Molecular Defense Response of Pine Trees (Pinus spp.) to the Parasitic Nematode Bursaphelenchus xylophilus

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.


Introduction
Pine wilt disease (PWD) is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, a migratory endoparasitic nematode that infects the stem of conifer trees and affects mainly species of the genus Pinus. This nematode is transmitted to healthy trees through the insect vector Monochamus spp. during feeding on young branches. After entering the tree, PWN migrates through the resin canals and feeds on plant stem tissues, causing a progressive blockage of water flow and leading to tree death. Visible PWD symptoms include needle chlorosis and wilting [1,2].
In North America, where PWN is native, little damage is caused by PWD, as pine species are mostly resistant to the nematode infection [1,2]. However, in the regions where PWN was introduced, namely in Eastern Asia and Western Europe countries [2][3][4][5][6], several pine species have shown high susceptibility to PWD and it has been causing serious damage to forest ecosystems, as well as significant economic losses for the forest products industry [3].
yunnanensis (Table 1). For P. thunbergii [35,36], P. massoniana [37], and P. pinaster [38] comparative analyses of differential gene expression were made between susceptible and selected resistant varieties or individuals. The experimental conditions varied among these studies, such as the age of the plants used (2 years old to adult trees), the use of seedlings or grafts, growth conditions (greenhouse, growth chamber, or field), the PWN isolates used (possibly with different levels of virulence), the amount of PWNs inoculated (500-30,000 PWNs), and sampling timepoints (Table 1). These differences may affect the plants' response to PWN inoculation, as well as disease progression, and consequently gene expression, protein, and metabolite profiles. Nevertheless, comparing the transcriptional response to PWN inoculation in several pine species allowed for the identification of several common molecular mechanisms involved both in the susceptible and resistant responses.

Phytohormone Signaling
Phytohormones are signaling molecules with vital roles in plant development and response to stress, including response to biotic stresses. The recognition of a pathogen or pest and subsequent trigger of PTI or ETI by the plant cell leads to the accumulation of hormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) [50]. These three hormones Cells 2022, 11, 3208 4 of 22 are well described as having major roles in initiating downstream immune responses. SA is generally associated with response to biotrophic and hemi-biotrophic pathogens, while JA and ET have been associated with response to herbivory and necrotrophic pathogens, and the activation of these pathways is considered mutually exclusive [50,51]. However, this dichotomy is not always observed in species other than Arabidopsis, and JA has been implicated in the response to a wider range of pathogens and pests in monocots and gymnosperms [52]. Other hormones such as auxins, gibberellins, cytokinins, brassinosteroids or abscisic acid (ABA), which are usually linked to plant development or response to abiotic stresses, can also play roles in plant defense response against biotic stresses [53]. Despite the importance of phytohormones in plant immune response, little emphasis has been given to their role in pine response to PWN inoculation except in a few studies with P. pinaster [38,54].
The differential expression of genes encoding JA biosynthesis enzymes was observed in P. pinaster after inoculation, both in susceptible and resistant plants [38,46]. However, 12-oxophytodienoate reductase 3 (OPR3) was the only gene reported by both studies as being upregulated, soon after inoculation (24-72 h post-inoculation, hpi). Other OPRs, allene oxide synthases (AOS), linoleate 13S-lipoxygenases (LOX), and phospholipase A2 (PLA2G), were upregulated in inoculated P. pinaster plants, particularly in resistant plants [38]. Genes responsive to JA were also induced after inoculation, such as the transcription factors ethylene response factors (ERFs), jasmonate-ZIM domain (JAZ/Tify) and MYC2, as well as the genes pathogenesis-related protein 4 (PR-4), pathogenesis-related protein 5 (PR-5), and chitinases, with higher levels of expression in resistant plants. This suggests that JA has an essential role in response to PWN in P. pinaster and may be important for resistance. Hormone quantification showed that JA levels were higher in inoculated plants when compared with controls at 72 hpi, but no differences were found in the more bioactive jasmonateisoleucine (JA-Ile) [38]. On the other hand, Rodrigues et al. [54] reported higher levels of methyl jasmonate (MeJA) in susceptible plants at the same timepoint. Therefore, the higher activation of genes related to JA biosynthesis and response in resistant plants were not explained by higher levels of JA or the quantified conjugated forms. In other pine species, the upregulation of JA responsive genes after inoculation has also been reported. One JAZ/Tify transcription factor, the JA responsive PR-3, PR-4, and PR-5, and JA biosynthesis genes were upregulated in P. massoniana susceptible plants 24 hpi [14,41]. The genes PR-2, PR-3, PR-4, and PR-5 were also upregulated in susceptible P. densiflora plants [44], as well as in susceptible and resistant P. thunbergii [35,36]. These results indicate that the JA pathway might have a central role in response to PWN in several pine species, but further studies are needed. Complex responses resulting from less-known crosstalk mechanisms between JA and other hormone signaling pathways may possibly help to explain its role in PWD.
In Arabidopsis, JA defense response consists of two antagonistic branches, regulated by distinct transcription factors, the MYC-branch and the ERF-branch [55,56]. While ET is necessary for the activation of the ERF-branch, usually in response to necrotrophic pathogens, ABA inhibits this defense response and leads to the activation of the MYCbranch in response to herbivory [50,[55][56][57]. ABA responsive genes were reported as upregulated in susceptible P. pinaster 24 hpi [48] and susceptible P. densiflora after inoculation [45], as well as in the resistant species Pinus yunnanensis [49]. In P. pinaster, the upregulation of PYL4, which encodes a protein crucial for the ABA-JA crosstalk, and PP2C, involved in ABA signaling, was only observed in resistant plants [38]. Furthermore, several NAC transcription factor genes, involved in the ABA-JA crosstalk, were more expressed in resistant plants. This seems to suggest a role for ABA in response and resistance to PWN. On the other hand, ERF genes were also upregulated in P. pinaster at 72 hpi [38] and two 1-aminocyclopropane-1-carboxylic acid (ACC) synthases, encoding an enzyme essential in the synthesis of ET, were upregulated in P. yunnanensis 24 hpi [49], suggesting a role for ET in PWN defense response. ET quantification has been reported for P. thunbergii, which showed an increase in ET levels only 2 weeks after inoculation. Increased ET levels were associated with the formation of embolisms in the xylem and consequent wilting symptoms [58,59]. In turn, ABA quantification in P. pinaster showed that, although no differences were observed between resistant, susceptible, and control plants at 72 hpi [38,54], ABA levels were lower in resistant plants at 48 hpi [54]. Therefore, the importance of ABA and ET pathways in pine response to PWN are still unclear.
The upregulation of genes with roles in the synthesis and accumulation of SA, namely enhanced disease susceptibility 1 (EDS1), phytoalexin deficient 4 (PAD4) and senescence associated gene 101 (SAG101), as well as several SA responsive genes, such as WRKY transcription factors (e.g., WRKY23, WRKY50, WRKY51) and PR-1, was observed in susceptible P. pinaster plants 72 hpi [38]. Furthermore, SA quantification revealed that susceptible P. pinaster plants had significantly higher levels of this hormone at 48 hpi and 72 hpi [38,54], suggesting that the SA pathway is associated with P. pinaster susceptibility to PWN. WRKY transcription factors, namely WRKY6 and WRKY51, were reported also as upregulated in susceptible P. densiflora trees [45], while PR-1 genes were more expressed in susceptible P. thunbergii plants when compared to resistant ones [35,36], suggesting that the activation of the SA pathway may also have a role in susceptibility in pine species other than P. pinaster. The activation of the SA defense pathway may inhibit JA response, in a manner that is independent of JA levels [51], as SA seems to suppress the JA defense response mostly at the transcription level in Arabidopsis. In this model species, SA influences the activity and localization of transcriptional regulators with roles in JA signaling and induces negative regulators of JA responsive genes, such as the WRKY transcription factors. Although further research is needed to understand whether SA inhibits the JA pathway in susceptible pine plants, SA/JA crosstalk may have an important role in the outcome of infected pine trees, with the activation of the SA defense pathway as a crucial step for susceptibility.
Overall, the role of phytohormones in PWN response and resistance needs further clarification in the pine species of interest. Quantification of hormones at several timepoints after PWN inoculation, as well as investigating the effect of hormone application in the plant response, might help in understanding their relevance in PWD.

Secondary Metabolism
The importance of secondary metabolites in plant defense response, and in particular in conifers' defense response, has been well described for the interaction with several pests and pathogens [60][61][62]. The expression of genes encoding secondary metabolites' biosynthetic enzymes in response to PWN inoculation has also been reported for a number of pine species. Quantification of these metabolites in PWD-susceptible and -resistant species has been described, as well as the effect of some of these compounds on PWN mobility and survival ( Table 2). Secondary metabolites include terpenes and phenylpropanoids, among other compounds.
Several terpene synthases genes were more expressed in resistant P. massoniana plants than in susceptible ones after PWN inoculation, including (-)-limonene synthase, (-)-β-pinene synthase, (+)-α-pinene synthase, and longifolene synthase [37,63]. Two of these terpene synthases, namely α-pinene (PmTPS4) and longifolene (PmTPS21) synthases, were further characterized [63]. The enzyme α-pinene synthase produced the monoterpenes α-pinene, β-pinene, β-myrcene, and D-limonene, while longifolene synthase produced the sesquiterpene longifolene and the monoterpene α-pinene. All of these compounds had an inhibitory effect on PWN survival when applied separately to PWN in vitro cultures, and a stronger effect when applied in combination. Therefore, the higher expression levels of terpene synthase genes probably result in the increased synthesis of terpene compounds with nematicidal effect, leading to resistance in P. massoniana. Other terpene compounds extracted from P. massoniana were shown to have a repellent effect on PWN (Table 2) [64]. However, no information about the concentration of such terpene compounds in resistant or susceptible P. massoniana plants is yet available.
In other pine species, the upregulation of terpene synthases genes after PWN inoculation was also observed. Shin et al. [44] reported the upregulation of limonene cyclase in susceptible P. densiflora plants, while α-pinene synthase was upregulated in the resistant species P. pinea [65]. In P. pinaster, genes involved in the synthesis of diterpenes, such as bifunctional abietadiene synthase (AS) and bifunctional levopimaradiene synthase (LPS), as well as genes involved in the sesquiterpenes biosynthesis pathway, such as (-)-germacrene D-synthase (GERD), had higher expression levels in resistant plants than in susceptible ones [38]. Despite these reports of differential gene expression in inoculated pine plants, connecting it to increased levels of terpene compounds has proven difficult. For susceptible P. pinaster and P. sylvestris plants, or for the resistant P. pinea, P. halepensis, or P. radiata, no alterations were found in the concentration of volatile and non-volatile terpenes after inoculation with PWN [68,69] at any of the studied timepoints (3 hpi to 2 months post-inoculation). However, an increase in diterpenes and sesquiterpenes was observed in P. pinea and P. halepensis after mechanical wounding, mimicking the insect vector feeding [68], while a significant increase in P. pinea limonene concentration was observed after feeding by M. galloprovincialis [70]. In P. pinaster, feeding by M. galloprovincialis caused an increase in several terpene compounds in susceptible plants, mainly in β-pinene, α-pinene, β-caryophyllene, Cells 2022, 11, 3208 7 of 22 and germacrene D, while a slight increase in β-myrcene and limonene was observed [70]. As PWNs enter the tree stem through wounds made during M. galloprovincialis feeding, the amount of terpene compounds produced and their relative proportions after wounding may impact the success of PWN infestation and consequently the resistance/susceptibility phenotypic outcome after infection. Studies to link gene expression and the synthesis of terpene compounds are still missing. Moreover, characterizing the enzymes with terpene synthase functions encoded by genes upregulated after wounding or PWN inoculation, similar to what was described for two P. massoniana enzymes [63], would elucidate their relevance and the role of their products in resistance to PWN.

Phenylpropanoids Biosynthesis
Phenylpropanoids have long been recognized for their roles in plant response to abiotic and biotic stresses, being key elements in resistance to pests and pathogens [62,71,72]. The phenylpropanoid biosynthesis pathway branches out into several pathways, such as flavonoid and stilbenoid biosynthesis. Therefore, this class of secondary metabolites includes a vast variety of compounds, such as flavonoids, isoflavonoids, anthocyanidins, stilbenes, tannins, suberin, lignans, and lignin [71]. The synthesis of these compounds is frequently induced by pathogens or pests in a large variety of plants. In PWD, several phenylpropanoids have been quantified in PWN-susceptible and -resistant species [66,69,73,74] and the effect of some of these compounds on PWN survival has been studied (Table 2) [64,66,67]. Furthermore, a large number of genes involved in phenylpropanoids biosynthesis were upregulated after PWN inoculation in various pine species, revealing its importance in pine response to PWN (Table 3). Table 3. Expression of genes related to secondary metabolism pathways in several pine species after inoculation with pinewood nematode.

Pathway
Genes P. densiflora P. massoniana P. thunbergii P. pinaster P. pinea P. yunnanensis P. strobus  The constitutive levels of total phenolic content have been measured in several resistant and susceptible pine species in an attempt to associate these levels with the phenotypic outcome after PWN inoculation [69,73,74]. However, high levels of phenolics have been found both in resistant species, such as P. pinea, P. halepensis, and P. radiata, and in the susceptible species P. pinaster. Furthermore, results were not in accordance across studies, possibly due to the use of plants with different ages (2-3 y.o. in Nunes da Silva et al. [69]; 12 y.o. in Trindade et al. [74]) and the collection of data at different timepoints (24-72 hpi in Pimentel et al. [73] and Trindade et al. [74]; 2 months post-inoculation in Nunes da Silva et al. [69]). Therefore, measurement of total phenolics seems of little value to discriminate between PWN-resistant and -susceptible phenotypes. Instead, the synthesis of specific phenolic compounds may be linked to resistance [64,66,74].
The first genes in the phenylpropanoid biosynthesis pathway, such as phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), caffeoyl-CoA O-methyltransferase (CCoAOMT) or caffeic acid O-methyltransferase (COMT), were upregulated in several susceptible pine species after PWN inoculation, namely in P. densiflora, P. massoniana and P. pinaster (Table 3) [38,40,44]. PAL and 4CL were also upregulated in the resistant species P. strobus [66] and more expressed in P. pinaster-resistant plants than in susceptible ones [38], suggesting that the activation of the phenylpropanoid biosynthesis pathway is a common response to PWN and may be important in resistance.
Several genes of the flavonoid biosynthesis pathway, such as chalcone synthases (CHS), were also induced by PWN inoculation in the susceptible species P. densiflora, P. massoniana, P. thunbergii, and P. pinaster, as well as in the resistant P. pinea [36,[38][39][40]44,46,48]. In P. pinaster and P. densiflora, a higher expression of flavonoid biosynthesis pathway genes was observed in resistant varieties [38,75], suggesting that the synthesis of flavonoids may have a role in PWN resistance. Accordingly, high-constitutive and PWN-induced levels of the flavonoids taxifolin and rutin were detected in the resistant species P. halepensis, while the susceptible P. pinaster and P. sylvestris had lower levels of these compounds [74]. Furthermore, levels of total flavonoids decreased in susceptible P. massoniana plants after PWN inoculation [41]. In many plant-nematode interactions, the activation of the flavonoid biosynthesis pathway has been associated with resistance to nematodes [72]. Products of this pathway have been shown to be toxic to several nematode species. For instance, naringenin, the product of CHS, caused a reduction in burrowing nematodes' (Radopholus similis) egg hatching, while kaempferol and quercetin repelled both root-knot nematodes (Meloidogyne incognita) and burrowing nematodes [76]. These compounds, and others with roles in plant resistance to parasitic nematodes (see [72]), may also affect PWN. Therefore, the levels of flavonoids should be further investigated in resistant pine species and varieties, as well as the toxicity and repellent effect of such compounds on PWNs.
Pinosylvin synthase (STS), a stilbene biosynthesis gene, was upregulated in P. densiflora and P. pinaster inoculated plants [44,46], and in the resistant species P. yunnanensis and P. strobus [49,66]. In P. strobus, an increase in the pinosylvin derivates dihydropinosylvin monomethyl ether (DPME) and pinosylvin monomethyl ether (PME) was observed together with STS upregulation, while in the susceptible species P. koraiensis and P. densiflora PME was not detectable and DPME was present only in trace amounts in P. koraiensis [66]. These compounds were shown to be toxic to PWN in in vitro assays, affecting nematode mobility and survival [64,66]. Interestingly, PME was more toxic to adult PWNs, while DPME was more toxic to juveniles [66]. High-constitutive levels of another stilbene, resveratrol, were observed in P. pinea and P. halepensis [74]. Levels of this compound also increased after PWN inoculation in P. halepensis. Therefore, the synthesis of stilbenoid compounds seems to be relevant in achieving resistance in some pine species.
Genes involved in the synthesis of lignans were upregulated in P. massoniana [40], while the synthesis of (+)-seoisolariciresinol was induced by PWN inoculation in P. halepensis [66]. Furthermore, the lignans (-)-nortrachelogenin and (+)-pinoresinol have been shown to be nematicidal for PWN [64]. However, the role of lignan compounds in PWN response has not been much explored, both in resistant and susceptible pine species or varieties.
Genes encoding transcription factors likely to be involved in the regulation of the phenylpropanoid, flavonoid and anthocyanin pathways, such as bHLH, MYB, or WRKY [71], were upregulated after PWN inoculation in P. densiflora, P. pinaster, P. pinea, P. yunnanensis, and P. strobus [38,45,49,66], supporting the importance of these pathways in pine response to PWN. However, most of the phenylpropanoid compounds reported were associated with resistance in one or few pine species. It is possible that each species may depend on a different combination of phenylpropanoids to achieve resistance, which may be speciesspecific. On the other hand, considering the existing variety of such compounds in plants, the lack of overlap between species may be simply due to the lack of extensive data. Further research focusing on the quantification of the several classes of phenylpropanoids before and after PWN inoculation, guided by the transcriptomics studies available, might provide new insights into the conserved and unique resistance mechanisms in the different pine species and varieties.

Lignin Biosynthesis and Cell Wall Reinforcement
Lignin is another product of the phenylpropanoid biosynthesis pathway. Several genes encoding enzymes specific to lignin biosynthesis were upregulated in P. densiflora, P. massoniana, P. thunbergii, P. pinaster, and P. yunnanensis in the initial response after PWN inoculation (24-72 hpi) (Table 3) [35,37,38,44,46,49]. Moreover, higher expression of genes involved in this pathway seems to be associated with the resistance phenotype in susceptible pine species. For instance, peroxidase genes, encoding an enzyme involved in the last step of lignin synthesis, were more expressed in resistant plants of P. massoniana, P. pinaster, and P. thunbergii [35,37,38]. Higher levels of lignin in cell walls around the inoculation zone were in fact associated with resistance in P. pinaster and P. thunbergii [38,77]. Furthermore, this lignin accumulation has been linked to a limitation in PWN migration in P. thunbergii stem tissues [77]. Therefore, increased lignification around the inoculation zone seems to be a conserved defense mechanism in resistant varieties within susceptible pine species. This can interfere with PWN migration as observed in P. thunbergii, but possibly also with PWN ability to digest plant cell walls and feed on their content [26]. It remains to be elucidated if constitutive levels of lignin in the stem also vary within pine species and if these levels can also influence the plant's phenotype after PWN inoculation. Cell wall reinforcement may also result from the cross-linking of the hydroxyproline-rich glycoproteins extensins (HRPG), catalyzed by peroxidases, which has also been associated with increased resistance to pathogens. Cross-linking of structural cell wall proteins such as extensins is the first histochemical modification observed in cell walls damaged by PWN, followed by lignification [77,78].

Oxidative Stress Response
During nematode infection, plants are typically under intense oxidative stress, where the reactive oxygen species (ROS) can be generated as part of the plant defense mechanisms, by dying plant cells, or by the nematodes themselves [26]. After nematode recognition, ROS produced by the plant act as signaling molecules in the activation of the defense response, having a role in the strengthening of plant cell walls via cross-linking, and may have a toxic effect on nematodes [25,26]. However, ROS are toxic to plant cells and may lead to their death if not transformed into innocuous molecules. Accordingly, during PWN infection, genes involved in ROS detoxification were differentially expressed after PWN inoculation in P. densiflora, P. massoniana, P. thunbergii, P. pinaster, and P. yunnanensis (Table 4) [35][36][37][38]40,44,46,49]. Furthermore, a few genes were reported to be more expressed in resistant than in susceptible plants, such as peroxidases [35,37,38], catalases [36,37], glutathione Stransferase [38], superoxide dismutase, and glutathione reductase (Table 4) [37]. In accordance with these gene expression results, the proteins glutathione S-transferase and superoxide dismutase were detected in higher levels in resistant P. massoniana plants 2 weeks postinoculation [42].  [37]. At 24 hpi, the levels of H 2 O 2 were higher in resistant plants when compared with susceptible ones, which was inverted at 15 and 30 dpi, when a steep increase was observed in susceptible plants. Thus, there seems to be a more efficient ROS detoxification in resistant plants, especially in more advanced phases of the disease, probably due to the action of the enzymes encoded by the genes more expressed in resistant P. massoniana plants.
Although ROS quantification has not been reported for other pine species infected with PWN, it is plausible that similar processes have a role in their resistance. In P. pinaster, genes encoding for aldehyde oxidase (GLOX) enzymes, which produce H 2 O 2 , had considerably higher expression levels in resistant plants in an early stage of infection (72 hpi) [38], which is consistent with the higher levels reported for resistant P. massoniana in a similar stage of infection (24 hpi). The production of H 2 O 2 in this early stage may promote the cross-linking in the plant cell wall, increasing its strength and impairing PWN migration and feeding.
At the metabolomics level also, the importance of cell redox homeostasis in resistance has been highlighted [47]. Pinus pinaster-resistant plants accumulated osmolytes after inoculation (e.g., fucose, GABA, trehalose), which are involved in protecting the cells from oxidative damage. Considering the importance that ROS detoxification seems to have in achieving resistance, it would be interesting to measure ROS at several timepoints after PWN inoculation in several pine species of interest, comparing resistant and susceptible varieties. Quantifying the enzymes encoded by the differentially expressed genes reported and correlating them with ROS concentration should highlight which enzymes are important for pine response and resistance to PWN.
The chitinases PR-3 and chitin-binding PR-4 were more expressed in resistant P. thunbergii [36] and P. pinaster [38] when compared to susceptible plants. Chitin is a main component of nematode eggshell [26,80] and possibly the pharyngeal lumen walls [29], suggesting that chitinases may compromise egg integrity and embryo development, as well as PWN feeding. Treatment with chitinase plant extracts caused premature egg hatching and increased juvenile mortality in the root-knot nematode Meloidogyne hapla [81]. Assessing the effects of chitinase extracts from pine trees, especially extracts from resistant varieties, in the several life stages of PWN would elucidate their role in PWD resistance. PR-5, which has antifungal activity, and PR-10 were also more expressed in resistant P. pinaster [38] and P. thunbergii [35] plants, respectively. It is unknown if these proteins have an impact on nematode growth, multiplication, or spread. Evaluating the effects of pine extracts of these proteins, similarly to what is here suggested for chitinases, would clarify this topic.
Other genes previously associated with plant defense response, such as mannose/glucosespecific lectin and ricin B-related lectin, were also upregulated early after inoculation (24-72 hpi in P. pinaster, P. pinea, and P. massoniana) [40,48]. Plant lectins have been shown to interact with mono-or oligosaccharides from several pests and pathogens and some were reported as toxic [82]. Interestingly, some lectins are involved in A. thaliana defense response against the root-knot nematode M. incognita [83].

Resistance Genes
Plant resistance genes are receptors that detect effectors released by a pathogen, parasite, or pest, starting a highly specific defense response that is effective in stopping the spread and multiplication of the invading organism. Resistance genes include the membrane receptors RLKs or RLPs, which recognize apoplastic effectors, and more often the intracellular receptors NLRs, which identify cytoplasmic effectors. These receptors may interact with the effector directly or indirectly by monitoring alterations caused by the effector in a plant co-factor [28,30]. Several studies have shown important roles of resistance genes in plant interactions with parasitic nematodes and herbivorous insects [29][30][31][32]. For instance, the RLP receptor Cf-2 recognizes the potato cyst nematode Globodera rostochiensis apoplastic effector venom allergen-like protein 1 (VAP1), indirectly, while the NLR receptor Gpa2 recognizes the potato cyst nematode Globodera pallida cytoplasmic effector GpRBP-1 [30]. The VAP1 gene was also identified in the PWN genome and the knockdown of this gene resulted in significantly lower PWN migration in the stem of pine seedlings when compared to wildtype PWN [84]. This suggests that VAP1 also acts as an effector in PWN-pine interactions by suppressing pine defense response. Another apoplastic effector, BxSapB1, has also been described recently [85], without which PWN has lower virulence than wildtype PWN. Furthermore, a NAMP has been characterized, namely BxCDP1, which leads to the initiation of PTI in a brassinosteroid-insensitive 1-associated kinase 1 (BAK1)-dependent manner [86].
The receptors involved in the recognition of these NAMP and effectors, and subsequent activation of pine defense response, are unknown. It is possible that different receptors participate in the activation of the immune response in resistant and susceptible plants, or that resistance genes detect PWN effectors and activate the more robust ETI in resistant plants. This is supported by the differential expression of distinct RLK/RLP and NLR receptor genes after PWN inoculation when comparing resistant and susceptible P. pinaster plants, as well as the downregulation of NLRs and resistance genes in susceptible P. massoniana [38,41]. Furthermore, two NLR receptor proteins were constitutively more expressed in resistant P. massoniana plants [43]. The activation of the ETI usually takes place in plants adapted to the pathogen or pest, implying a coevolution of the two organisms and often an arms race [28]. As PWN is an invasive parasite, susceptible pine species have not evolved in the presence of PWN. Therefore, genetic resistance to PWD occurring in natural stands has likely evolved due to the selective pressures of another pathogen or pest. In fact, one gene may confer resistance to more than one organism, as in the case of Mi-1.2, which confers resistance to root-knot nematodes (Meloidogyne spp.) [87], the potato aphid (Macrosiphum euphorbiae) [88], the white fly (Bemisia tabaci) [89], and the tomato psyllid (Bactericera cockerelli) [90]. To better understand pine response and resistance to PWN, it would be interesting to investigate if ETI is activated in resistant plants, what effectors may be recognized by resistance genes, and what receptors are involved in these responses.

Post-Transcriptional Regulation Mediated by Small RNAs in Pine Response to PWN
Small RNAs (sRNAs) are a class of non-coding RNAs, with 20 to 35 nucleotides, that are key players in post-transcriptional and transcriptional gene silencing [91]. MicroRNAs (miRNAs), one of the best studied sRNA classes, are mostly involved in post-transcriptional gene silencing by guiding the cleavage or translation inhibition of complementary target transcripts [92] with roles in a variety of processes, including plant development, as well as response to abiotic and biotic stresses [93,94]. During plant defense response, sRNAs are known to be involved in the regulation of plant hormone synthesis and signaling, callose deposition, expression of NLR receptors, and of other resistance proteins, ROS detoxification, and secondary metabolites synthesis [95], being essential players in PTI and ETI immune responses. After nematode infection, several host sRNAs have also been associated with resistance traits. Transcription factors and hormone signaling genes are examples of sRNA targets associated with plant defense mechanisms against parasitic nematodes [96,97]. The great majority of studies on sRNAs involved in plant response to nematode diseases focus on sedentary endoparasitic nematodes and their involvement in feeding sites' development [96,[98][99][100]. Nevertheless, the first steps have been taken to understand the role of miRNAs in PWD response.

MicroRNA-Mediated Response to PWN
The first report of miRNAs differentially expressed in response to PWD was in P. massoniana [101]. These authors showed that in P. massoniana inoculated with PWN, several miRNAs were differentially expressed in the needles during the first 3 days post-inoculation (dpi). The predicted targets for these miRNAs were associated with plant hormone signaling (e.g., zeatin synthesis and ethylene signaling), RNA transport, splicing, and fatty acid metabolism, among other processes. The expression of hormone signaling predicted targets showed a significant increase at 2 dpi followed by a decrease at 3 dpi and negatively correlated with the expression of their corresponding miRNAs. The impact of the infection on hormone synthesis and accumulation was confirmed by the quantification of indole acetic acid (IAA) and zeatin contents in P. massoniana needles, which were shown to initially decrease (3 dpi), then increase (9 dpi), and finally decrease significantly at 14 dpi [101]. The suppression of growth-related hormone signaling and synthesis at 14 dpi was suggested by the authors to be a consequence of the damage caused by PWN. On the other hand, the initial decrease in hormone content (3 dpi) likely results from growth-defense trade-offs, suggesting that the tree has relocated its energy for the defense response to the detriment of growth and development [102]. However, the expression of miRNAs involved in the regulation of plant immune response was not detected in this study, probably because only the pine needles were sampled, while PWN only infects stem tissues.
A recent study focusing on the quantification of P. pinaster stem miRNAs has, in fact, identified different miRNAs which are predictably defense-related [103]. MicroRNAs differentially expressed after inoculation targeted JA responsive genes, RLK and NLR receptor genes, transcription factors WRKY, genes involved in ROS detoxification, such as thioredoxins and peroxiredoxins, and genes involved in terpene biosynthesis, such as LPS. When comparing resistant and susceptible plants, only 8 miRNAs were differentially expressed, and their targets included genes possibly involved in the activation of plant defense response, such as RLKs and GDP-L-fucose synthase 2, ROS detoxification and JA signaling pathway. Many of the post-transcriptionally regulated pathways and genes have been shown to be PWN-responsive in several pine species, as described above. Targeting of JA regulators by miR166, miR947, miRnovel_43f, and miRnovel_110; regulation of RLKs, essential for PTI activation, by miR166h, miR951f, and miR947f; increase of oxidoreductase activity in resistant plants possibly involving miR3627m; and increased terpene synthesis as a consequence of miRNAs' differential expression, have been suggested as important post-transcriptional events for P. pinaster resistance to PWN. In addition, trans-acting shortinterfering RNAs (tasiRNAs), a class of siRNAs derived from specific miRNA cleaved transcripts, have also been predicted to originate from miR11532 and miR947f activity [103]. The predicted targets for these tasiRNAs seem to be involved in plant hormone signal transduction, plant-pathogen interaction, and flavonoid biosynthesis pathways, adding another regulatory layer to the control of the defense response pathways.

Trans-Kingdom RNA Silencing in PWD
In recent years, great interest has been shown in trans-kingdom sRNAs and their potential for new plant protection technologies against pathogens and pests. These sRNAs can perform regulatory functions after being transferred between distantly related organisms [104]. Plant-originated sRNAs can target pathogen housekeeping genes, effectors, pathogenicity, or development-related genes, for instance, and the pathogen sRNAs can suppress host immunity [99,105]. Naturally occurring trans-kingdom sRNAs can, therefore, be considered an addition to the well-known innate immunity zig-zag arms race between hosts and pathogens [106].
Possible targets for PWN and P. pinaster miRNAs in P. pinaster and PWN transcriptomes, respectively, have been identified [103]. Pinus pinaster genes putatively regulated by PWN trans-kingdom miRNAs are involved in transcriptional processes, protein synthesis and assembly, and immune response-related processes (e.g., isoprenoid biosynthesis and regulation of ABA-activated signaling pathway). The targeting of those genes has been supported by degradome data and suggests that PWN enhances its pathogenicity by weakening P. pinaster cell functions and preventing the establishment of a timely and effective defense response. On the other hand, pine miRNAs may target PWN response to stimuli, transcriptional response, detoxification of plant xenobiotics (e.g., cytochrome P450 or epoxide hydrolase genes) and digestion of plant tissues (e.g., peptidases and lysosomal enzymes) [103], possibly affecting nematode pathogenicity.
Trans-kingdom RNA silencing opens new doors for the employment of technologies that use RNA-interference (RNAi) to regulate plant resistance, such as host-induced gene silencing (HIGS), or spray-induced gene silencing (SIGS) [107]. In HIGS, pathogen genes are silenced by expressing complementary double-stranded RNAs (dsRNA) or artificial miRNAs in the host plant, while in SIGS, the dsRNA is sprayed on the infected plant, having similar silencing results. Interestingly, the silencing of some PWN genes predicted to be targeted by P. pinaster miRNAs through the external application of double-stranded RNA (dsRNA) efficiently reduced PWN pathogenicity (e.g., cytochrome P450 [108][109][110]; the peptidases cathepsin [111] and cysteine peptidase BxCAT2 [112]). This may be particularly important in the control of more virulent strains of PWN. By comparing 4 different isolates of PWN, Shynia et al. [112] have identified Bx-GH30 and Bx-CAT2 as proteins highly secreted by virulent PWN isolates, showing their contribution to isolate virulence in a host species and possible association with pine wilt disease. These effectors are good examples of potential candidates to be targeted aiming at nematode control strategies, and other candidates have been reported (e.g., [112][113][114][115]). Although naturally occurring trans-kingdom RNAi transference was never observed for plant-nematode interactions, the transference of artificial RNAi from the plant host to root-knot nematodes, and subsequent gene silencing, has been previously reported (e.g., [116,117]). For instance, HIGS has been successfully used to knock down several effector genes in M. incognita [116,118]. Therefore, P. pinaster miRNAs that putatively target PWN pathogenicity genes should be further validated and their potential application in HIGS or SIGS should be investigated [104,107].

Defense Response Induced by the Application of Phytohormones and Secondary Metabolites
In recent years, the application of elicitors to pine trees in order to induce plant immunity prior to PWN infection has been investigated as a PWD control method [119][120][121][122]. Elicitors can be a variety of substances, such as plant hormones, purified molecules derived from pathogens or pests, or synthetic molecules, which induce plant defense response [123]. These substances have the potential to be used as biocontrol agents, as they are more economical and eco-friendly approaches than the traditional use of insecticides against the insect vector or trunk injection of nematicides. A few elicitors have been evaluated for PWD with positive results in reducing disease progression or PWN multiplication, namely methyl salicylate (MeSA) and chitosan [119][120][121][122].
The application of MeSA in P. densiflora and P. thunbergii seedling leaves in the form of spray one and two weeks before PWN inoculation significantly decreased disease progression when compared to seedlings without treatment [119,120,124]. Treatment of P. densiflora plants with MeSA and subsequent inoculation with PWN seem to induce genes and pathways similar to those previously associated with PWN resistance, such as PR-1, PR-2, PR-5, peroxidases, extensins, flavonoid biosynthesis genes, and genes involved in ROS detoxification [119,124]. The expression levels of these genes were much higher in plants treated with MeSA than in non-treated plants, reinforcing their importance in achieving resistance to PWD.
Similarly, the application of chitosan, a compound derived from chitin, in the soil increased P. pinaster resistance to PWN, as shown by the significantly lower number of PWNs observed in treated plants at several timepoints after inoculation when compared to untreated ones [121,122]. Chitosan application induced catalase activity, an enzyme involved in ROS detoxification, as well as the production of phenolic compounds, anthocyanins (flavonoids), carotenoids (terpenes), and lignin [122]. Therefore, application of chitosan and MeSA induced similar pathways that seem to be crucial for PWD resistance in pine trees.
On the other hand, the trunk injection of MeJA had a small effect on improving P. densiflora resistance to PWN [124]. However, spraying P. massoniana seedlings with MeJA seems to induce the production of diterpenes and deter the insect vector Monochamus alternatus from feeding on the stems of elicited plants [125]. These observations show that MeJA has also the potential to be used as a control compound at the level of the host-insect vector interaction. Further studies are needed to confirm that MeJA application has the same effect on other pine trees and insects of the genus Monochamus.

Conclusions and Future Perspectives
After PWN infection, pine trees undergo a significant transcriptional change that may be followed by changes in the synthesis of proteins and metabolites (e.g. [47,63,66]). We presented an overview of the defense response pathways induced in several pine species by PWN inoculation, revealing that at least part of this defense seems to be conserved ( Figure 1). More importantly, the analysis of more resistant pine varieties and families highlighted several resistance mechanisms, many of which were also conserved among pine species. ROS detoxification, limiting PWN migration and feeding through cell-wall reinforcement, including lignification, and production of secondary metabolites that affect PWN mobility, impairing PWN development and feeding through the action of chitinases, or directly influencing PWN survival by producing secondary metabolites with nematicidal effects, are likely to act together in achieving resistance to PWN. Small RNAs seem to be important players in regulating several of these pathways, as well as plant growth, during PWN infection. Moreover, naturally occurring trans-kingdom miRNA transference between PWN and its host may be an important process in repressing pine defense response or PWN pathogenicity.
To better understand resistance mechanisms, it is important to complement transcriptomics studies with proteomics and metabolomics, since differences in mRNA levels do not always translate into protein abundance or activity due to variations in translational efficiency, which may be caused by post-transcriptional regulation, mRNA secondary structures or availability of cellular resources, and post-translational modifications, which influence protein structure or function. Extending these studies to a higher number of pine varieties and families selected for PWD resistance would allow us to distinguish between candidate genes specific to varieties from more general candidate genes. Functional analysis of candidate genes in model systems can confirm their role in resistance and give details about their functions [9]. After confirming their function, the overexpression, knockdown or knockout of the candidate genes may be explored aiming at obtaining resistance to PWN. However, genetically engineering conifer species is difficult, and many times optimized for a specific clonal background [126]. On the other hand, in many countries strict regulations prevent the plantation of genetically engineered trees in the fields. An alternative could be to use CRISPR/Cas9-directed genome editing technologies that are more leniently regulated in several countries, including Japan. Furthermore, genome editing may be more efficient and promising results have been obtained in P. radiata [127]. and biomarkers. Interesting results have been obtained in detecting pine trees with PWD in the forest, using unmanned aerial vehicles with multispectral cameras, and machine learning algorithms [129]. The adaption of these methods in the screening of plant traits in large-scale inoculation assays could be of great relevance and should be further explored. In conclusion, the emerging knowledge on PWD is of great relevance for its control and should be explored in the near future taking advantage of the powerful highthroughput technologies and tools now available for precision plant breeding. Figure 1. Representation of pine defense response to pinewood nematode (PWN). Pattern-triggered immunity (PTI) is initiated after recognition of nematode-associated molecular patterns (NAMPs), such as BxCDP1, or damage-associated molecular patterns (DAMPs) from damaged plant cells, by cell surface receptor-like kinases (RLKs) or receptor-like proteins (RLPs). PTI leads to a transcriptional reprograming of the plant cell, which includes the activation of hormone pathways, such as jasmonic acid (JA) and salicylic acid (SA) pathways, the expression of pathogenesis-related genes (PR), genes involved in oxidative stress response, and genes encoding enzymes involved in the biosynthesis of secondary metabolites (terpenes and phenylpropanoids). In susceptible plants, it is possible that the activation of the SA pathway inhibits the JA pathway. PWN releases effectors, such as the apoplastic VAP1 and BxSapB1, that repress the plant defense response. These effectors may be recognized by resistance genes, whether directly or indirectly through the monitoring of a co-factor protein that is altered by the effector. Effectors can be recognized by cell surface receptors, when they are apoplastic, or internal nucleotide-binding/leucine-rich-repeat (NLR) receptors, when they are injected into the cytoplasm by the PWN. Recognition of effectors by resistance genes initiates a stronger and more sustained defense response, the effector-triggered immunity (ETI).  Patterntriggered immunity (PTI) is initiated after recognition of nematode-associated molecular patterns (NAMPs), such as BxCDP1, or damage-associated molecular patterns (DAMPs) from damaged plant cells, by cell surface receptor-like kinases (RLKs) or receptor-like proteins (RLPs). PTI leads to a transcriptional reprograming of the plant cell, which includes the activation of hormone pathways, such as jasmonic acid (JA) and salicylic acid (SA) pathways, the expression of pathogenesis-related genes (PR), genes involved in oxidative stress response, and genes encoding enzymes involved in the biosynthesis of secondary metabolites (terpenes and phenylpropanoids). In susceptible plants, it is possible that the activation of the SA pathway inhibits the JA pathway. PWN releases effectors, such as the apoplastic VAP1 and BxSapB1, that repress the plant defense response. These effectors may be recognized by resistance genes, whether directly or indirectly through the monitoring of a co-factor protein that is altered by the effector. Effectors can be recognized by cell surface receptors, when they are apoplastic, or internal nucleotide-binding/leucine-rich-repeat (NLR) receptors, when they are injected into the cytoplasm by the PWN. Recognition of effectors by resistance genes initiates a stronger and more sustained defense response, the effector-triggered immunity (ETI). Furthermore, the search for SNPs in candidate genes for resistance and association with phenotype after PWN inoculation will be key to the development of molecular markers useful for accelerating breeding programs through marker-assisted selection. Developing biomarkers for resistance may also aid in the selection of trees with constitutive higher concentrations of specific secondary metabolites associated with resistance. The use of high-throughput phenomics could increase the precision, efficiency, and speed of the plant screening for resistant and susceptible phenotypes [128]. In combination with genomics and metabolomics, it would improve the identification of molecular markers and biomarkers. Interesting results have been obtained in detecting pine trees with PWD in the forest, using unmanned aerial vehicles with multispectral cameras, and machine learning algorithms [129]. The adaption of these methods in the screening of plant traits in large-scale inoculation assays could be of great relevance and should be further explored. In conclusion, the emerging knowledge on PWD is of great relevance for its control and should be explored in the near future taking advantage of the powerful high-throughput technologies and tools now available for precision plant breeding.