MAPPING OF THE ENTOMOCIDAL FRAGMENT OF SPODOPTERA-SPECIFIC BACILLUS-THURINGIENSIS TOXIN CRYIC

Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic c leavage sites in the CryI proteins facilitates the expression of activ e toxins in transgenic plants to obtain protection from various insect s. However, the engineering of CryIC toxins has, thus far, failed to y ield applicable resistance to armyworms of Spodoptera species represen ting common insect pests worldwide. To improve the production of recom binant CryIC toxins, we established a CryIC consensus sequence by comp arative analysis of three cryIC genes and tested the stability and pro tease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resist ant CryIC core toxin were mapped to amino acid residues 128 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera mid gut proteases may further shorten the C-terminus of CryIC toxin to res idue A615. However, C-terminal truncation of CryIC to residue L614, an d a mutation causing amino acid replacement I610T, abolished the insec ticidal activity of CryIC toxin to S. littoralis larvae, as well as it s resistance to trypsin and Spodoptera midgut proteases. Because no Cr yIC toxin carrying a proteolytically processed N-terminus could be sta bly expressed in bacteria, our data indicate that, in contrast to othe r CryI poteins, an entomocidal fragment located between amino acid pos itions 1 and 627 is required for stable production of recombinant CryI C toxins.