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J. Biol. Chem., Vol. 279, Issue 16, 15779-15786, April 16, 2004

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The Role of Bacillus thuringiensis Cry1C and Cry1E Separate Structural Domains in the Interaction with Spodoptera littoralis Gut Epithelial Cells^*

Dror Avisar, Menahem Keller, Ehud Gazit¶, Evgenia Prudovsky, Baruch Sneh, and Aviah Zilberstein||

From the Department of Plant Sciences and ^¶Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, the Department of Field and Garden Crops, The Volcani Center, POB 6, Bet Dagan 50250, Israel

The Bacillus thuringiensis -endotoxins Cry1C and Cry1E share toxicity against several important lepidopteran species. Their combined use to delay development of resistance in target insects depends on their differential interaction with the gut epithelial cells. The three structural domains and combinations of two consecutive domains of Cry1C and Cry1E were separately expressed in Escherichia coli, and their interactions with the brush border membrane vesicles (BBMV) of Cry1E-tolerant and -susceptible Spodoptera littoralis larvae were studied. About 80% reduction in binding of Cry1E and each of its separate domains to BBMV of Cry1E-tolerant larvae was observed, whereas Cry1C was toxic to all larvae and bound equally to BBMV derived from both Cry1E-tolerant and -susceptible larvae. These results suggest differential interactions of the two toxins with BBMV encompassing all three domains. Comparable binding assays performed with fluorescent Cry1C and Cry1C domain II showed that Cry1C has higher B_max and lower K_d than Cry1C domain II and further supported the existence of toxin multisite interactions. Competitive binding assays were used to estimate the sequence of interaction events. Cry1C domain II could compete with domain III binding, whereas domain III did not interfere with domain II binding, indicating sequential interactions of domain III and then domain II with the same membrane site. No competition between domain II of Cry1C and Cry1E was observed, confirming the existence of different domain II binding sites for the two toxins. Taken together, all three domains specifically interact with the epithelial cell membrane. The folding of the three-domain toxin probably dictates the sequence of interaction events.

Received for publication, November 18, 2003 , and in revised form, February 2, 2004.

^* This research was supported by USA-Israel Binational Agricultural Research and Development Fund Research Grant Award Is-2629-95 and United States-Israel Binational Science Foundation (Jerusalem, Israel) (BSF) Grant 2001235. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article is dedicated to the memory of Jeff Schell, who initiated our Cry1C study.

^|| To whom correspondence should be addressed: Dept. of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-3-6407410; Fax: 972-3-6409380; E-mail: aviah{at}post.tau.ac.il.

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				D. Avisar, M. Segal, B. Sneh, and A. Zilberstein Cell-cycle-dependent resistance to Bacillus thuringiensis Cry1C toxin in Sf9 cells J. Cell Sci., July 15, 2005; 118(14): 3163 - 3171. [Abstract] [Full Text] [PDF]