Binding analyses of Bacillus thuringiensis Cry delta-endotoxins using brush border membrane vesicles of Ostrinia nubilalis

Transgenic corn expressing the Bacillus thuringiensis CrylAb gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascert ained whether CrylF, Cry9C, or Cry9E recognizes the CrylAb binding site on the O. nubilalis brush border by three approaches. An optical biosensor tec hnology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with CrylAb reduced BBMV binding to immobilized CrylAb, wher eas preincubation with CrylF, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a CrylF-coated surface was reduced when vesicles were prei ncubated in CrylF or CrylAb but not Cry9C or Cry9E, A radioligand approach measured I-125-CrylAb toxin binding to BBMV in the presence of homologous ( CrylAb) and heterologous (CrylAc, CrylF, Cry9C, or Cry9E) toxins. Unlabeled CrylAc effectively competed for I-125-CrylAb binding in a manner comparabl e to CrylAb itself, Unlabeled Cry9C and Cry9E toxins did not inhibit I-125- CrylAb binding to BBMV. CrylF inhibited (125)-CrylAb binding at concentrati ons greater than 500 nM. CrylF had low-level affinity for the CrylAb bindin g site. Ligand blot analysis identified CrylAb, CrylAc, and CrylF binding p roteins in BBMV. The major CrylAb signals on ligand blots were at 145 kDa a nd 154 kDa, but a strong signal was present at 220 kDa and a weak signal wa s present at 167 kDa. CrylAc and CrylF binding proteins were detected at 22 0 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 Mfa pr otein. We speculate that isoforms of aminopeptidase and cadherin in the bru sh border membrane serve as CrylAb, CrylAc, and CrylF binding proteins.