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Pinus massoniana Lamb. is a crucial timber and resin conifer in China, but its plantation industry is threatened by outbreaks of pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (pinewood nematode; PWN). However, as of yet, there is no comprehensive analysis of NBS-LRR genes in P. massoniana involved in its defense against PWN. In this study, 507 NBS genes were identified in the transcriptome of resistant and susceptible P. masoniana inoculated with the PWN. The phylogenetic analysis and expression profiles of resistant and susceptible P. massoniana revealed that the up-regulated PmNBS-LRR97 gene was involved in conferring resistance to PWN. The results of real-time quantitative PCR (qRT-PCR) showed that PmNBS-LRR97 was significantly up-regulated after PWN infection, especially in the stems. Subcellular localization indicated that PmNBS-LRR97 located to the cell membrane. PmNBS-LRR97 significantly activated the expression of reactive oxygen species (ROS)-related genes in P. massoniana. In addition, the overexpression of PmNBS-LRR97 was capable of promoting the production of ROS, aiding in plant growth and development. In summary, PmNBS-LRR97 participates in the defense response to PWN and plays an active role in conferring resistance in P. massoniana. This finding provides new insight into the regulatory mechanism of the R gene in P. massoniana. [ABSTRACT FROM AUTHOR]