title: Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.
abstract: To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay 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 residue. 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 CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua.
authors.affiliation: Department of Molecular Biology, Centre for Plant Breeding and Reproduction Research, Wageningen, The Netherlands. R.A.deMaagd@CPRO.DLO.NL
authors.forename: R A
authors.initials: RA
authors.lastname: de Maagd
authors.forename: M S
authors.initials: MS
authors.lastname: Kwa
authors.forename: H
authors.initials: H
authors.lastname: van der Klei
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
authors.forename: B
authors.initials: B
authors.lastname: Schipper
authors.forename: J M
authors.initials: JM
authors.lastname: Vlak
authors.forename: W J
authors.initials: WJ
authors.lastname: Stiekema
authors.forename: D
authors.initials: D
authors.lastname: Bosch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism; D006460:Hemolysin Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism / Q000382:microbiology*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis