Screening of effector genes of Bursaphelenchus xylophilus and the function of Bx-Hh-grl

  Objective  The cloning and function of effector gene of pine wood nematode (Bursaphelenchus xylophilus) were studied to reveal the effects of Bx-Hh-grl on the pathogenicity of the nematode, and to provide theoretical basis for the control of the nematode.

  Method  Black pine (Pinus thunbergia) was inoculated with Bx1022 strain. Twenty days later, total RNA of the nematodes was extracted for transcriptome sequencing, and the transcriptome was set as the transcriptome of phytophagous phase. The total RNA of Bx1022 cultured on Botrytis cinerea was extracted for transcriptome sequencing, and the transcriptome was set as the transcriptome of mycophagous phase. The differentially expressed gene Bx-Hh-grl was screened by comparative analysis of the two transcriptomes. The transmembrane domain and signal peptide of Bx-Hh-grl encoding protein were predicted. The expression sites of Bx-Hh-grl were detected by in-situ hybridization. The expression of Bx-Hh-grl was interfered by RNAi to verify the important role of Bx-Hh-grl on the pathogenicity of pine wood nematode. Q-PCR confirmed that the RNAi effect was significant. The RNAi treated pine wood nematodes were inoculated into branches of 3-year-old black pine. The untreated pine wood nematodes (CK group) and ddH₂O treated pine tree nematodes (Mock group) were used as the control groups to compare the differences of the incidences of black pines and the pathogenicity of differently treated pine wood nematodes.

  Result  The differentially expressed gene Bx-Hh-grl was screened out from the transcriptomes of phytophagous phase and mycophagous phase, and the protein encoded by Bx-Hh-grl had transmembrane domain and signal peptide. The results of in-situ hybridization showed that Bx-Hh-grl was expressed in the esophageal gland of pine wood nematode, which was consistent with the effector gene characteristics. Q-PCR results showed that the expression of Bx-Hh-grl in pine wood nematode was down-regulated after RNAi treatment, and the effect of RNAi treatment was significant. Inoculation experiments showed that the symptoms of Bx-Hh-grl-RNAi nematodes treated group showed significantly later than the CK group, and the Mock group remained healthy all the time.

  Conclusion  Bx-Hh-grl is an effector gene related to the pathogenicity of pine wood nematode. Understanding the function of Bx-Hh-grl is beneficial to further clarify the pathogenicity of pine wood nematode, and lay a theoretical foundation for reducing the damage and controlling the nematode.