Certain strains of the gram-positive sporulating bacterium Bacillus thuringiensis (Bt)produce proteins with toxic activity against insect larvae. After ingestion and binding to specific sites on the midgut epithelium, 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 agricultural pests, much less is known about their binding targets in the insect membrane, or the molecular interactions leading to pore formation and death. 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. These investigators have previously shown that most of this resistance is due to a knockout of a cadherin-like protein, HevCaLP, by insertion of a transposable element fragment into the coding sequence of the gene. They have also identied two additional resistance genes that are unlinked to the HevCaLP gene. This project will characterize the interactions of all three genes by rigorously determining the relationship between genotypes and resistance phenotypes. Methods to be employed include construction and analysis of three-generation pedigrees based on crosses between resistant and susceptible strains; linkage map construction with AFLPs; QTL analysis; growth inhibition and mortality bioassays; and (with collaborators) measurement of toxin binding phenotypes of larval midgut membranes. This project will be the first to characterize the interaction between different genes conferring insect resistance to a bacterial toxin, and will provide important tools for future determination of the molecular identity of the newly-discovered genes.
This project will contribute molecular and genetic tools that can be applied to ensure the continued sustainability of safe and effective control of insect pests by Bt transgenic crops, with a reduction of chemical insecticide use in US agroecosystems. Undergraduates from Clemson University and minority institutions in South Carolina will participate in the research. Results from this project will be featured in the activities of the South Carolina DNA Learning Center, recently established to provide outreach education in biotechnology and modern genetics to K-12 teachers, non-formal educators, students, and the general public.
