Fluorescent-based assays establish Manduca sexta Bt-R1a cadherin as a receptor for multiple Bacillus thuringiensis Cry1A toxins in Drosophila S2 cells

A fluorescence-based approach was developed to analyze in vivo the function of Manduca sexta cadherin (Bt-R-1) as a Cry1 toxin receptor. We cloned a Bt-R-1a cDNA that differs from Bt-R-1 by 37 nucleotides and two amino acids and expressed it transiently in Drosophila melanogaster Schneider 2 (S2) cells. Cells expressing Bt-R-1a bound Cry1Aa, Cry1Ab, and Cry1Ac toxins on ligand blots, and in saturation binding assays. More Cry1Ab was bound relative to Cry1Aa and Cry1Ac, though each CryIA toxin bound with high-affinity (Kd values from 1.7 to 3.3 nM). Using fluorescent microscopy and flow cytometry assays, we show that Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1Ba, killed S2 cells expressing Bt-R-1a cadherin. These results demonstrate that M. sexta cadherin Bt-R-1a functions as a receptor for the Cry1A toxins in vivo and validates our cytotoxicity assay for future receptor studies. (C) 2003 Elsevier Ltd. All rights reserved.