Identification and Characterization of NAC Transcription Factors Involved in Pine Wilt Nematode Resistance in Pinus massoniana

<i>Pinus massoniana</i> Lamb. is an economically important conifer native to China. However, it is highly susceptible to the pine wood nematode (<i>Bursaphelenchus xylophilus</i>, PWN), the causal agent of pine wilt disease (PWD), resulting in substantial ecological and economic losses. To elucidate potential molecular defense mechanisms, 50 NAC (NAM, ATAF1/2, and CUC2) transcription factors (PmNACs) were identified in the <i>P. massoniana</i> genome. Phylogenetic analysis divided these PmNACs into seven subfamilies, and motif analysis identified ten conserved motifs associated with stress responses. Twenty-three genes were selected for expression analysis in various tissues and under exogenous salicylic acid (SA), methyl jasmonate (MeJA), and PWN infection. Six genes (<i>PmNAC1</i>, <i>PmNAC8</i>, <i>PmNAC9</i>, <i>PmNAC17</i>, <i>PmNAC18</i>, and <i>PmNAC20</i>) were significantly up-regulated by both hormonal treatment and PWN infection, implying their involvement in JA/SA-mediated immune pathways. Functional characterization showed <i>PmNAC8</i> is a nuclear-localized transcription factor with autoactivation activity. Furthermore, transient overexpression of <i>PmNAC8</i> in <i>Nicotiana benthamiana</i> induced reactive oxygen species (ROS) accumulation and necrotic lesions. Collectively, these results elucidate NAC-mediated defense responses to PWN infection in <i>P. massoniana</i> and identify candidate genes for developing PWD-resistant pine varieties.