title: Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins.
abstract: In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins.
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Tanaka
authors.affiliation: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Miyamoto
authors.affiliation: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Noda
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Endo
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kikuta
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan. Electronic address: ryoichi@cc.tuat.ac.jp.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Sato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000472:pathogenicity; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000633:toxicity*; D012831:Bombyx / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D003001:Cloning, Molecular; D004731:Endotoxins / Q000096:biosynthesis / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000633:toxicity*; D054884:Host-Pathogen Interactions; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007305:Insecticide Resistance / Q000235:genetics* / Q000276:immunology; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000633:toxicity; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C095416:Cry toxin receptors; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: ABC transporter C2; Bacillus thuringiensis; BmABCC2; Bombyx mori; Cry1A; Domain II; Receptor