Identification, purification and cloning of a high-affinity invertebrate protocadherin receptor BT-R(2) from the pink bollworm (Pectinophora gossypiella) for Bacillus thuringiensis Cry1A toxins.

摘要

Pink bollworm (PBW) is a highly destructive cotton pest. Formulations of the most widely used microbial pesticide, B. thuringiensis (Bt) have been used for more than three decades as biological insecticides to control P. gossypiella. Recently, seed of transgenic cotton cultivers carrying the toxin genes of Bt, which have a high degree of resistance to the PBW, have been developed and sold commercially. Despite broad interest in controlling PBW using Bt toxins, little is known about the molecular mechanism of their toxicity and the insect receptor molecules that bind these toxins in the PBW.; The purpose of this dissertation was to identify, clone and characterize the receptor for Cry1A toxins in the PBW. Cry1Aa, Cry1Ab and Cry1Ac that are toxic to PBW were used in ligand-blot experiments to detect specific binding proteins in midgut brush border membrane vesicles (BBMV) of PBW. All three toxins bind to a protein of ∼200 kDa and Cry1Ac also binds to an ∼120-kDa protein. The three toxins bind specifically, saturably and with high affinity to BBMV. A competition ligand blot using unlabeled Cry1Ac to compete with 125I-Cry1Ac shows the disappearance of the 200-kDa protein but not the 120-kDa proteins. These results suggest that the 200-kDa protein is the common membrane binding receptor for Cry1Aa, Cry1Ab and Cry1Ac toxins. Consequently, Cry1Ac binding to the 200-kDa proteins is more likely to be mediating toxicity in PBW than binding to the 120-kDa proteins. The 200-kDa was immunoprecipitated by Cry1Ab and focused over 2-D gels as one single spot that binds 125I-Cry1Ab or Ac. The 200-kDa protein, which binds Cry1A toxins, has been cloned and expressed in E. coli and mammalian cell cultures. The receptor, named BT-R₂, is an epithelial invertebrate protocadherin, termed E-IVP, and showed 60% similarity to the previously cloned receptor BT-R₁ that binds Cry1A toxins in the tobacco hornworm. The receptor contains two leucine zippers that probably function in receptor aggregation and form a nutrient channel. The transfected COS-7 cells expressed the receptor on the surface, bound the Cry1Ab toxin and cross-reacted with BT-R₁ antiserum. Collectively, these results demonstrate that BT-R ₂ in the pink bollworm is the common high-affinity receptor for the Cry1A family of toxins.