title: Three toxins, two receptors, one mechanism: Mode of action of Cry1A toxins from Bacillus thuringiensis in Heliothis virescens.
abstract: Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity.
authors.affiliation: Department of Entomology, Max-Planck Institute for Chemical Ecology, Hans-Knoell-Str. 8, 07745, Jena, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Bretschneider
authors.affiliation: Department of Entomology, Max-Planck Institute for Chemical Ecology, Hans-Knoell-Str. 8, 07745, Jena, Germany.
authors.forename: David G
authors.initials: DG
authors.lastname: Heckel
authors.affiliation: Department of Entomology, Max-Planck Institute for Chemical Ecology, Hans-Knoell-Str. 8, 07745, Jena, Germany. Electronic address: ypauchet@ice.mpg.de.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Pauchet
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D015820:Cadherins / Q000235:genetics* / Q000378:metabolism; D004731:Endotoxins / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000494:pharmacology*; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000382:microbiology; D009036:Moths / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000382:microbiology; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D015820:Cadherins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: ABC transporter; Bt Cry1A toxin; Cadherin; Heliothis virescens; Insecticide resistance