Pine wilt disease (PWD), caused by the plant–parasitic nematode Bursaphelenchus xylophilus, has become a severe environmental problem in the Iberian Peninsula with devastating effects in Pinus pinaster forests. Despite the high levels of this species' susceptibility, previous studies reported heritable resistance in P. pinaster trees. Understanding the basis of this resistance can be of extreme relevance for future programs aiming at reducing the disease impact on P. pinaster forests. In this study, we highlighted the mechanisms possibly involved in P. pinaster resistance to PWD, by comparing the transcriptional changes between resistant and susceptible plants after infection. Our analysis revealed a higher number of differentially expressed genes (DEGs) in resistant plants (1,916) when compared with susceptible plants (1,226). Resistance to PWN is mediated by the induction of the jasmonic acid (JA) defense pathway, secondary metabolism pathways, lignin synthesis, oxidative stress response genes, and resistance genes. Quantification of the acetyl bromide-soluble lignin confirmed a significant increase of cell wall lignification of stem tissues around the inoculation zone in resistant plants. In addition to less lignified cell walls, susceptibility to the pine wood nematode seems associated with the activation of the salicylic acid (SA) defense pathway at 72 hpi, as revealed by the higher SA levels in the tissues of susceptible plants. Cell wall reinforcement and hormone signaling mechanisms seem therefore essential for a resistance response.

Pine wilt disease (PWD) is a severe environmental problem in Eastern Asia and Western Europe, devastating large forest areas and causing significant economic losses. This disease is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, a parasitic migratory nematode that infects the stem of conifer trees. Here we review what is currently known about the molecular defense response in pine trees after infection with PWN, focusing on common responses in different species. By giving particular emphasis to resistance mechanisms reported for selected varieties and families, we identified shared genes and pathways associated with resistance, including the activation of oxidative stress response, cell wall lignification, and biosynthesis of terpenoids and phenylpropanoids. The role of post-transcriptional regulation by small RNAs in pine response to PWN infection is also discussed, as well as the possible implementation of innovative RNA-interference technologies, with a focus on trans-kingdom small RNAs. Finally, the defense response induced by elicitors applied to pine plants before PWN infection to prompt resistance is reviewed. Perspectives about the impact of these findings and future research approaches are discussed.

Pine wilt disease (PWD), caused by the parasitic nematode Bursaphelenchus xylophilus, or pinewood nematode (PWN), is a serious threat to pine forests in Europe. Pinus pinaster is highly susceptible to the disease and it is currently the most affected European pine species. In this work, we investigated the role of small RNAs (sRNAs) in regulating P. pinaster–PWN interaction in an early stage of infection. After performing an artificial PWN inoculation assay, we have identified 105 plant microRNAs (miRNAs) responsive to PWN. Based on their predicted targets, part of these miRNAs was associated with roles in jasmonate-response pathway, ROS detoxification, and terpenoid biosynthesis. Furthermore, by comparing resistant and susceptible plants, eight miRNAs with putative functions in plant defence and resistance to PWN have been identified. Finally, we explored the possibility of bidirectional trans-kingdom RNA silencing, identifying several P. pinaster genes putatively targeted by PWN miRNAs, which was supported by degradome analysis. Targets for P. pinaster miRNAs were also predicted in PWN, suggesting a role for trans-kingdom miRNA transfer and gene silencing both in PWN parasitism as in P. pinaster resistance to PWD. Our results provide new insights into previously unexplored roles of sRNA post-transcriptional regulation in P. pinaster response and resistance to PWN.

Embryogenesis is a brief but potentially critical phase in the life cycle of a tree for adaptive phenotypic plasticity. Using somatic embryogenesis in maritime pine (Pinus pinaster Ait.), we found that temperature during the maturation phase affects embryo development and postembryonic tree growth for up to 3 yr. We examined whether this somatic stress memory could stem from temperature- and/or development-induced changes in DNA methylation. For this, we developed a 200 mb custom sequence capture bisulfite analysis of genes and promoters to identify differentially methylated cytosines (DMCs) between temperature treatments (18, 23, and 28 °C) and developmental stages (immature and cotyledonary embryos, shoot apical meristem of 2-yr-old plants) and investigate if these differences can be mitotically transmitted from embryonic to postembryonic development (epigenetic memory). We revealed a high prevalence of temperature-induced DMCs in genes (8% to 14%) compared to promoters (<1%) in all 3 cytosine contexts. Developmental DMCs showed a comparable pattern but only in the CG context and with a strong trend toward hypomethylation, particularly in the promoters. A high percentage of DMCs induced by developmental transitions were found memorized in genes (up to 45%–50%) and promoters (up to 90%). By contrast, temperature-induced memory was lower and confined to genes after both embryonic (up to 14%) and postembryonic development (up to 8%). Using stringent criteria, we identified 10 genes involved in defense responses and adaptation, embryo development, and chromatin regulation that are candidates for the establishment of a persistent epigenetic memory of temperature sensed during embryo maturation in maritime pine. Here, we provide evidence that DNA methylation marks established during the embryonic phase are transmitted to the postembryonic plant development phase.