; j, EG11936; k, EG11934; l, EG11935; m, EG11937.

FIG. 2.

Schematic diagram of the apparent cry gene operons cloned from B. thuringiensis strains EG5899, EG9444, and EG4851. The restriction map for the EG4851 cry genes shows the restriction sites used to generate knockout mutations within the cry coding regions.

FIG. 3.

Nucleotide sequence and translation of the apparent ET84-ET80-ET76 operon from B. thuringiensis strain EG4851.

FIG. 4.

SDS-PAGE analysis of knockout mutations within the ET84, ET80, and ET76 coding regions. Lanes: MW, molecular mass standards (in kilodaltons); 1, EG10650; 2, EG11658; 3, EG12156; 4, EG12158; 5, SIC8004; 6, SIC8006; 7, SIC8008; 8, SIC8096.

FIG. 5.

Bioassay evaluation of binary toxins against WCR larvae. (A) SDS-PAGE analysis of the binary toxins produced by the recombinant B. thuringiensis strains SIC8004 (lane 2, ET76 and ET80), EG11648 (lane 3, ET71 and ET79), and EG11936 (lane 4, ET39 and ET74). A purified Cry3Bb1 variant (lane 1) was used as a standard in the bioassays. (B) Chart plotting decreasing mean larval mass with increasing binary toxin concentration. The untreated control (UTC) yielded a mean larval mass of 0.4 mg.