DOMAIN-III SUBSTITUTION IN BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN CRYIA(B) RESULTS IN SUPERIOR TOXICITY FOR SPODOPTERA-EXIGUA AND ALTERED MEMBRANE-PROTEIN RECOGNITION

To test our hypothesis that substitution of domain III of Bacillus thu ringiensis delta-endotoxin (Cry) proteins might improve toxicity to pe st insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening as say was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid r esidue, Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA( b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than C ryIC, In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain II I substitution appeared not to affect binding-site specificity. Howeve r, binding to a 200-kDa protein by CryIA(b) in preparations of solubil ized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution, A reciprocal hybrid contain ing domains I and II of CryIC and domain III of CryIA(b) did bind to t he 200-kDa protein, confirming that domain III of CryIA(b) was essenti al for this reaction. These results show that domain III of CryIC prot ein plays an important role in the level of toxicity to S. exigua, tha t substitution of domain III may be a powerful tool to increase the re pertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. e xigua.