uses a variant Bt Cry3Bb1 insecticidal protein (Donovan et al., 1992). Cry3Bb1 is known to be biologically active The corn rootworm (CRW; Diabrotica spp.) is one of the most against several species within the Coleopteran family serious pests of corn in the USA. Chemical insecticides and crop rotation have been the only two options available to growers for Chrysomelidae, including the western corn rootworm, managing CRW. Unfortunately, both of these tactics can be ineffective Diabrotica virgifera virgifera LeConte (Rupar et al., as a result of either resistance or behavioral modifications. In this 1991). The biological activity of this protein against D. paper, we describe transgenic maize (Zea mays L.) hybrids that control virgifera virgifera suggested its potential use in creating CRW. These hybrids were created with a Cry3Bb1 Bacillus thurintransgenic plants expressing Cry3Bb1 that would confer giensis (Bt) variant that is approximately eight times more lethal to protection to corn root tissue from larval feeding damcorn rootworm larvae than the wild-type protein. A DNA vector age. To further augment protection of the root system containing the modified cry3Bb1 gene was placed under control of a from larval feeding damage, modifications were introroot-enhanced promoter (4-AS1) and was introduced into embryonic duced in the cry3Bb1 gene that gave rise to an amino acid maize cells by microprojectile bombardment. Described here is the variant Cry3Bb1 protein with an eight-fold increase in molecular genetic characterization, protein expression levels, and field performance of the recently commercialized MON863 hybrids.

The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is uniquely toxic to Diabrotica undecimpunctata, the Southern corn rootworm, while retaining activity against Leptinotarsa decemlineata, the Colorado potato beetle. The crystal structure of the delta-endotoxin Cry3Bb1 has been refined using data collected to 2.4 A resolution, with a residual R factor of 17.5% and an R(free) of 25.3%. The structure is made up of three domains: I, a seven-helix bundle (residues 64-294); II, a three-sheet domain (residues 295-502); and III, a beta-sandwich domain (residues 503-652). The monomers in the orthorhombic C222(1) crystal lattice form a dimeric quaternary structure across a crystallographic twofold axis, with a channel formed involving interactions between domains I and III. There are 23 hydrogen bonds between the two monomers conferring structural stability on the dimer. It has been demonstrated that Cry3Bb1 and the similar toxin Cry3A form oligomers in solution. The structural results presented here indicate that the interactions between domains I and III could be responsible for the initial higher order structure and have implications for the biological activity of these toxins. There are seven additional single amino-acid residues in the sequence of Cry3Bb1 compared with that of Cry3A; one in domain I, two in domain II and four in domain III, which also shows the largest conformational difference between the two proteins. These changes can be implicated in the selectivity differences noted for these two delta-endotoxins.

No abstract

Bioassay screening of Bacillus thuringiensis culture supernatants identified strain EG2158 as having larvicidal activity against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Ion-exchange fractionation of the EG2158 culture supernatant resulted in the identification of a protein designated Sip1A (secreted insecticidal protein) of approximately 38 kDa having activity against Colorado potato beetle (CPB). An oligonucleotide probe based on the N-terminal sequence of the purified Sip1A protein was used to isolate the sip1A gene. The sequence of the Sip1A protein, as deduced from the sequence of the cloned sip1A gene, contained 367 residues (41,492 Da). Recombinant B. thuringiensis and Escherichia coli harboring cloned sip1A produced Sip1A protein which had insecticidal activity against larvae of CPB, southern corn rootworm (Diabrotica undecimpunctata howardi), and western corn rootworm (Diabrotica virgifera virgifera).