Identification of an α-galactosidase with high affinity and synergistic activity against Bacillus thuringiensis App6Aa2 toxin in Bursaphelenchus xylophilus

Abstract

The App6Aa2 toxin, derived from Bacillus thuringiensis, is nematicidal and highly toxic to Bursaphelenchus xylophilus. Receptors play a critical role in the mechanism of B. thuringiensis crystal toxin toxicity, yet the specific binding receptors for App6Aa2 in B. xylophilus have not been identified. This study identified a GPI-anchored protein, α-galactosidase (BxGal), in B. xylophilus as a potential binding protein. Western blotting and ELISA assays confirmed a high binding affinity between BxGal and App6Aa2 (Kd = 24.5 ± 11.2 nM). Remarkably, combining App6Aa2 with the BxGal protein produced a synergistic effect, significantly increasing nematode mortality from 25 % to 81 % (P < 0.05), thereby enhancing the toxicity of App6Aa2 against B. xylophilus. Besides, RNAi silencing of BxGal in nematodes had no specific effect on App6Aa2 toxicity at high concentrations, while mortality increased slightly at lower concentrations. These findings indicate that BxGal is a high-affinity binding protein for App6Aa2; it does not function as the primary receptor and warrants further investigation into its role in modulating nematode susceptibility to App6Aa2.