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Applied and Environmental Microbiology, January 2001, p. 323-329, Vol. 67, No. 1
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.1.323-329.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Importance of Cry1 delta -Endotoxin Domain II Loops for Binding Specificity in Heliothis virescens (L.)

Juan Luis Jurat-Fuentes1 and Michael J. Adang1,2,*

Department of Entomology1 and Department of Biochemistry and Molecular Biology,2 University of Georgia, Athens, Georgia 30602-2603

Received 13 April 2000/Accepted 2 November 2000

We constructed a model for Bacillus thuringiensis Cry1 toxin binding to midgut membrane vesicles from Heliothis virescens. Brush border membrane vesicle binding assays were performed with five Cry1 toxins that share homologies in domain II loops. Cry1Ab, Cry1Ac, Cry1Ja, and Cry1Fa competed with 125I-Cry1Aa, evidence that each toxin binds to the Cry1Aa binding site in H. virescens. Cry1Ac competed with high affinity (competition constant [Kcom] = 1.1 nM) for 125I-Cry1Ab binding sites. Cry1Aa, Cry1Fa, and Cry1Ja also competed for 125I-Cry1Ab binding sites, though the Kcom values ranged from 179 to 304 nM. Cry1Ab competed for 125I-Cry1Ac binding sites (Kcom = 73.6 nM) with higher affinity than Cry1Aa, Cry1Fa, or Cry1Ja. Neither Cry1Ea nor Cry2Aa competed with any of the 125I-Cry1A toxins. Ligand blots prepared from membrane vesicles were probed with Cry1 toxins to expand the model of Cry1 receptors in H. virescens. Three Cry1A toxins, Cry1Fa, and Cry1Ja recognized 170- and 110-kDa proteins that are probably aminopeptidases. Cry1Ab and Cry1Ac, and to some extent Cry1Fa, also recognized a 130-kDa molecule. Our vesicle binding and ligand blotting results support a determinant role for domain II loops in Cry toxin specificity for H. virescens. The shared binding properties for these Cry1 toxins correlate with observed cross-resistance in H. virescens.

* Corresponding author. Mailing address: Department of Entomology, University of Georgia, Biosciences Building, 125 Cedar St., Athens, GA 30602-2603. Phone: (706) 542-2436. Fax: (706) 542-2640. E-mail: adang{at}arches.uga.edu.

Applied and Environmental Microbiology, January 2001, p. 323-329, Vol. 67, No. 1
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.1.323-329.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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