Domain III of Cry1Ac Is Critical to Binding and Toxicity against Soybean Looper (Chrysodeixis includens) but Not to Velvetbean Caterpillar (Anticarsia gemmatalis).

Rubina Mushtaq,Juan Jurat-Fuentes,Abdul Shakoori

doi:10.3390/toxins10030095

Rubina Mushtaq, Juan Jurat-Fuentes + Show 1 more

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https://doi.org/10.3390/toxins10030095

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Abstract

Insecticidal proteins Cry1Ac and Cry2Ac7 from the bacterium Bacillus thuringiensis (Bt) belong to the three-domain family of Bt toxins. Commercial transgenic soybean hybrids produce Cry1Ac to control the larvae of the soybean looper (Chrysodeixis includens) and the velvet bean caterpillar (Anticarsia gemmatalis). The specificity of Cry1Ac is determined by loops extending from domain II and regions of domain III in the three-dimensional structure 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 parental toxins. Bioassays with H1.2Ac revealed toxicity against the larvae of A. gemmatalis but not against 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 in A. gemmatalis was specific and saturable. Results from competition binding assays supported the finding that Cry1Ac specificity against A. gemmatalis is mainly dictated by domain II. Taken together, these distinct interactions with binding sites may help explain the differential susceptibility to Cry1Ac in C. includens and A. gemmatalis, and guide the design of improved toxins against soybean pests.

Highlights

Cry proteins from the bacterium Bacillus thuringiensis (Bt) are used in sprays or produced by transgenic crops for environmentally friendly pest control [1]
256–461, respectively, and domain III encompassed amino acids 464–609 (Figure 1). This third domain of Cry1Ac was substituted with domain III of Cry2Ac7, which spanned from amino acids 485–623 (Figure 1)
A. gemmatalis and C. includens [15], yet production of a single toxin greatly increases the probability of resistance evolution in target insects

Summary

Introduction

Cry proteins from the bacterium Bacillus thuringiensis (Bt) are used in sprays or produced by transgenic crops for environmentally friendly pest control [1]. The vast majority of Cry proteins present a shared structural three-domain organization, supporting a similar mode of action. The steps involved in the mode of action of Cry1Ac and other three-domain Bt toxins have been thoroughly reviewed [2]. The Cry1Ac toxin is processed in the midgut fluids to an active core that recognizes binding sites in proteins in the midgut brush border epithelium. This binding is conducive to further processing of the toxin and formation of an oligomer, which inserts in the membrane of midgut cells to form a pore through the insertion of amphipathic alpha helices in domain I.

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