Cinnamyl Alcohol Dehydrogenase Gene Regulates Bursaphelenchus xylophilus Reproduction and Development

Abstract

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a globally distributed destructive disease of pine forest. To study the PWD pathogenic mechanism, the cinnamyl alcohol dehydrogenase gene (BxCAD-1) from B. xylophilus was selected. The BxCAD-1 gene was amplified by PCR from the cDNA library of B. xylophilus and cloned into the expression vector pET-15b to construct the recombinant vector pET-15b-BxCAD-1. The recombinant cinnamyl alcohol dehydrogenase protein was purified by Ni-NTA affinity chromatography from Escherichia coli BL21 (DE3) harboring pET-15b-BxCAD-1 induced by IPTG. The effects of pH, temperature, metal ions and substrates on the activity of BxCAD-1 were determined, showing the highest catalytic activity at pH 8.0 and 40 °C with cinnamyl alcohol as substrate and Zn2+ as an activator. To elucidate the functions of BxCAD-1 in B. xylophilus, the expression of the gene was down-regulated by RNA interference. Results showed that the movement, feeding, reproduction, spawning rate, hatching rate, lifespan, infectivity and sensitivity to ethanol decreased compared with negative controls. RNA interference also affected the development of B. xylophilus from the larval stage to the adult stage. In situ hybridization showed that the gene was expressed in the digestive tract of male and female adults. This study revealed a promising target for PWD control.