title: Synthesis and toxicity of full-length and truncated bacterial CryIVD mosquitocidal proteins expressed in lepidopteran cells using a baculovirus vector.
abstract: Full-length (72K) and truncated (61K) CryIVD mosquitocidal proteins of Bacillus thuringiensis (Bt) were expressed in Spodoptera frugiperda cells and larvae of Trichoplusia ni using a baculovirus vector to investigate the role of CryIVD peptides in toxicity as well as to evaluate further the baculovirus/lepidopteran system for expressing Bt proteins. The cryIVD genes were inserted into the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) under control of the polyhedrin promoter by recombination in S. frugiperda cells between a transfer vector carrying the Bt genes and vDA26Z, a recombinant AcMNPV carrying the Escherichia coli beta-galactosidase gene under control of the DA26 promoter. Recombinant AcMNPVs carrying the genes were detected as blue occlusion body-negative plaques in monolayers of S. frugiperda cells grown in the presence of X-Gal. Infection of S. frugiperda cells and T. ni larvae with plaque-purified recombinant virus, expressing either the full-length or truncated CryIVD protein, resulted in the synthesis of proteins of the expected size, as confirmed by immunoblot analyses, and their crystallization into cuboidal inclusions in the cytoplasm. Infected cells and purified inclusions from the virus (AcCryIVD) expressing the full-length protein were highly toxic to mosquito larvae, but similar preparations from the virus (AcCryIVD-C) expressing the truncated protein with a 9.6K deletion at the N terminus were non-toxic. Proteolysis with trypsin of CryIVD proteins produced by Bt and the recombinant AcMNPVs yielded peptides corresponding in size, showing that synthesis of mosquitocidal Bt proteins in lepidopteran cells occurred. The lack of toxicity of the truncated CryIVD protein, which like the toxic full-length protein yielded a 34K protein on proteolysis that has been implicated in toxicity, indicates that by itself this protein is non-toxic. These results demonstrate the utility of the baculovirus system for expression of mosquitocidal Bt proteins and for investigation of their mode of action.
authors.affiliation: Department of Entomology, University of California, Riverside 92521.
authors.forename: Y
authors.initials: Y
authors.lastname: Pang
authors.forename: R
authors.initials: R
authors.lastname: Frutos
authors.forename: B A
authors.initials: BA
authors.lastname: Federici
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000633:toxicity; D001427:Bacterial Toxins / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004731:Endotoxins; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006460:Hemolysin Proteins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009036:Moths; D010572:Pest Control, Biological / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010948:Viral Plaque Assay; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D001616:beta-Galactosidase