title: Knockdown of aminopeptidase-N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein Cry1Ac.
abstract: Aminopeptidase-N (APN) and cadherin proteins located at the midgut epithelium of Helicoverpa armigera have been implicated as receptors for the Cry1A subfamily of insecticidal proteins of Bacillus thuringiensis. Ligand blot analysis with heterologously expressed and purified H. armigera Bt receptor with three closely related Cry1A proteins tentatively identified HaAPN1 as an interacting ligand. However, to date there is no direct evidence of APN being a functional receptor to Cry1Ac in H. armigera. Sf21 insect cells expressing HaAPN1 displayed aberrant cell morphology upon overlaying with Cry1Ac protein. Down-regulating expression of HaAPN1 by RNA interference using double-stranded RNA correlated with a corresponding reduction in the sensitivity of HaAPN1-expressing cells to Cry1Ac protein. This clearly establishes that insect cells expressing the receptor recruit sensitivity to the insecticidal protein Cry1Ac, and their susceptibility is directly dependent on the amount of HaAPN1 protein expressed. Most importantly, silencing of HaAPN1 in H. armigera in vivo by RNA interference resulted in reduced transcript levels and a corresponding decrease in the susceptibility of larvae to Cry1Ac. BIAcore analysis of HaAPN1/Cry1Ac interaction further established HaAPN1 as a ligand for Cry1Ac. This is the first functional demonstration of insect aminopeptidase-N of H. armigera being a receptor of Cry1Ac protein of B. thuringiensis.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067, India.
authors.forename: Swaminathan
authors.initials: S
authors.lastname: Sivakumar
authors.forename: Raman
authors.initials: R
authors.lastname: Rajagopal
authors.forename: G Raja
authors.initials: GR
authors.lastname: Venkatesh
authors.forename: Anand
authors.initials: A
authors.lastname: Srivastava
authors.forename: Raj K
authors.initials: RK
authors.lastname: Bhatnagar
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D018826:CD13 Antigens / Q000502:physiology*; D004731:Endotoxins / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000494:pharmacology*; D019476:Insect Proteins / Q000302:isolation & purification / Q000502:physiology; D007306:Insecticides / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects; D007915:Lepidoptera / Q000201:enzymology*; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000502:physiology; D018411:Spodoptera; D014162:Transfection
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C095416:Cry toxin receptors; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007306:Insecticides; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens