Binding analyses of Bacillus thuringiensis Cry delta-endotoxins using brush border membrane vesicles of Ostrinia nubilalis.
Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit (125)I-Cry1Ab binding to BBMV. Cry1F inhibited (125)I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.
Hua
G
G
Departments of Entomology, Athens, Georgia 30602-2605, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
Masson
L
L
Jurat-Fuentes
J L
JL
Schwab
G
G
Adang
M J
MJ
D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins; D001665:Binding Sites; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism* / Q000633:toxicity; D006460:Hemolysin Proteins; D015151:Immunoblotting; D007915:Lepidoptera / Q000378:metabolism*; D008024:Ligands; D008871:Microvilli / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D022161:Transport Vesicles / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008024:Ligands; C030956:insecticidal crystal protein, Bacillus Thuringiensis