Domains of Bacillus thuringiensis crystal proteins involved in insecticidal activity

The expected increase in application of Bacillus thuringiensis (Bt) in crop protection makes it necessary to anticipate the development of Bt-resistant insects. To safeguard the long-term use of Bt-based insecticides, we studied the mode of action of Bt crystal proteins. CryIA(b), CryIC and CryIE are all toxic to lepidoptera, but each protein has its own specificity profile. In order to identify regions in the proteins involved in insecticidal specificity, hybrids between the genes cryIA(b) cryIC and cryIE were generated. Plasmids were constructed carrying two truncated crystal protein genes. In vivo recombination between these genes resulted in hybrids encoding full-length 140 kDa protoxins. In this manner, sets of CryIA(b)-CryIE, CryIC-CryIE and CryIE-CryIC hybrid proteins were isolated. The analysis of the CryIC-CryIE hybrids showed that domain II is involved in receptor binding, but that domain II of CryIC is not sufficient for the high activity of this protein towards Spodoptera exigua and Mamestra brassicae. Biochemical evidence and insect toxicity assays indicate that all CryIA(b)-CryIE hybrids are malfolded and precipitate as insoluble aggregates