title: Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system.
abstract: Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera.
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: wangjing610610@163.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: zhn@njau.edu.cn.
authors.forename: Haonan
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: 2013202034@njau.edu.cn.
authors.forename: Huidong
authors.initials: H
authors.lastname: Wang
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: 2015102101@njau.edu.cn.
authors.forename: Shan
authors.initials: S
authors.lastname: Zhao
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: 2015202036@njau.edu.cn.
authors.forename: Yayun
authors.initials: Y
authors.lastname: Zuo
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: yhyang@njau.edu.cn.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Yang
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: wyd@njau.edu.cn.
authors.forename: Yidong
authors.initials: Y
authors.lastname: Wu
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004731:Endotoxins / Q000378:metabolism*; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D006460:Hemolysin Proteins / Q000378:metabolism*; D007814:Larva / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000382:microbiology; D009036:Moths / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000382:microbiology*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D015820:Cadherins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Bt receptor; CRISPR/Cas9; Cadherin; Helicoverpa armigera; Knockout