Enhanced toxicity of Bacillus thuringiensis Cry3A 8-endotoxin in coleopterans by mutagenesis in recetor binding loop (original) (raw)

We used site-directed mutagenesis to modify the cally cleaved from an inactive protoxin, to an active toxin Bacillus thuringiensis cry3A gene in amino acid residues 350-form within the insect midgut. The activated toxin binds to 354. Two mutant toxins, A1 (Ra4sA,Yss0F, Y3sIF). and A2 receptors in the midgut and is believed to integrate into the _._ _ (R_sA,AYsso,AYssl), showed significantly improved toxicity lipid bilayer of the brush border membrane. Ion channels are O "" against Tenebrio molitor (yellow mealworm). The mutant toxin formed, causing midgut cells to lose their membrane potential. A1 was also more potent against both Leptinotarsa decemlineata If the rate of damage to the midgut exceeds the rate of repair, (Colorado potato beetle) and Cho,somela scripts (cottonwood lesions form. bacteria invade the hemocele, and death results leaf beetle), while A2 displayed enhanced toxicity only in L. from bacterial septicemia. The fi-endotoxins from B.t. comdecemlineata. Competitive binding assays of L. decemlineata prise a group of over 100 related proteins [5}, which were t5 brush border membrane vesicles (BBMV) revealed that binding previously categorized by insecticidal activity [6] but currently affinities for the AI and A2 mutant toxins were ca. 2.5-fold cq" I-higher than for the wild-type Cry3 toxin. S mar bind ng assays by amino acid similarity [7]. The spectrum of toxicity for each O with C. scripta BBMV revealed a ca. 5-fold lower dissociation toxin is relatively narrow, while the collective activity of this rate for the Al mutant as compared to that of Cry3A. group of pesticidal toxins now spans seven orders of insect © 2000 Federation of European Biochemical Societies. and several other invertebrate groups including nematodes. mites and protozoans [8].

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