title: In vivo and in vitro binding of Vip3Aa to Spodoptera frugiperda midgut and characterization of binding sites by (125)I radiolabeling.
abstract: Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the binding protocol for the (125)I-Vip3Aa to S. frugiperda brush border membrane vesicles (BBMV) allowed the determination of binding parameters of Vip3A proteins for the first time. Heterologous competition using Vip3Ad, Vip3Ae, and Vip3Af as competitor proteins showed that they share the same binding site with Vip3Aa. In contrast, when using Cry1Ab and Cry1Ac as competitors, no competitive binding was observed, which makes them appropriate candidates to be used in combination with Vip3A proteins in transgenic crops.
authors.affiliation: Departamento de Genética, Facultad de CC. Biológicas, Universidad de Valencia, Valencia, Spain.
authors.forename: Maissa
authors.initials: M
authors.lastname: Chakroun
authors.affiliation: Departamento de Genética, Facultad de CC. Biológicas, Universidad de Valencia, Valencia, Spain juan.ferre@uv.es.
authors.forename: Juan
authors.initials: J
authors.lastname: Ferré
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D004064:Digestive System / Q000187:drug effects / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000648:ultrastructure; D006460:Hemolysin Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007814:Larva / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007457:Iodine Radioisotopes; C447139:Vip3A protein, Bacillus thuringiensis; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014357:Trypsin