Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica

• Published in: Journal of invertebrate pathology 2013-06, Vol.113 (2), p.123-128
• Main Authors: Yamaguchi, Takuya, Bando, Hisanori, Asano, Shin-ichiro
• Format: Article
• Language: English
• Subjects: adults; alkaline phosphatase; Amino Acid Sequence; amino acid sequences; Animals; Bacillus thuringiensis; Bacterial Proteins - metabolism; beta-glucosidase; Biological and medical sciences; Biological control; chromatography; Coleoptera - metabolism; Control; Cry8; Diptera; DNA primers; Endotoxins - metabolism; Fundamental and applied biological sciences. Psychology; genes; Glucosidase; Glucosidases - chemistry; Glucosidases - isolation & purification; Glucosidases - metabolism; Hemolysin Proteins - metabolism; Insect Proteins - chemistry; Insect Proteins - isolation & purification; Insect Proteins - metabolism; insecticidal properties; insects; Japanese beetle; larvae; Lepidoptera; mechanism of action; membrane alanyl aminopeptidase; Molecular Sequence Data; Peptide Mapping; Pest Control, Biological; Phytopathology. Animal pests. Plant and forest protection; polymerase chain reaction; Popillia japonica; Protozoa. Invertebrates; Receptor; receptors; Sequence Alignment; Sequence Analysis, Protein; toxins
• ISSN: 0022-2011; 1096-0805
• DOI: 10.1016/j.jip.2013.03.006

[Display omitted] •The Cry8Da binding protein was purified by column chromatography.•Three internal amino acid sequences of the Cry8Da binding protein were determined.•The internal amino acid sequences shared homology with Coleopteran β-glucosidases.•The fraction containing the 150kDa Cry8Da binding protein had β-glucosidase activity.•Partial β-glucosidase genes were amplified by PCR using degenerate primers. Cry8Da from Bacillus thuringiensis galleriae SDS-502 has insecticidal activity against both the larvae and adult Japanese beetle (Popillia japonica Newman). The receptor determines the specificity of the insecticidal activity of Cry proteins and hence, in order to reveal the mode of action of Cry toxin, receptor identification is a necessary step. However, a receptor for Cry8-type toxin has not been identified in the Scarabaeidae family of insects. Therefore, we aimed to identify the receptor of Cry8Da toxin in adult P. japonica BBMV. A ligand blot showed the Cry8Da toxin only bound to a 150kDa protein in the BBMV of adult P. japonica. In order to identify the Cry8Da toxin binding protein, it was purified by column chromatography and three internal amino acid sequences were determined. Two of the three internal amino acid sequences shared homology with Coleopteran β-glucosidases. In addition, the fraction containing the Cry8Da toxin binding protein had β-glucosidase activity but no aminopeptidase N and alkaline phosphatase activity, both of which are commonly reported as receptors for Cry toxins in Lepidopteran and Dipteran insects. The β-glucosidase homologous genes could be amplified by PCR using degenerate oligonucleotide primers designed from a conserved sequence of Coleopteran β-glucosidases and an internal amino acid sequence of the Cry8Da toxin binding protein. Taken together, the β-glucosidase in adult P. japonica BBMV is the receptor for B. thuringiensis Cry8Da toxin.