Abstract
Cry1A insecticidal toxins bind sequentially to different larval gut proteins facilitating oligomerization, membrane insertion and pore formation. Cry1Ac interaction with cadherin triggers oligomerization. However, a mutation in an ABC transporter gene (ABCC2) is linked to Cry1Ac resistance in Plutella xylostella. Cry1AcMod, engineered to lack helix α-1, was able to form oligomers without cadherinbinding and effectively countered Cry1Ac resistance linked to ABCC2. Here we analyzed Cry1Ac and Cry1AcMod binding and oligomerization by western blots using brush border membrane vesicles (BBMV) from a strain of P. xylostella susceptible to Cry1Ac (Geneva 88) and a strain with resistance to Cry1Ac (NO-QAGE) linked to an ABCC2 mutation. Resistance correlated with lack of specific binding and reduced oligomerization of Cry1Ac in BBMV from NO-QAGE. In contrast, Cry1AcMod bound specifically and still formed oligomers in BBMV from both strains. We compared association of pre-formed Cry1Ac oligomer, obtained by incubating Cry1Ac toxin with a Manduca sexta cadherin fragment, with BBMV from both strains. Our results show that pre-formed oligomers associate more efficiently with BBMV from Geneva 88 than with BBMV from NO-QAGE, indicating that the ABCC2 mutation also affects the association of Cry1Ac oligomer with the membrane. These data indicate, for the first time, that ABCC2 facilitates Cry1Ac oligomerization and oligomer membrane insertion in P. xylostella.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Bacillus thuringiensis Toxins
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Bacterial Proteins / chemistry*
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Bacterial Proteins / pharmacology
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Biological Control Agents / chemistry*
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Biological Control Agents / metabolism
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Cell Membrane / chemistry
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Cell-Derived Microparticles / chemistry
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Cell-Derived Microparticles / metabolism
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Endotoxins / chemistry*
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Endotoxins / genetics
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Endotoxins / metabolism
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Endotoxins / pharmacology
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Hemolysin Proteins / chemistry*
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Hemolysin Proteins / genetics
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Hemolysin Proteins / metabolism
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Hemolysin Proteins / pharmacology
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Insect Proteins / chemistry*
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Insect Proteins / genetics
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Insect Proteins / metabolism
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Insecticide Resistance
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Insecticides / chemistry*
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Insecticides / metabolism
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Larva / chemistry
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Larva / drug effects*
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Larva / metabolism
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Manduca / drug effects
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Microvilli / chemistry
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Microvilli / drug effects
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Microvilli / metabolism
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Moths / drug effects
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Multidrug Resistance-Associated Protein 2
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Multidrug Resistance-Associated Proteins / chemistry*
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Multidrug Resistance-Associated Proteins / genetics
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Multidrug Resistance-Associated Proteins / metabolism
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Mutation
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Protein Binding
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Protein Engineering
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Protein Isoforms
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Protein Multimerization
Substances
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Bacillus thuringiensis Toxins
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Bacterial Proteins
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Biological Control Agents
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Endotoxins
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Hemolysin Proteins
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Insect Proteins
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Insecticides
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Multidrug Resistance-Associated Protein 2
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Multidrug Resistance-Associated Proteins
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Protein Isoforms
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insecticidal crystal protein, Bacillus Thuringiensis