Production of Bacillus thuringiensis Recombinant Cry Proteins and Analysis of Mode of Action of their Toxicity

Abstract

The Cry insecticidal proteins of Bacillus thuringiensis (Bt) are produced by transgenic crops for effective and environmentally-safe insect pest control. These transgenic Bt crops are considered to be the most successful agricultural biotechnology for insect control, yet their sustainability is threatened by the evolution of resistance in targeted pests. The evidence suggests that this resistance is probably due to the alterations in recognition of Cry toxin receptors in the insect midgut membrane. Consequently, information is needed on determinants of receptor recognition for designing improved toxins and to adopt effective insect resistance management practices. In this study four Cry proteins, Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 were expressed either in E. coli expression system or in native Bt strain. Insoluble Cry1Ac and Cry2Ac7 proteins were refolded and purified prior to use. Bioassays of Cry1Ac (C-terminally truncated version) and Cry2Ac7 protoxins were performed with an armyworm, Spodoptera litura Fabricius (Lepidoptera: Noctuidae), a polyphagous cosmopolitan insect which is a serious crop pest of many Asian countries including Pakistan. Cry2Ac7 was found to be toxic to this pest whereas tnCry1ac (truncated Cry1Ac) was nontoxic. To activate the protoxins, the proteins were trypsin digested and further purified by anion-exchange chromatography. Trypsin activated proteins (toxins) were assayed against velvetbean caterpillar, Anticarsia gemmatalis and soybean looper, Chrysodeixes includens (Pseudoplusia includens) which are lepidopteran pests of crops of great economic importance especially soybean. All these proteins were found to be toxic to both of these pests. Amongst all these toxins Cry1Ac was the most highly toxic protein. In general, C. includens larvae were always less susceptible compared to larvae of A. gemmatalis to these toxins which is in agreement with the previous findings of the relative susceptibilities of these soybean pests for Bt pesticides. The Bt insecticidal proteins Cry1Ac and Cry2Ac7 belong to the three domain family of Bt toxins. Commercial transgenic soybean hybrids produce Cry1Ac to control larvae of the soybean looper and the velvetbean caterpillar. Specificity of Cry proteins is known to be majorly determined by domain II and domain III of the toxin. In this study, we constructed a hybrid toxin (H1.2Ac) containing domains I and II of Cry1Ac and domain III of Cry2Ac7, in an attempt to obtain a protein with enhanced toxicity compared to parent toxins. H1.2Ac protein was expressed in E. coli expression system and was refolded and purified using Histagged chromatography. Recombinant H1.2Ac protein was also trypsin activated prior to the bioassays. Bioassays with H1.2Ac revealed toxicity to larvae of A. gemmatalis but not to C. includens. Saturation binding assays with radiolabeled toxins and midgut brush border membrane vesicles demonstrated no specific H1.2Ac binding to C. includens, while binding to A. gemmatalis was specific and saturable. The competition binding assays showed that Cry1Ac specificity against A. gemmatalis was mainly dictated by domain II. The binding assay in the presence of N-acetylgalactosamine (GalNAc) further clarifies the significance of domain III of Cry1Ac in binding to the receptors of C. includens. Taken together, these distinct interactions with binding sites explain the differential susceptibility of C. includens and A. gemmatalis to Cry1Ac and may provide guidelines for designing the improved toxins against soybean pests. The significance of this work is in identifying Cry2Ac7 as toxic to S. litura and Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 as toxic to C. includens and A. gemmatalis. Cry1Ie2 can be pyramided with any or all of Cry1Ac, Cry2Ac7 and Cry1Fa proteins in transgenic plants to enhance their efficiency to combat insect pests. While the present study presents evidence for the importance of Cry1Ac domain III for toxicity against C. includens, further research would be needed to identify lethal Cry1Ac receptors and determine their interactions with domains II and/or III of Cry1Ac. This information contributes to the design of more active insecticidal proteins against this pest and our general understanding of the Cry mode of action in Lepidoptera.