Since the causative pathogen was identified [1], many researchers have characterized the physiological changes associated with progression of pine wilt disease, and by the mid-1990s, more than two thousand papers on the disease had been published [5]. Many symptoms associated with PWN infection, including decreased photosynthesis, denaturation of xylem and cortex parenchyma cells, traumatic resin canal formation, cambium destruction, production of phytotoxic substances and ethylene, reduced water potential and transpiration in leaves and heat pulse velocity have been studied (for review, see reference [6]). Based on the anatomical and biochemical evidence gathered during these 40 years, development of symptoms is thought to occur in two stages, early and advanced stages [6]. In the early stage, a small number of nematodes migrate and symptoms such as necrosis and destruction of cortex and phloem tissue and cambium, destruction of cortex resin canals, formation of wound periderm in cortex parenchyma around resin canals, and ethylene release all occur near the inoculation site. In the advanced stage, ethylene production is enhanced and coincides with the broad destruction of cortex parenchyma, cambial destruction, and cavitation-induced embolism of the tracheids in the xylem. The cavitation-induced embolism causes a decrease in leaf water potential and cessation of photosynthesis. After cessation of photosynthesis, severe symptoms develop rapidly with a burst in the nematode population; this population increase results in wilting and eventual tree death. Resistance against PWN infection depends on stopping the symptoms at the early stage or preventing the progression of infection to the advanced stage. While the physiological changes that occur as pine wilt disease progresses have been characterized anatomically and biochemically, molecular genetic events such as changes in transcript profiles that may be associated with the physiological changes in either resistant or susceptible trees remain poorly understood.

Recently, the gene expression profiles of resistant (resistant variety of P. thunbergii 'Sendai-290'; resistant rank 1) and non-resistant Japanese black pine (P. thunbergii) trees were assessed using the LongSAGE technique on stems collected 3 days after PWN inoculation [7]. The researchers found that catalase and proteins in the dienelactone hydrolase family were highly expressed in resistant trees, but not non-resistant trees, whereas pathogenesis-related (PR)-1, 2, 3, leucoanthocyanidin dioxygenase and cell wall-related genes were expressed at higher levels in the non-resistant trees. Although the study assessed a difference of one time point in the defense responses of resistant and non-resistant P. thunbergii following PWN inoculation, the defense response is continuous, and the differences in resistance and susceptibility do not depend only upon qualitative differences in the activated defense genes, but also on differences in the timing and magnitude of their expression [8]. To characterize the differences in transcript profiles of resistant and susceptible trees as pine wilt symptoms develop, it is necessary to sample resistant and susceptible individuals over a defined time course.

Subtractive suppression hybridization (SSH) is a powerful tool for gene expression profiling that effectively identifies genes differentially expressed under different conditions or in different tissues [9]. This method is relatively simple and easy, it can be used with species for which there is little or no genomic information, and the cDNAs isolated are typically longer than 100 bp and can be effectively annotated using comparative genomics (e.g., BLAST analysis). This method is often used to isolate plant genes specifically expressed in response to pathogen infection and to identify differences in the transcript profiles of infected resistant and susceptible plants [10-14]. SSH selection reduces the cloning of abundantly expressed housekeeping genes or genes commonly expressed in both "tester" and "driver" samples, and therefore normalizes the expressed cDNA profiles during library construction. As a result, SSH selection significantly enhances the chances of cloning differentially expressed genes.

There was a significant difference in transcript diversity between resistant and susceptible trees after infection with PWN. The percentage of unique sequences in susceptible libraries ranged from 71.59 to 27.17%, whereas the percentage of unique sequences in resistant libraries ranged from 72.82 to 96.11%. Although transcript diversity of the susceptible library at 3 dpi was higher than the susceptible libraries at 1 and 7 dpi, transcript diversity in resistant libraries was higher than susceptible libraries at all time points after PWN infection. According to the GO classification of the differentially expressed transcripts, a large percentage in susceptible trees was involved in stress/defense response categories such as the response to stimulus, multi-organism process, and immune system process within the biological process category and the enzyme inhibitor activity, carbohydrate binding, pattern binding, and hydrolase activity within the molecular function category. Furthermore, a large percentage of transcripts in the cellular component category were in the cytoplasmic membrane-bounded vesicle subcategory. On the other hand, a large percentage of transcripts in resistant SSH libraries was categorized into the cellular process, metabolic process and response to stimulus subcategories of the biological process category and in the ligand binding and transferase activity subcategories of the molecular function category. Many transcripts recovered in libraries from resistant trees were assigned to the cellular component subcategory and further classified into organelle subcategories of plastid, chloroplast, and mitochondrion. The difference in transcript diversity between libraries from resistant and susceptible trees revealed by the GO classification indicated a qualitative difference in the stress/defense response of resistant and susceptible trees to PWN infection.

Resistance and susceptibility do not depend only upon qualitative differences in the activated defense genes, but also on differences in the timing and magnitude of their expression [8]. The gene regulation patterns of pathogenesis related defense proteins such as PR-1b, PR-2, PR-3, PR-4, PR-5, PR-6 and antimicrobial peptide indicated temporal and quantitative differences between resistant and susceptible trees in response to PWN infection. In regulating the plant defense response, most pathogenesis related proteins are induced through the action of the signaling compounds salicylic acid (SA), jasmonic acid (JA), or ethylene [16]. PR-1b, PR-2 and PR-5 genes are SA-responsive genes and also indicators of systemic acquired resistance; additionally, the PR-6 gene is a JA and ethylene responsive gene [17]. Although the relationship between phytohormones and the defense response in the PWN-nematode interaction is not clear from this study, it is interesting that expression of pathogenesis related genes associated with phytohormones such as SA and JA/ethylene and with antimicrobial activities were induced more quickly and to a higher level in susceptible than in resistant trees.

The same three phytohormones, SA, JA, and ethylene, are important for both basal and R-gene mediated defense responses to foliar pathogens and insects. The JA and ethylene signaling pathways seem to work synergistically, whereas the SA and JA/ethylene signaling pathways inhibit each other, and negative cross-talk exists between SA and JA/ethylene signaling pathways [18]. In interactions between Hero A-resistant tomatoes and cyst nematode, expression of SA-responsive genes PR-1 and PR-5 is a hallmark of the resistant cultivar, and expression of JA-dependent PR-6 is higher in the susceptible cultivar, indicating that SA plays some role in the resistance to the nematode and that JA and ethylene in susceptible tomatoes are likely to interfere with the SA-inducible resistance pathway [19,20]. The involvement of SA in resistance and expression of SA-responsive genes in resistant plants has been observed in interactions between other plant species and nematodes (e.g., an Arabidopsis thaliana mutant [21] and a root knot nematode resistant peanut [13]). In our study, the higher induction of both SA-responsive genes such as PR-1b, PR-2, PR-5 and JA/ethylene-responsive genes such as PR-6 in susceptible trees indicates that the defense response mediated by these phytohormones was not very effective in controlling PWN infestations.

We detected notable levels of putative HRGPs, extensin and peroxidase (PR-9) in resistant trees at 7 dpi and 14 dpi, though expression in susceptible trees was higher than in resistant trees at 1 and 3 dpi. Cell wall-mediated resistance is the first line of plant defense against pathogens, and the components of plant cell wall are modified by production of reactive oxygen species (ROS) such as H₂O₂ during attack by pathogens [22]. The structural cell wall proteins extensin and HRGPs play an essential role in biotic and abiotic stress responses due to their oxidative cross-linking, which contributes to the strength of cell walls and is catalyzed by an oxidizing system based on peroxidase and H₂O₂ [23-25]. The cross-linking of HRGPs and extensin is involved in cell wall-mediated resistance and has a major role in arresting pathogens at the site of entry [23]; these proteins accumulate in the walls of a number of plant species during interactions with microbes [26]. A number of reports describe the response of plant cell wall HRGPs and extensin to pathogens such as viruses, bacteria, and fungi (for review, see reference [27]). In plant-nematode interactions, high extensin gene expression was observed in the cortical region of tobacco at 7 and 14 days after inoculation with root knot nematodes [28]. In rhg1 resistant soybean, extensin was identified as one of the genes characteristically expressed in syncytia after inoculation with soybean cyst nematode, indicating that altered cell wall composition is important in the defense response [29]. Also, in Mi resistant tomato, extensin and glycosyltransferase may play a role in cell wall synthesis, which is an essential defense against root knot nematode [30,31]. In anatomical studies of PWN infection, Ishida et al. [32] inoculated virulent nematode (B. xylophilus) isolate S6-1 and avirulent nematode (B. mucronatus) isolate B. m to Japanese black pine, and observed accumulation of lignin- and suberin-like substances around the resin canals in the cortex with both nematode isolates at 7 dpi. Kusumoto et al. [33] also inoculated a virulent nematode (B. xylophilus) isolate, Ka-4, to Japanese black pine and found more frequent accumulation of phenolic compounds around the cortex resin canals in resistant trees at 7 dpi and 14 dpi after inoculation with PWN (B. xylophilus), and suggested that this accumulation was a very effective defense against infection due to restricting PWN migration. Although the relationship between HRGPs or extensin and other substances in the cell wall was not examined in this study, it is possible that upregulation of expression of cell wall-related genes such as those for HRGPs or extensin and oxidative cross-linking of these proteins by peroxidases plays a role in the effective defense response of trees resistant to PWN infection at 7 dpi and 14 dpi.

PR-10 was also one of the characteristically significantly upregulated genes in libraries in the 7 dpi subtraction library from resistant trees. Although the biological function of PR-10 remains unclear, many PR-10 genes are upregulated when plants are exposed to abiotic stresses, such as SA, CuCl₂, H₂O₂, cold, darkness and wounding [34], and biotic stresses, such as viruses [35], bacteria [34,36], fungi [37-40] and insects [41,42]. We observed synchronized expression of PR10 with peroxidase in resistant trees, which indicates this gene may be induced by ROS such as H₂O₂. However, PR10 (CpPRI) acts against a digestive proteinase from the root knot nematode Meloidogyne incognita [43]. Therefore, PR10 may act as a proteinase against cellulases, beta-1,3-glucanase, and pectate lyases secreted from PWN [44-46].

Heat shock protein (HSP) ESTs were characteristically recovered in libraries from resistant trees. In particular, stable HSP70 expression in infected resistant trees was validated by qRT-PCR. HSP family members, which consist of HSP70, HSP60 and HSP90, are required for folding of nascent proteins and intracellular transportation in addition to stress responses, and are collectively called molecular chaperones [47]. In the interaction between soybean and soybean cyst nematode, Klink et al. [48] observed the induction of HSP70 and ROS responsive genes such as lipoxygenase and superoxidase dismutase isolated from 3 dpi syncytial cells showing an incompatible response to soybean cyst nematode infection, and suggested that HSP70 may be involved in maintaining a properly functioning environment for other defense responses. In the present study, it is unclear how HSP70 is involved in the defense response to PWN infection.

Three ESTs--putatively encoding CYP450, metallothionein-like protein, and XET--were detected in resistant SSH libraries at 1 dpi and 3 dpi, which depend on the genes significantly downregulated in susceptible trees at 1 and 3 dpi. In plants, CYP450 monooxygenases play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products [49]. In conifers, diterpene resin acids are prominent defense compounds against insect pests and pathogens in conifers [50-52], and CYP450s are involved in the formation of a suite of diterpene resin acids of conifer oleoresin; they oxidize abietadienol and abietadienal to abietic acid in vitro and in vivo [53,54]. Keeling and Bohlmann [50,51] indicated that oleoresin may contain specific components that are toxic or deterrent to insect herbivores or may affect adults or broods physiologically and thus prevent successful colonization or reproduction. The downregulation of CYP450 observed in the present study may cause a reduction in diterpene resin acids in pine trees infested with PWN. Consequently, PWN may expand its invasion and habitat, and the rapid reduction in CYP450 expression in susceptible trees may trigger PWN expansion.

Metallothioneins are involved in ROS scavenging, and in rice, downregulation of metallothionein expression is observed during the oxidative burst phase in elicitor-treated cells, and metallothionein expression is important for defense signaling [55,56]. The metallothionein expression we observed indicated that ROS accumulation and defense signaling may have been induced by 1 dpi in susceptible trees infected with PWN, whereas it may not have been induced much in resistant trees 3 dpi later; this provides evidence for rapid induction of defense response genes such as those encoding pathogenesis related proteins in susceptible trees.

XET action is thought to regulate wall loosening during turgor-driven expansion by rearranging load-bearing xyloglucan cross-links between cellulose microfibrils, and its activity and expression have been detected in growing tissues [57-64]. We found that XET was downregulated in both resistant and susceptible trees following PWN infection, and its regulation was induced relatively early, by 1 dpi. These findings suggest that the expansion of cell walls in xylem, phloem or both is inhibited by the downregulated XET during PWN infection; alternatively, the cell wall may be immobilized by the cross-linking of HRGPs or extensin.