Molecular characterization and functional analysis of genes mediating emamectin benzoate action to the pinewood nematode (Bursaphelenchus xylophilus)

Abstract

Emamectin benzoate (EB) is a highly effective and low-toxicity pesticide for the control of Bursaphelenchus xylophilus. However, its action mechanism in B. xylophilus has not yet been verified. Here, the genes (Bxy-glc-1, Bxy-glc-2, Bxy-glc-4, and Bxy-avr-14) encoding the glutamate-gated chloride channel (GluCl) of B. xylophilus were analysed and cloned. Functional validation of the target genes was conducted using RNAi and pathogenicity detection assays. The results of the bioinformatics analysis showed that the four GluCl genes contained the Cys-loop region and three transmembrane structural domains. Molecular docking and molecular dynamics simulation predictions revealed that BXY-GLC-2, BXY-GLC-4, and BXY-GLC-1 all had strong binding affinities to EB, and BXY-AVR-14 had no binding affinity to EB. The expression and in situ hybridisation of Bxy-glc-1, Bxy-glc-2, Bxy-glc-4, and Bxy-avr-14 was significantly higher in adult B. xylophilus than at other developmental stages. Interference of Bxy-glc-1, Bxy-glc-2, and Bxy-glc-4 significantly reduced adult mortality relative to the control group, and interference of Bxy-avr-14 did not have a significant on adult mortality. Adult mortality was lowest in the combined Bxy-glc-2 + Bxy-glc-4 treatment group, followed by the Bxy-glc-1 + Bxy-glc-2 and Bxy-glc-1 + Bxy-glc-4 groups. No significant changes were observed in the mortality rate of the Bxy-avr-14 group and the combination of the other three genes. The dsBxy-glc-1, dsBxy-glc-2, and dsBxy-glc-4 groups accelerated the progression of pine wilt disease induced by EB relative to the sole EB-treated group. Our results confirmed that Bxy-glc-1, Bxy-glc-2, and Bxy-glc-4 are target genes of GluCl in B. xylophilus.