Pinus massoniana is a pioneer species for afforestation timber and oleoresin, while epidemics of pinewood nematode (PWN; Bursaphelenchus xylophilus) are causing a serious biotic disaster for P. massoniana in China. Importantly, resistant P. massoniana could leak copious oleoresin terpenoids to build particular defense fronts for survival when attacked by PWN. However, the defense mechanisms regulating this process remain unknown. Here, PmCYP720B11v2, a cytochrome P450 monooxygenase gene, was first identified and functionally characterized from resistant P. massoniana following PWN inoculation. The tissue-specific expression pattern and localization of PmCYP720B11v2 at the transcript and protein levels in resistant P. massoniana indicated that its upregulation in the stem supported its involvement in the metabolic processes of diterpene biosynthesis as a positive part of the defense against PWN attack. Furthermore, overexpression of PmCYP720B11v2 may enhance the growth and development of plants. In addition, PmCYP720B11v2 activated the metabolic flux of antioxidases and stress-responsive proteins under drought conditions and improved drought stress tolerance. Our results provide new insights into the favorable role of PmCYP720B11v2 in diterpene defense mechanisms in response to PWN attack in resistant P. massoniana and provide a novel metabolic engineering scenario to reform the stress tolerance potential of tobacco.

Abies beshanzuensis, an extremely rare and critically endangered plant with only three wild adult trees globally, is strongly mycorrhizal-dependent, leading to difficulties in protection and artificial breeding without symbiosis. Root hair morphogenesis plays an important role in the survival of mycorrhizal symbionts. Due to the lack of an effective genome and transcriptome of A. beshanzuensis, the molecular signals involved in the root hair development remain unknown, which hinders its endangered mechanism analysis and protection. Herein, transcriptomes of radicles with root hair (RH1) and without root hair (RH0) from A. beshanzuensis in vitro plantlets were primarily established. Functional annotation and differentially expressed gene (DEG) analysis showed that the two phenotypes have highly differentially expressed gene clusters. Transcriptome divergence identified hormone and sugar signaling primarily involved in root hair morphogenesis of A. beshanzuensis. Weighted correlation network analysis (WGCNA) coupled with quantitative real-time PCR (qRT-PCR) found that two hormone–sucrose–root hair modules were linked by IAA17, and SUS was positioned in the center of the regulation network, co-expressed with SRK2E in hormone transduction and key genes related to root hair morphogenesis. Our results contribute to better understanding of the molecular mechanisms of root hair development and offer new insights into deciphering the survival mechanism of A. beshanzuensis and other endangered species, utilizing root hair as a compensatory strategy instead of poor mycorrhizal growth.

Ubiquitins (Ubs) play a crucial role in plant–pathogen interactions, particularly the RPL40 family, which is essential for protein synthesis. While Pinus massoniana is highly susceptible to pine wilt disease (PWD) caused by Bursaphelenchus xylophilus, the defense mechanisms mediated by RPL40s remain poorly understood. Here, we performed a genome-wide identification of the ubiquitin and ubiquitin-like gene family (PmUBQs) in P. massoniana. We identified 30 PmUBQ genes unevenly distributed across 11 chromosomes, which were classified into six subfamilies based on phylogenetic analysis. An analysis of promoter regions indicated that the PmUBQ genes were enriched with cis-acting elements associated with stress responses, hormone regulation, and development. Specifically, two group II members, PmRPL40-1 and PmRPL40-2, located on chromosomes 12 and 11, respectively, were identified and exhibited distinct responses to B. xylophilus infection in resistant and susceptible P. massoniana. PmRPL40-1 was significantly highly expressed in the 15 days post-inoculation, while PmRPL40-2 was downregulated on day 1 and then upregulated. Moreover, both genes showed peak divergence at 15 days post-inoculation; the expression levels of PmRPL40-1 and PmRPL40-2 in resistant P. massoniana were approximately 1.8- and 3.7-fold higher, respectively, than in susceptible P. massoniana. These patterns suggest that PmRPL40s might be involved in the rapid activation of defense responses and late-stage cell repair. Notably, transient overexpression of PmRPL40-1 in P. massoniana led to a significant 1.6-fold increase in the jasmonic acid (JA) content (p < 0.0001). These findings reveal the key PmUBQ genes and suggest that PmRPL40s contribute to PWD resistance potentially through the modulation of JA signaling, offering potential targets for molecular breeding in P. massoniana.

Pathogenesis-related protein 1 (PR1) plays important roles in plant responses to both biotic and abiotic stresses; however, its role in mediating defense against pine wood nematode in Pinus massoniana remains unclear. In this study, a total of 63 PR1 family members were identified in P. massoniana using bioinformatics approaches and were named PmPR1-1 to PmPR1-63 based on their phylogenetic relationships. Phylogenetic analysis showed that these members were distributed among four of the six subfamilies. Most of the encoded proteins were hydrophilic, with lengths ranging from 131 to 406 amino acids. Their promoter regions contained multiple cis-acting elements associated with phytohormone signaling and stress responses, and some members formed gene clusters on chromosomes 2, 5, and 9. qRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis showed that the clustered genes PmPR1-46, PmPR1-55, PmPR1-56, and PmPR1-61 were significantly upregulated in the early stage of pine wood nematode inoculation in both resistant and susceptible P. massoniana plants, with higher expression levels in resistant plants. Transient overexpression of PmPR1-61 increased SOD and PPO activities as well as proline content while decreasing CAT activity. These results suggest that the PmPR1 family may be involved in the defense response of P. massoniana against pine wood nematode. Among them, PmPR1-55, PmPR1-56, and PmPR1-61 represent candidate resistance genes worthy of further investigation and provide valuable gene resources for elucidating resistance mechanisms and supporting molecular breeding in P. massoniana.

Pine wilt disease (PWD), caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus ), poses a severe threat to global pine forests. Plants can enhance their resistance to external stresses by eliciting secondary metabolites; however, the transcriptional regulatory mechanisms governing this defence response in pines remain largely unexplored. Here, we report a novel defence regulatory module in Pinus massoniana . PmWRKY‐10 may participate in PWN resistance by regulating the biosynthesis of the nematicidal metabolite pinosylvin monomethyl ether (PME). We first demonstrated that PME accumulation is closely linked to resistance in P. massoniana and provided evidence that PME exhibits direct and potent nematicidal activity against PWN in vitro. By integrating multi‐omics profiling with weighted gene co‐expression network analysis (WGCNA), we identified PmPMT2‐1 as a key candidate associated with PME biosynthesis and accumulation, while recognizing that it is unlikely to function as the sole rate‐limiting determinant. Functional analyses demonstrated that PmPMT2‐1 overexpression promoted PME accumulation and enhanced resistance, whereas silencing the gene impaired both processes. Mechanistically, we identified PmWRKY‐10 as an upstream transcriptional activator that directly binds to the W‐box element in the PmPMT2‐1 promoter, inducing its transcription. Our work delineates a complete PmWRKY‐10 → PmPMT2‐1 →PME transcriptional‐metabolic pathway that is essential for stilbene‐based resistance in pine. This discovery not only provides fundamental insights into the inducible defence mechanisms of conifers but also identifies key genetic targets for breeding resistant varieties and developing sustainable forest management strategies.