Calcofluor disrupts binding of Bt toxin Cry1Ac to midgut receptors in Trichoplusia ni

Abstract

The parasporal crystal proteins (Cry proteins) from the soil bacterium Bacillus thuringiensis (Bt) are major insecticidal toxins in formulated Bt sprays and in current transgenic Bt crops widely used in agriculture. To understand the modes of action of Cry proteins and mechanisms of Cry resistance in insects, it is important to understand the specific interaction of Cry proteins with the specific receptors in the insect midgut. Previous studies have found that the fluorescent brightener Calcofluor could significantly reduce the insecticidal activity of Cry1Ac in the cabbage looper, Trichoplusia ni. In this study, the effects of Calcofluor in T. ni larvae on the structure of the midgut, the composition and abundance of midgut brush border membrane proteins, and the binding of midgut brush border membranes with Cry1Ac were examined. Finally, the inhibiting activity of Calcofluor on the binding of Cry1Ac to midgut binding sites was determined. The results from this study indicated that Calcofluor blocks the binding of Cry1Ac to the midgut binding sites by competitively binding the carbohydrate moieties that are involved in the specific binding of Cry1Ac to the midgut, which consequently inhibits the toxicity of Cry1Ac in larvae. Therefore, this study revealed that carbohydrate moieties on insect midgut brush border membranes play crucially important roles in the functional specific binding of Cry1Ac to the midgut receptors in the pathway of toxicity.