Abstract
Bacillus thuringiensis Cry protein exerts its toxic effect through a receptor-mediated process. Both aminopeptidases and cadherin proteins were identified as putative Cry1A receptors from Heliothis virescens and Manduca sexta. The importance of cadherin was implied by its correlation with a Cry1Ac resistant H. virescens strain (Gahan, L. J., Gould, F., and Heckel, D. G. (2001) Science 293, 857-860). In this study, the Cry1Ac toxin-binding region in H. virescens cadherin was mapped to a 40-amino-acid fragment, from amino acids 1422 to 1440. This site overlaps with a Cry1Ab toxin-binding site, amino acids 1363-1464 recently reported in M. sexta (Hua, G., Jurat-Fuentes, J. L., and Adang, M. J. (2004) J. Biol. Chem. 279, 28051-28056). Further, feeding of the anti-H. virescens cadherin antiserum or the partial cadherins, which contain the toxin-binding region, in combination with Cry1Ab/Cry1Ac reduced insect mortality by 25.5-55.6% to first instar H. virescens and M. sexta larvae, suggesting a critical function for this cadherin domain in insect toxicity. Mutations in this region, to which the Cry1Ac binds through its loop 3, resulted in the loss of toxin binding. For the first time, we show that the cadherin amino acids Leu(1425) and Phe(1429) are critical for Cry1Ac toxin interaction, and if substituted with charged amino acids, result in the loss of toxin binding, with a K(D) of < 10(-5) m. Mutation of Gln(1430) to an alanine, however, increased the Cry1Ac affinity 10-fold primarily due to an increase on rate. The L1425R mutant can result from a single nucleotide mutation, CTG --> CGG, suggesting that these mutants, which have decreased toxin binding, may lead to Cry1A resistance in insects.
Highlights
Bacillus thuringiensis, a Gram-positive bacterium that produces crystal inclusions during its sporulation stage of growth, The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY692445
Since only 4 amino acids were lacking at the N terminus, we used PCR to obtain the 5Ј-end of the clone using sequences from the pFR-CAD-partial and a published report [18], which became available while this project was underway
Aminopeptidases, and other proteins/lipids are identified as Cry1A toxin-binding molecules from different insect species [5,6,7,8,9, 11, 13, 18, 33,34,35,36]
Summary
A Gram-positive bacterium that produces crystal inclusions during its sporulation stage of growth, The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY692445. One major step in this process involves toxin binding to membrane receptors. First demonstrated in a laboratory-selected H. virescens strain [18], such mutations have been shown in fieldisolated Pectinophora gossypiella [19], leading to resistant insects. In both insect species, resistance to Cry1A toxins results from either mutations to premature stop codons or deletions in the cadherin gene. Resistance to Cry1A toxins results from either mutations to premature stop codons or deletions in the cadherin gene These resistant insects show significant fitness costs [20], resulting in poor larval survival on cotton plants. This high fitness costs suggest that these deletions likely affect cadherin function
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