Bacillus thuringiensis ( Bt) toxins are organic insecticides that have been genetically engineered into a variety of agricultural crops that have become widely commercialized. The increasing rate at which farmers are utilizing Bt -crops leads to the fundamental concern that targeted insects will develop Bt -resistance. Strategies for resistance management have been devised and are currently undertaken, however, given our limited understanding of how Bt toxins operate these strategies may not provide long-term solutions. Insect studies have shown that after ingestion by a susceptible host, toxin becomes solubilized and proteolytically activated in the gut. Active toxin binds to gut receptors and inserts itself into the membrane leading to cell lysis and death. While we understand the general mode of Bt action, there is still very little information regarding the genes required for Bt -toxicity. Our lab is interested in defining the molecular mechanisms underlying Bt -toxicity and resistance in the nematode Caenorhabditis elegans . We have taken a genetic approach in C. elegans to identify molecular components required for Bt -toxicity. C. elegans is susceptible to a class of Bt toxins and we have identified five genes that confer resistance to one Bt toxin, Cry5B. Two of these genes,
bre-3 and
bre-5 , have been cloned, and I am currently investigating where these proteins are expressed in the wild-type animal.
bre-3 encodes a novel protein containing multiple potential transmembrane domains and
bre-5 encodes a likely glycosyltransferase. RNAi experiments conducted for both genes demonstrate the null phenotype of
bre-3 and
bre-5 is Bt -resistance with no other detectable phenotypes. Antibodies directed against
bre-5 were created and used to show that
bre-5 localizes to a perinuclear compartment that might be ER/golgi in most and/or all cells of early and late embryos. This localization pattern is consistent with where a carbohydrate modification enzyme would be expected to function. Further experiments with antibodies are underway to localize the
bre-3 protein. Cloning of the
bre-2 gene is currently in progress and close to being identified. Three-factor mapping and deficiency data position
bre-2 to the right of
unc-64 on the right arm of chromosome III. Transgenic rescue experiments are ongoing to determine the molecular identity of this gene. Concomitantly, we are performing RNAi experiments of the predicted genes in this region to clone
bre-2 .