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Applied and Environmental Microbiology, September 2002, p. 4583-4592, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4583-4592.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

Neema Agrawal, Pawan Malhotra, and Raj K. Bhatnagar*

International Center for Genetic Engineering and Biotechnology, New Delhi-110067, India

Received 29 January 2002/ Accepted 29 May 2002

Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.

* Corresponding author. Mailing address: Insect Resistance Group, ICGEB, P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi-110067, India. Phone: 91-11-6195007. Fax: 91-11-616 2316. E-mail: raj{at}icgeb.res.in.

Applied and Environmental Microbiology, September 2002, p. 4583-4592, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4583-4592.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pigott, C. R., Ellar, D. J. (2007). Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity. Microbiol. Mol. Biol. Rev. 71: 255-281 [Abstract] [Full Text]  
  • Avisar, D., Segal, M., Sneh, B., Zilberstein, A. (2005). Cell-cycle-dependent resistance to Bacillus thuringiensis Cry1C toxin in Sf9 cells. J. Cell Sci. 118: 3163-3171 [Abstract] [Full Text]  
  • Avisar, D., Keller, M., Gazit, E., Prudovsky, E., Sneh, B., Zilberstein, A. (2004). The Role of Bacillus thuringiensis Cry1C and Cry1E Separate Structural Domains in the Interaction with Spodoptera littoralis Gut Epithelial Cells. J. Biol. Chem. 279: 15779-15786 [Abstract] [Full Text]  
  • Rajagopal, R., Sivakumar, S., Agrawal, N., Malhotra, P., Bhatnagar, R. K. (2002). Silencing of Midgut Aminopeptidase N of Spodoptera litura by Double-stranded RNA Establishes Its Role as Bacillus thuringiensis Toxin Receptor. J. Biol. Chem. 277: 46849-46851 [Abstract] [Full Text]  


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