Evolution of Bacillus thuringiensis Cry toxins insecticidal activity.
Bottom Line: 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.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.
Affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México. Apdo. postal 510-3, Cuernavaca 62250, Morelos, Mexico.Show MeSH
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Mentions: We have revised several examples of evolved Cry toxins with improved performance in controlling different insect pests. Some of these Cry mutants show novel insecticidal activity, others improved toxicity against a specific target and others were shown to be active against resistant insects to Cry toxins. Nevertheless, as pointed before, most of these Cry mutants were the result of analysing few mutants in different insect species but not from a high through output system that could detect improved mutants from a large number of variants. We believe that the evolution of Cry toxicity using high through output systems is likely to provide toxins that will perform better in controlling insect pests. In Fig. 3 we propose a general strategy for in vitro evolution of toxicity of Cry toxins. The rational behind this strategy is that Cry toxin mutants with improved binding affinities to either BBMV from the target insect or isolated insect Cry-binding proteins, will provide mutants that are likely to show enhanced insecticidal activity. The first step is the construction of cry gene library of variants that could then be screened for binding to BBMV or toxin-receptors from the target insect. Several methods for creating variability could be exploited depending on the binding selection procedure. Using general mutagenesis strategies as gene shuffling or prone PCR can explore the whole gene cry sequence including domain I. As discussed earlier, there are examples of domain I mutations that enhance Cry toxicity presumably by enhancing membrane partioning into the membrane (Mandal et al., 2007; Alzate et al., 2010). Gene libraries could also be created by shuffling domain III among different Cry toxins or by shuffling domain II loop regions that are likely to provide Cry toxins with improved toxicity or altered specificity. Finally, gene libraries could be created by mutation of receptor binding epitopes like domain II loop regions or residues of domain III β16. In the second step the cry gene libraries are cloned into phagemid vectors for the display of Cry mutants in the phage particles. The third step is the screening of libraries by biopanning against BBMV or pure receptor molecules. The fourth step is the selection of Cry mutants with enhanced binding to BBMV or receptors. Finally, the fifth step is the determination of toxicity of Cry toxins with improved binding against the target insect. Improved variants could be the substrate for additional mutagenesis, binding selection and bioassays.
Affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México. Apdo. postal 510-3, Cuernavaca 62250, Morelos, Mexico.