Evolution of Bacillus thuringiensis Cry toxins insecticidal activity

Abstract

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.

Fig. 1

Binding regions of monomeric and oligomeric forms mapped in Cry1Ab toxin to Manduca sexta receptors, cadherin, alkaline phosphatase (ALP), and aminopeptidase-N (APN). The monomeric form depicted corresponds to the three-dimensional structure of Cry1Aa (pdb 1CIY) and the oligomeric structure corresponds to Cry4Ba trimeric structure (pdb 1W99).

Fig. 2

Representation of Cry toxin regions where mutations enhanced insecticidal activity in different Cry toxins. The three-dimensional structure of Cry1Aa toxin (pdb 1CIY) is depicted.

Fig. 3

General strategy for in vitro evolution of toxicity of Cry toxins. Five steps are proposed for in vitro evolution of Cry toxins, 1. Construction of gene libraries with Cry variants obtained by different mutagenesis strategies (prone PCR, gene shuffling, domain III swapping, domain II loop 2 swapping and mutagenesis of receptor binding regions); 2. Display of gene libraries on phage; 3. Biopanning of phage display libraries using brush border membrane vesicles of insect of interest or purified receptors (cadherin is shown as example); 4. Selection of variants with improved binding characteristics; 5. Toxicity assays against the target insect to select Cry toxins with improved insecticidal activity.