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Pinus, a conifer, dominates the world’s forest ecosystems. But it is seriously infected with pine wood nematode (PWN). Transcription factors (TFs) are key regulators in regulating plant resistance. However, the molecular mechanism of TFs remains thus far unresolved in P. thunbergii inoculated with Bursaphelenchus xylophilus. Here, we used RNA-seq technology to identify differentially expressed TFs in resistant and susceptible pines. The results show that a total of 186 differentially expressed transcription factors (DETFs), including 99 upregulated and 87 downregulated genes were identified. Gene ontology (GO) enrichment showed that the highly enriched differentially expressed TFs were responsible for secondary biosynthetic processes. According to KEGG pathway analysis, the differentially expressed TFs were related to chaperones and folding catalysts, phenylpropanoid biosynthesis, and protein processing in the endoplasmic reticulum. Many TFs such as NAC, LBD, MYB, bHLH, and WRKY were determined to be quite abundant in the DETFs. Moreover, the NAC transcription factor PtNAC9 was upregulated in PWN-resistant and susceptible P. thunbergii and especially distinctly upregulated in resistant pines. By purifying recombinant PtNAC9 protein in vitro, we found that overexpression of PtNAC9 at the early stage of B. xylophilus infection could reduce the degree of disease. We also demonstrated the content of salicylic acid (SA) and the related genes were increased in the PtNAC9 protein-treated plants. These results could be helpful in enhancing our understanding of the resistance mechanism underlying different resistant pine.

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT–qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the β-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01–2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application.

The pinewood nematode, Bursaphelenchus xylophilus, has been determined as one of the world’s top ten plant-parasitic nematodes. It causes pine wilt, a progressive disease that affects the economy and ecologically sustainable development in East Asia. B. xylophilus secretes pathogenic proteins into host plant tissues to promote infection. However, little is known about the interaction between B. xylophilus and pines. Previous studies reported transthyretin proteins in some species and their strong correlation with immune evasion, which has also been poorly studied in B. xylophilus. In this study, we cloned and functionally validated the B. xylophilus pathogenic protein BxTTR-52, containing a transthyretin domain. An in situ hybridization assay demonstrated that BxTTR-52 was expressed mainly in the esophageal glands of B. xylophilus. Confocal microscopy revealed that BxTTR-52-RFP localized to the nucleus, cytoplasm, and plasma membrane. BxTTR-52 recombinant proteins produced by Escherichia coli could be suppressed by hydrogen peroxide and antioxidant enzymes in pines. Moreover, silencing BxTTR-52 significantly attenuated the morbidity of Pinus thunbergii infected with B. xylophilus. It also suppressed the expression of pathogenesis-related genes in P. thunbergii. These results suggest that BxTTR-52 suppresses the plant immune response in the host pines and might contribute to the pathogenicity of B. xylophilus in the early infection stages.

Bursaphelenchus xylophilus is the most economically important species of migratory plant-parasitic nematodes (PPNs) and causes severe damage to forestry in China. The successful infection of B. xylophilus relies on the secretion of a repertoire of effector proteins. The effectors, which suppress the host pine immune response, are key to the facilitation of B. xylophilus parasitism. An exhaustive list of candidate effectors of B. xylophilus was predicted, but not all have been identified and characterized. Here, an effector, named BxSCD3, has been implicated in the suppression of host immunity. BxSCD3 could suppress pathogen-associated molecular patterns (PAMPs) PsXEG1- and INF1-triggered cell death when it was secreted into the intracellular space in Nicotiana benthamiana. BxSCD3 was highly up-regulated in the early infection stages of B. xylophilus. BxSCD3 does not affect B. xylophilus reproduction, either at the mycophagous stage or the phytophagous stage, but it contributes to the virulence of B. xylophilus. Moreover, BxSCD3 significantly influenced the relative expression levels of defense-related (PR) genes PtPR-3 and PtPR-6 in Pinus thunbergii in the early infection stage. These results suggest that BxSCD3 is an important toxic factor and plays a key role in the interaction between B. xylophilus and host pine.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, that causes pine wilt disease (PWD), has severely damaged forests and ecosystems in countries of East Asia and Europe. Since the high propagation rate of PWD is the main cause of the devastating disease, it is very important to interfere with the spread of PWN mediated by its insect vector (Monochamus alternatus). In order to explore new methods and approaches for the prevention and control of pine wood nematode disease, chemicals with attraction or repellency to pine wood nematode and stable diffusion or volatilization in infected wood were screened out to interfere and hinder the pupa chamber effect, the aggregation behavior of PWN to pupa chamber in M. alternatus. In the present study, 22 chemicals were inoculated on water agar medium and infected wood to verify the tropism of PWN toward the chemicals from the aspects of attraction and repellency. The results indicate that linoleic acid, oleic acid, diacetyl and β-myrcene have attractive effects on PWN, while kerosene and β-humulene have repellent effects on PWN and are able to stably permeate and volatilize within pine trees. This research shows that some chemicals can affect the movement behavior of PWN and interfere with its migration to the pupal chamber in M. alternatus, thus reducing the dispersal efficiency of PWNs carried by insect vectors. This chemically based strategy would provide novel approaches for effective controlling of pine wilt disease.

The pine wood nematode (PWN) Bursaphelenchus xylophilus is recognized as a major invasive species in many countries and causes widespread mortality in pine trees. Pine wood nematode disease (PWD) has spread northward from southern China to several areas of Liaoning Province, which has temperatures outside of the optimal range for this disease. To determine whether obvious variations in the population adaptability of PWN are involved in its rapid spread from southern to northern China, this study compared the differences in morphology of eight southern strains and eight northern strains and the pathogenicity of the 16 strains to Pinus thunbergii, the pine species that is the most susceptible to PWD in China, and to P. tabuliformis, the main PWN host in northern Liaoning Province. The southern-strain females were smaller than the northern-strain females, except for strain GD32. The size differences between the males of the different strains were not significant. The difference in pathogenicity between the northern and southern strains to P. tabuliformis was more significant than the difference in their pathogenicity to P. thunbergii. The pathogenicity differentiation among northern strains was lower than that among southern strains, and the northern strains showed stronger pathogenicity to P. tabuliformis. The P. tabuliformis inoculation experiment showed that the pathogenicity of GD32, JS27, FJ14, LN13, and LN06 was significantly higher than that of FJ13. The results suggest that some PWN populations in the southern region, which are better adapted to P. tabuliformis, were likely directly transmitted to the northern region, resulting in the spread of PWD in the northern region. The spread of PWN from the south did not necessarily require a process of adaptation to the host or to the northern climate.

Stenotrophomonas maltophilia influences the reproduction, pathogenicity, and gene expression of aseptic Bursaphelenchus xylophilus after inoculation of aseptic Pinus massoniana. Pine wilt disease is a destructive pine forest disease caused by B. xylophilus, and its pathogenesis is unclear. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. S. maltophilia is one of the most dominant bacteria in B. xylophilus, and its effect is ambiguous. This study aims to explore the role of S. maltophilia in pine wilt disease. The reproduction and virulence of aseptic B. xylophilus and B. xylophilus containing S. maltophilia were examined by inoculating aseptic P. massoniana seedlings. The gene expressions of two nematode treatments were identified by transcriptome sequencing. The reproduction and virulence of B. xylophilus containing S. maltophilia were stronger than that of aseptic nematodes. There were 4240 differentially expressed genes between aseptic B. xylophilus and B. xylophilus containing S. maltophilia after inoculation of aseptic P. massoniana, including 1147 upregulated genes and 2763 downregulated genes. These differentially expressed genes were significantly enriched in some immune-related gene ontology (GO) categories, such as membrane, transporter activity, metabolic processes, and many immune-related pathways, such as the wnt, rap1, PI3K-Akt, cAMP, cGMP-PKG, MAPK, ECM-receptor interaction, and calcium signaling pathways. The polyubiquitin-rich gene, leucine-rich repeat serine/threonine-protein kinase gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, acetyl-CoA carboxylase gene, and heat shock protein genes were the key genes associated with immune resistance. Moreover, there were four cell wall hydrolase genes, thirty-six detoxification- and pathogenesis-related protein genes, one effector gene and ten cathepsin L-like cysteine proteinase genes that were differentially expressed. After inoculation of the host pine, S. maltophilia could affect the virulence and reproduction of B. xylophilus by regulating the expression of parasitic, immune, and pathogenicity genes of B. xylophilus.

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. Accurately differentiating B. xylophilus from other nematodes species, especially its related species B. mucronatus, is important for pine wood nematode detection. Thus, we attempted to identify a specific protein in the pine wood nematode using proteomics technology. Here, we compared the proteomes of B. xylophilus and B. mucronatus using Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization -time-of-flight/time-of-flight (MALDI-TOF/TOF-MS) technologies. In total, 15 highly expressed proteins were identified in B. xylophilus compared with B. mucronatus. Subsequently, the specificity of the proteins identified was confirmed by PCR using the genomic DNA of other nematode species. Finally, a gene encoding a specific protein (Bx-Prx) was obtained. This gene was cloned and expressed in E. coli. The in situ hybridisation pattern of Bx-Prx showed that it was expressed strongly in the tail of B. xylophilus. RNAi was used to assess the function of Bx-Prx, the results indicated that the gene was associated with the reproduction and pathogenicity of B. xylophilus. This discovery provides fundamental information for identifying B. xylophilus via a molecular approach. Moreover, the purified recombinant protein has potential as a candidate diagnostic antigen of pine wilt disease, which may lead to a new immunological detection method for the pine wood nematode.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD) and causes huge economic losses in pine forests and shows a remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus. Our previous studies showed that autophagy exists in PWN. Therefore, we tested the effects of autophagy inducer rapamycin on PWN and the results revealed that the feeding rate and reproduction were significantly promoted on fungal mats. The gene expression patterns of BxATG1 and BxATG8 under the different stress were determined by quantitative reverse transcription PCR (qRT-PCR). We tested the effects of RNA interference on BxATG1 and BxATG8 in PWN during different periods of infection in Pinus thunbergii. The results revealed that BxATG1 and BxATG8 may play roles in allowing PWN to adapt to changing environmental conditions and the virulence of PWN was influenced by the silence of autophagy-related genes BxATG1 and BxATG8. These results provided fundamental information on the relationship between autophagy and PWN, and on better understanding of gene function of BxATG1 and BxATG8 in PWN.

The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH). BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi) on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD.