Certain strains of the gram-positive sporulating bacterium Bacillus thuringiensis (Bt) produce protein toxins with insecticidal activity against Lepidoptera (the insect Order containing butterflies and moths). After binding to specific sites on the midgut epithelium of larvae, the toxins insert into the lipid bilayer to create pores, which eventually lyse the epithelial cells and kill the insect. Although Bt toxins have received a great deal of study because of their potential use in biological control of insect pests, little is known about the toxin binding targets in the insect membrane, or their molecular interaction with toxin that leads to pore formation. Toxin-resistant strains of lepidopteran species that are normally susceptible offer unique opportunities for studying the toxin mode of action. The noctuid moth Heliothis virescens is typically highly susceptible to the Bt toxin Cry1Ac, but the laboratory selected YHD2 strain is up to 10,000-fold resistant. Approximately 80% of the resistance can be accounted for by a single locus, BtR-4, which has been mapped on Linkage Group 9 of H.virescens. BtR-4 is the most potent Bt-resistance gene known in any insect. Moreover, the resistant YHD2-type allele at the BtR-4 locus is present in field populations of H.virescens at the surprisingly high frequency of 10-3. Yet in no insect species is the identity, protein product, or normal physiological function of any Bt resistance gene known.
The long-term goal is to use a genetic approach on Bt-resistant insect strains to study the mechanism of toxin action. Techniques of positional (map-based) cloning will be used to isolate, sequence, and characterize the BtR-4 gene. The specific objectives are to 1) localize BtR-4 to within a 0.5 cM region of Linkage Group 9 by fine-scale mapping, 2) assemble a contig of BAC (bacterial artificial chromosome) clones spanning the BtR-4 region, 3) isolate and sequence expressed sequences within the contig, and 4) confirm one of these candidates as the actual BtR-4 gene by sequence comparison and experimental approaches.
This project will produce the first Bt resistance gene cloned from any species, an advance that will enable a more detailed molecular characterization of the mechanism of Bt toxin action. In the process, it will serve as a pioneering illustration of the feasibility of the map-based cloning approach in a nondipteran insect. The location and identity of the other expressed sequences within the contig covering the BtR-4 region will comprise one of the first detailed physical maps of a chromosomal region from a Lepidopteran. The physical and genetic maps resulting from this project, along with the vast data available for Dipteran species such as Drosophila melanogaster, will enable the first comparison at this level of genomics between two Orders within the insects.
