The HevCaLP protein mediates binding specificity of the Cry1A class of Bacillus thuringiensis toxins in Heliothis virescens.
Retrotransposon-mediated disruption of the BtR-4 gene encoding the Heliothis virescens cadherin-like protein (HevCaLP) is linked to high levels of resistance in the YHD2 strain to Cry1Ac toxin from Bacillus thuringiensis. This suggests that HevCaLP functions as a Cry1Ac toxin receptor on the surface of midgut cells in susceptible larvae and that the BtR-4 gene disruption eliminates this protein in resistant larvae. However, Cry1Ac toxin binding to HevCaLP is yet to be reported. We used the polymerase chain reaction and immunoblotting as tools to discriminate between individual H. virescens larval midguts from susceptible (YDK) and resistant (CXC, KCBhyb, and YHD2-B) strains according to their BtR-4 gene disruption genotype and phenotype. This approach allowed us to test the correlation between BtR-4 gene disruption, lack of HevCaLP, and altered Cry1A toxin binding. Toxin-binding assays using brush border membrane vesicles revealed that a wild-type BtR-4 allele is necessary for HevCaLP production and Cry1Aa toxin binding, while most of Cry1Ab and Cry1Ac binding was independent of the BtR-4 genotype. Moreover, toxin competition experiments show that KCBhyb midguts lacking HevCaLP are more similar to midguts of the original YHD2 strain than to the current YHD2-B strain. This resolves discrepancies in published studies of Cry1A binding in YHD2 and supports our earlier suggestion that a separate genetic change occurred in YHD2 after appearance of the cadherin disruption, conferring even higher resistance in the resulting YHD2-B strain as well as a large reduction in Cry1Ab and Cry1Ac binding.
Jurat-Fuentes
Juan L
JL
Department of Entomology and Biochemistry, University of Georgia, Athens, Georgia 30602-2279, USA.
Gahan
Linda J
LJ
Gould
Fred L
FL
Heckel
David G
DG
Adang
Michael J
MJ
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism; D001667:Binding, Competitive; D015820:Cadherins / Q000235:genetics / Q000502:physiology*; D004731:Endotoxins / Q000378:metabolism; D005838:Genotype; D006460:Hemolysin Proteins; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D007457:Iodine Radioisotopes / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D009036:Moths / Q000382:microbiology*; D010641:Phenotype; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015820:Cadherins; C095416:Cry toxin receptors; D004731:Endotoxins; D006460:Hemolysin Proteins; C433228:HevCaLP protein, Heliothis virescens; D019476:Insect Proteins; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis