Pine wood nematode (PWN) causes serious diseases in conifers, especially pine species. To investigate the transcriptomic profiles of genes involved in pine-PWN interactions, two different pine species, namely, Pinus thunbergii and P. massoniana, were selected for this study. Weighted gene coexpression network analysis (WGCNA) was used to determine the relationship between changes in gene expression and the PWN population after PWN infection. PWN infection negatively affects the expression of most genes in pine trees, including plant defense-related genes such as genes related to plant hormone signal transduction, plant-pathogen interactions, and the MAPK signaling pathway in plants. However, the expression of chalcone synthase genes and their related genes were proportional to the changes in nematode populations, and chalcone synthase genes were dominant within the coexpression module enriched by genes highly correlated with the nematode population. Many genes that were closely related to chalcone synthase genes in the module were related to flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis. Pine trees could actively adjust their defense strategies in response to changes in the number of invasive PWNs, but the sustained expression of chalcone synthase genes should play an important role in the inhibition of PWN infection.

Melampsora larici-populina causes serious poplar foliar diseases called rust worldwide. Salicylic acid (SA) and jasmonic acid (JA) are important phytohormones that are related to plant defence responses. To investigate the transcriptome profiles of SA- and JA-related genes involved in poplar rust interaction, two tolerant poplars and one intolerant poplar were selected for this study. Weighted gene coexpression network analysis (WGCNA) was applied to characterize the changes in the transcriptome profiles and contents of SA and JA after infection with the virulent E4 race of M. larici-populina. In response to infection with the E4 race of M. larici-populina, tolerant symptoms were correlated with the expression of genes related to SA and JA biosynthesis, the levels of SA and JA, and the expression of defence-related genes downstream of SA and JA. Tolerant poplars could promptly regulate the occurrence of defence responses by activating or inhibiting SA or JA pathways in a timely manner, including regulating the expression of genes related to programmed cell death, such as Kunitz-type trypsin inhibitor (KTI), to limit the growth of E4 and protect themselves. WGCNA suggested that KTI might be regulated by a Cytochrome P450 family (CYP) gene. Some CYPs should play an important role in both JA- and SA-related pathways. In contrast, in intolerant poplar, the inhibition of SA-related defence signalling through increasing JA levels in the early stage led to continued inhibition of a large number of plant–pathogen interaction-related and signalling-related genes, including NBS-LRRs, EDS1, NDR1, WRKYs, and PRs. Therefore, timely activation or inhibition of the SA or JA pathways is the key difference between tolerant and intolerant poplars.

Temperatures directly influence the distribution and intensity of pine wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus. To date, however, little is known about the causation and mechanism of this influence. The molecular chaperone HSP90 is a key component that contributes to survival in the abiotic stress response. In this study, we investigated the relationship between the survival of B. xylophilus and the functionality of the HSP90 gene. Bx-HSP90 was cloned from a suppression subtractive hybridization library. In situ mRNA hybridization showed that Bx-HSP90 was constitutively expressed in response to all of the temperatures tested, and RT-PCR indicated that all of the temperatures could induce Bx-HSP90 transcription, with the highest transcript level detected at 30 °C. The suppression of the Bx-HSP90 transcript by RNA interference led to a 25% reduction in the number of nematodes at 30 °C after 44 h. Sharp declines in the survival of the RNAi-treated nematodes were observed after 8 days at 25 °C, 48 h at 30 °C and 24 h at 35 °C. Both heat shock and the knockdown of Bx-HSP90 hindered the growth of the B. xylophilus populations. The results indicate that Bx-HSP90 is essential for the survival of B. xylophilus, confirming the thermoregulatory function of the gene, and delineate the timeframe and temperature range within which the gene function occurs.

Rust caused by Melampsora larici-populina is one of the most damaging diseases of poplars. Rust is considered to be a model pathogen for genetic studies because both pathogen and host genomes are available. The poplar ‘Robusta’, whose general rust resistance is defeated by virulent rust E4, provides suitable host material for studies of the gene regulation involved in rust resistance/susceptibility. In this study, we investigated the microRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent rust. We were particularly interested in delineating the host-pathogen interactions with a specific focus on microRNAs (miRNAs). To study the susceptibility of poplar to M. larici-populina, small RNA (sRNA) libraries, a degradome cDNA library and digital gene expression libraries were constructed for rust-inoculated and rust-free susceptible poplar ‘Robusta’ leaves through high-throughput sequencing. Altogether, 12,722 regulating interactions were identified. The results delineated the framework of post-transcriptional regulation of gene expression in the susceptible poplar, which was infected by the virulent rust. The results indicated that pathogen-associated molecular patterns (PAMPs) and PAMP-triggered immunity were induced by the infection of virulent rust E4 and that miRNAs still functioned at this stage. After this stage, miRNA-regulated R genes, such as TIR-NBS-LRR and CC-NBS-LRR, were not fully functional. Additionally, the rust-responsive miRNAs did not regulate the signaling component genes related to the salicylic acid pathway or the hypersensitive response. We found that the defense-related post-transcriptional regulation of the susceptible poplar ‘Robusta’ functions normally only at the stage of PAMPs and PAMP-triggered immunity (PTI). More importantly, the miRNA-mediated post-transcriptional regulation of defense signal pathway genes were inactivated by the infection of virulent rust at the stage of effector-triggered susceptibility and during the following stages of salicylic acid and hypersensitive responses. This inactivation was the major characteristic of ‘Robusta’ susceptibility.

Background: Recently, pine wood nematode (PWN, Bursaphelenchus xylophilus) has been found in the extreme cold area of northeast China. The third-stage dispersal juvenile (DJ3) of PWN, which is a long-lived stress-resistant stage, plays an important role in the process of PWN spreading to low-temperature areas, as this stage can survive under unfavorable conditions. Results: Weighted correlation network analysis (WGCNA) was used to analyze the expression patterns of 15,889 genes included in 21 RNA-Seq results of PWN at DJ3 and the other 6 different stages, and a total of 12 coexpression modules were obtained. Among them, the magenta module has the highest correlation with DJ3, which included a total of 652 genes. KEGG enrichment analysis showed that most of the genes in the magenta module were involved in metabolic processes, which were related to autophagy and longevity regulation. These pathways included starch and sucrose metabolism, which contains trehalose metabolism. To explore the function of trehalose in DJ3 formation and survival under − 20 °C, a trehalose-6-phosphate synthase encoding gene (Bx-tps), a trehalose-6-phosphate phosphatase encoding gene (Bx-tpp) and 7 trehalase encoding genes (Bx-tres) were identified and investigated. The expression of these 9 genes was related to the formation of DJ3. A treatment under − 20 °C induced the accumulation of trehalose. The survival rate of DJ3 at -20 °C reduced after silencing of any of these trehalose metabolism genes. Further analysis suggested that two trehalose synthesis genes were highly correlated with DJ3 and might be involved in autophagy by regulating with energy conversion related genes. Conclusions: The above results indicated that trehalose metabolism promotes DJ3 formation and helps DJ3 survive at -20 °C. Although trehalose accumulation is favorable for DJ3 to cope with low-temperature stress, multiple trehalose metabolism genes need to work together. There may be a multi-path regulated physiological process involving trehalose synthesis genes under low-temperature stress resistance. This physiological process may regulate the formation and maintenance of DJ3 through autophagy and energy conversion.

Background: Pine trees challenged byBursaphelenchus xylophilusinvasion produce phytoalexins to combat this nematode. Nevertheless, the phytoalexins of Asian pine trees are ineffective againstB. xylophilus. The anti-phytoalexin genes ofB. xylophilusdisable almost all Asian pine phytoalexins, which has allowedB. xylophilusto devastate pine forests in eastern Asia over the last four decades. However, to date, the factors that stimulate anti-phytoalexin gene expression and the mechanisms by which these genes act are not well understood.Results: Here, we described anti-phytoalexin genes inB. xylophilususing transcriptomic and bioinformatics analyses. The genes that were induced by bothPinus massonianaand carvone and had similarly elevated expression trends were considered anti-phytoalexin genes. Altogether, 187 anti-phytoalexin genes were identified, including 4 cathepsin genes. KEGG pathway enrichment indicated that those cathepsins were related to the Lysosome pathway. Since cathepsins help to maintain metabolic homeostasis by participating in the degradation of heterophagic and autophagic material, the lysosomal cathepsin geneBx-cathepsin Wwas cloned and characterized. The results of the RNAi assessment indicated that the knockdown ofBx-cathepsin Wreduced the survival rates ofB. xylophilusunder carvone orP. massonianastress. The correlation betweenBx-cathepsin Wand the susceptibility of pines showed thatBx-cathepsin Wmight help improve the anti-phytotoxin ability ofB. xylophilus.Conclusions: The results indicated that the anti-phytoalexin geneBx-cathepsin Wsupported the survival ofB. xylophilusunderP. massonianaphytoalexin stress. The cDNA library sequencing, differentially expressed gene identification, and WGCNA algorithm analysis provided insight at a systemic level into the gene regulation ofB. xylophilusin response to the immune reaction ofP. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.