title: The Heliothis virescens cadherin protein expressed in Drosophila S2 cells functions as a receptor for Bacillus thuringiensis Cry1A but not Cry1Fa toxins.
abstract: Genetic knockout of the BtR4 gene encoding the Heliothis virescens cadherin-like protein (HevCaLP) is linked to resistance against Cry1Ac toxin from Bacillus thuringiensis. However, the functional Cry1Ac receptor role of this protein has not been established. We previously proposed HevCaLP as a shared binding site for B. thuringiensis (Bt) Cry1A and Cry1Fa toxins in the midgut epithelium of H. virescens larvae. Considering that Cry1Ac and Cry1Fa are coexpressed in second-generation transgenic cotton for enhanced control of Heliothine and Spodoptera species, our model suggests the possibility of evolution of cross resistance via alteration of HevCaLP. To test whether HevCaLP is a Cry1Ac and Cry1Fa receptor, HevCaLP was transiently expressed on the surface of Drosophila melanogaster Schneider 2 (S2) cells. Expressed HevCaLP bound [(125)I]Cry1A toxins under native (dot blot) and denaturing (ligand blot) conditions. Affinity pull-down assays demonstrated that Cry1Fa does not bind to HevCaLP expressed in S2 cells or in solubilized brush border membrane proteins. Using a fluorescence-based approach, we tested the ability of expressed HevCaLP to mediate toxicity of Cry1A and Cry1Fa toxins. Cry1A toxins killed S2 cells expressing HevCaLP, whereas Cry1Fa toxin did not. Our results demonstrate that HevCaLP is a functional Cry1A but not Cry1Fa receptor.
authors.affiliation: Department of Entomology, University of Georgia, Athens, Georgia 30602-2603, USA.
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: Jurat-Fuentes
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Adang
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism*; D016923:Cell Death; D004331:Drosophila melanogaster / Q000166:cytology*; D004731:Endotoxins / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006460:Hemolysin Proteins; D007457:Iodine Radioisotopes / Q000378:metabolism; D007915:Lepidoptera / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015820:Cadherins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D049452:Green Fluorescent Proteins