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Applied and Environmental Microbiology, December 2008, p. 7654-7659, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01373-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species

Carmen Sara Hernández-Rodríguez,¹ Adri Van Vliet,² Nadine Bautsoens,² Jeroen Van Rie,² and Juan Ferré^1*

Departamento de Genética, Facultad de CC. Biológicas, Universidad de Valencia, Valencia, Spain,¹ Bayer BioScience, Ghent, Belgium²

Received 19 June 2008/ Accepted 14 October 2008

For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with ¹²⁵I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with ¹²⁵I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K_d (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R_t (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.

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* Corresponding author. Mailing address: Departamento de Genética, Facultad de CC. Biológicas, Dr. Moliner 50, 46100-Burjassot, Valencia, Spain. Phone: (34) 96 354 4506. Fax: (34) 96 354 3029. E-mail: juan.ferre{at}uv.es

Published ahead of print on 17 October 2008.

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Applied and Environmental Microbiology, December 2008, p. 7654-7659, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01373-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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