41st Annual Meeting of the Society for Invertebrate Pathology
and 9th International Conference on Bacillus thuringiensis

The use of transgenic Bt-maize is increasing yearly (last year accounting for about 19% of the total maize planted area in the world) because of the efficient control of the corn borers, in especial Ostrinia nubilalis . Resistance to Bacillus thuringiensis (Bt) insecticidal toxins has been linked to the 12-domain cadherin locus in 3 lepidopteran species. The O. nubilalis cadherin gene has been revealed as a complex gene of about 20 kbp in length, with 34 introns. In the present work, we have studied the size polymorphism of the gene in a Spanish population, by amplifying the genomic sequence of the gene in 16 overlapping regions. The variability observed was not uniformly distributed, with a maximum in region 14 and a minimum (no polymorphism) in region 4. All this size variability must be due to changes in the intronic regions because we found no detectable size differences in mRNA. This variability can be useful to select appropriate polymorphic regions to be used as markers of this gene in experiments such us to determine the genetic linkage of the cadherin to Bt resistance traits.

Cry toxins produced by the bacterium Bacillus thuringiensis (Bt) are effective biological insecticides. Cadherin-like proteins serve as functional receptors for these toxins in Lepidoptera, but little is known in Coleoptera. We present the first report demonstrating a functional interaction between the coleopteran-specific Cry3Aa toxin and a coleopteran cadherin. This putative Cry3Aa receptor cadherin was cloned from Tenebrio molitor larval midgut mRNA and the predicted protein, TmCad1, shares similarity with lepidopteran cadherin Bt receptors in both domain structure and putative toxin binding region. A TmCad1 peptide (rTmCad1p) containing the putative toxin binding region specifically interacted with Cry3Aa and promoted the formation of Cry3Aa toxin oligomers. Moreover, functional analysis demonstrated that TmCad is a toxin binding protein that promotes Cry3Aa toxicity in T. molitor larvae. Our data suggests similarities between the mode of action of Cry toxins in Lepidoptera and Coleoptera.

An Ostrinia nubilalis colony was selected for resistance to Bacillus thuringiensis Cry1Ab protoxin. Previous work evidenced the implication of more than one genetic locus and the reduction of the cadherin receptor. We have now determined the contribution of the cadherin gene to the overall Cry1Ab resistance in this strain. Individual larval midguts from susceptible (Europe-S) and resistant (Europe-R) insects were used to prepare cDNAs from the cadherin gene. We found major mutations that suggested highly structural deficient proteins because they introduced premature termination codons (PTC) and/or large deletions (1383-1701 bp). In the resistant strain, these mutations were found in 13 out of 20 insects analyzed. In the susceptible strain, only one PTC was detected among the major mutations, but always in heterozygotes. To check for the contribution of the major mutations to the resistance, Europe-R insects were subjected to a high dose of Cry1Ab protoxin. The analysis of the survivors showed that major mutations were absent. These results support a polygenic inheritance of resistance in the Europe-R strain, in which mutations in the cadherin gene would contribute to resistance by means of an additive effect.