Toxicity, binding, and permeability analyses of four Bacillus thuringiensis Cry1 delta-endotoxins using brush border membrane vesicles of Spodoptera exigua and Spodoptera frugiperda

The binding and pore formation properties of four Bacillus thuringiensis Cr y1 toxins were analyzed by using brush border membrane vesicles from Spodop tera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nont oxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic t o S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was acti ve against S. frugiperda but only marginally active against S. exigua. Bioa ssays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the e ffects of iodination on toxin activity were different. The toxicities of I- labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly les s than the toxicities of the unlabeled toxins, while CrylCa retained its in secticidal activity when it was labeled with I-125. Binding assays showed t hat iodination prevented Cry1Fa from binding to Spodoptera brush border mem brane vesicles. I-125-labeled Cry1Ac, Cry1Bb, and CrylCa bound with high-af finities to brush border membrane vesicles from S. exigua and S. frugiperda . Competition binding experiments performed with heterologous toxins reveal ed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, a nd Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membran e vesicles and toxicity was detected, Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a li ght scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilize d brush border membrane vesicles, but the extent of permeabilization did no t necessarily correlate with in vivo toxicity.