title: Insecticidal Specificity of Cry1Ah to Helicoverpa armigera Is Determined by Binding of APN1 via Domain II Loops 2 and 3.
abstract: Bacillus thuringiensis Cry1Ah protein is highly toxic against Helicoverpa armigera but shows no toxicity against Bombyx mori larvae. In contrast, the closely related Cry1Ai toxin showed the opposite phenotype: high activity against B. mori but no toxicity against H. armigera. Analysis of binding of Cry1Ah to brush border membrane vesicle (BBMV) proteins from H. armigera and B. mori by surface plasmon resonance revealed association of toxin binding with insect specificity. Pulldown experiments identified aminopeptidase N1 (APN1) as a Cry1Ah binding protein that was not observed in the assays using B. mori BBMV proteins. The APN1 Cry1Ah binding region was narrowed to the region fromA548 toS798 (fragment H3) by expressing four different APN1 fragments in Escherichia coli and analyzing Cry1Ah binding by ligand blot. Binding competition experiments of Cry1Ah to APN1 fragment H3 using synthetic peptides corresponding to four predicted domain II loop regions showed that loop 2 and loop 3 have additive effects on binding to APN1 fragment H3. Moreover, switching of loop 2 and loop 3 regions from Cry1Ah to Cry1Ai toxins showed that loop 2 and loop 3 are both involved in specificity and toxicity against H. armigera IMPORTANCE: Domain II loop regions have been shown to be involved in binding to larval gut proteins mediating insect specificity. The modification of loop regions is a direct and effective method to construct new Cry toxin variants to increase toxicity or modify specificity. Our results show that the exchange of loop regions from one toxin into another is a successful scheme for modification of B. thuringiensis Cry toxin specificity.
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, People's Republic of China.
authors.forename: Zishan
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, People's Republic of China.
authors.forename: Yuxiao
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, People's Republic of China.
authors.forename: Gemei
authors.initials: G
authors.lastname: Liang
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, People's Republic of China.
authors.forename: Fuping
authors.initials: F
authors.lastname: Song
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, People's Republic of China.
authors.forename: Xueping
authors.initials: X
authors.lastname: Zhou
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, People's Republic of China jzhang@ippcaas.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology*; D001667:Binding, Competitive / Q000502:physiology*; D004731:Endotoxins / Q000235:genetics / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000235:genetics / Q000494:pharmacology*; D007306:Insecticides / Q000494:pharmacology*; D009036:Moths / Q000187:drug effects*; D011485:Protein Binding / Q000502:physiology; D000072417:Protein Domains; D013045:Species Specificity
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Bacillus thuringiensis; Cry1Ah; Cry1Ai; Helicoverpa armigera; aminopeptidase N; insecticidal specificity