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Gene expressions of Bx-tlp-1, Bx-tlp-2, Bx-cpi and Bx-cpl-1. CK-controls; 12 h-soaked for 12 h; 24 h-soaked for 24 h; 36 h-soaked for 36 h; 48 h-soaked for 48 h. There were three biological replications for each experiment. The error line means standard deviation of the mean.
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: Bursaphelenchus xylophilus is a nematode species that has damaged pine trees worldwide, but its pathogenesis has not been fully characterized. α-pinene helps protect host species during the early B. xylophilus infection and colonization stages. In this study, we identified potential molecular mimicry proteins based on a comparative transcriptomic...
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... contrast, the Bx-cpi expression level exhibited the opposite pattern over the same period. Additionally, the Bx-tlp-2 expression was extremely low (Figure 6). Therefore, the quantitative real-time PCR (qPCR) results were consistent with the transcriptomic data, confirming the accuracy of our experiments. ...
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Purpose Basing on semiquantitative assessment of 99mTc-MIBI uptake in parathyroids of secondary hyperparathyroidism (SHPT) patients from chronic renal failure, objective guidance could be given to improve the qualitative diagnosis accuracy of MIBI uptake.
Methods MIBI uptake intensiveness was semiquantitatively calculated with software ImageJ. MIBI...
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... An analysis of the expression of the potential molecular mimicry proteins in B. xylophilus infecting pine trees indicated that the temporal changes to the α-pinene content in the trees are consistent with the expression levels of the genes encoding a TLP (CPI) in B. xylophilus and P. massoniana. Thus, these genes are likely important for B. xylophilus infections of pine species (Meng et al., 2017(Meng et al., , 2020. ...
... The PCR and LAMP primers designed and used in this study ( Table 1) targeted B. xylophilus genes (Bx-tlp-1, Bx-tlp-2, and Bx-cpi) encoding previously identified potential molecular mimicry proteins (Meng et al., 2020). The PCR primers were designed using Primer 6.0. ...
... oxysporum Schltdl, B. cinerea, and Pestalotia diospyri (Kikuchi et al., 2009;Ye and Giblin-Davis, 2013;Meng et al., 2020). At least three replicates were analyzed for each assay. ...
The introduction of the pine wood nematode (Bursaphelenchus xylophilus) to new areas has affected the international forestry industry because this pathogen causes pine wilt disease (PWD). Therefore, methods for the accurate and reliable detection of B. xylophilus are essential for controlling and managing this pest. The PCR and Loop-Mediated Isothermal Amplification (LAMP) techniques developed in this study involve species-specific primer sets targeting B. xylophilus genes encoding potential molecular mimicry proteins (Bx-tlp-1, Bx-tlp-2, and Bx-cpi), which are associated with pathogenicity. The PCR and LAMP results revealed that the primers were specific for B. xylophilus Bx-tlp-1, Bx-tlp-2, and Bx-cpi. Moreover, our LAMP assay targeting Bx-tlp-1 conducted at 63°C detected B. xylophilus within 20 min and B. xylophilus from Monochamus alternatus or M. saltuarius within 30 min. The lower limits of detection for the LAMP and PCR assays were 10 pg and 10 ng genomic DNA, respectively, implying these assays may be useful for the rapid detection of B. xylophilus in pine forests. Designing primers specific for Bx-tlp-1, Bx-tlp-2, and Bx-cpi enabled the relatively rapid detection of B. xylophilus isolates as well as M. alternatus or M. saltuarius carrying B. xylophilus. These primers, which were designed following a thorough functional analysis of key B. xylophilus pathogenicity-related genes, may be useful for developing improved assays for the early diagnosis and prevention of PWD.
... Additionally, Meng et al. (2017) cloned the gene encoding Pm-tlp and revealed that its expression is associated with the expression of Bx-tlp-1 from B. xylophilus. The sequence homology between Bx-TLP-1 and Pm-TLP has been reported as having a higher sequence similarity between the Bx-TLP-1 protein sequence and the plant protein sequences, which indicated that they may have similar functions (Meng et al. 2017(Meng et al. , 2020. At the same time, studies demonstrated that the expression of Bx-tlp-1 is responsive to a-pinene (Meng et al. 2020). ...
... The sequence homology between Bx-TLP-1 and Pm-TLP has been reported as having a higher sequence similarity between the Bx-TLP-1 protein sequence and the plant protein sequences, which indicated that they may have similar functions (Meng et al. 2017(Meng et al. , 2020. At the same time, studies demonstrated that the expression of Bx-tlp-1 is responsive to a-pinene (Meng et al. 2020). Moreover, the treatments with dsRNA targeting Bx-tlp-1 decreased expression by 90%, with the silencing effect maintained even in the F3 offspring; the Bx-tlp-1 gene was associated with B. xylophilus pathogenicity (Meng et al. 2019). ...
... The flow rate was 1.5 ml/min, with an injection temperature of 250 C and a sample size of 0.5 ml. The samples were quantified on the basis of a standard curve (Meng et al. 2020). Gene expression was analyzed by qRT-PCR assay using the LightCycler 480 II system (Roche Diagnostics Ltd., Risch-Rotkreuz, Switzerland) with TB Green Premix Ex Taq (TaKaRa Bio, Shiga, Japan), which was used as described in Meng et al. (2019). ...
Pine wilt disease is a major forest disease worldwide, including in China, where it has severely damaged pine forest ecosystems, and the pathogen is pine wood nematode (Bursaphelenchus xylophilus). The thaumatin-like protein-1 gene (Bx-tlp-1) is a key gene associated with B. xylophilus pathogenicity, which is also responsive to α-pinene. In this study, an examination of Pinus massoniana seedlings infected by B. xylophilus revealed that monoterpene (sesquiterpene) levels peaked on days 15 and 27 (days 18 and 27). Meanwhile, P. massoniana Pm-tlp expression levels were high on days 3, 12, and 27, which were consistent with the expression of key enzymes genes in the terpene biosynthesis pathway. The functional similarity of B. xylophilus Bx-TLP-1 and P. massoniana Pm-TLP suggests Bx-TLP-1 and Pm-TLP may have similar roles in P. massoniana. There was also no secondary accumulation of terpenes in P. massoniana seedlings during B. xylophilus treated with dsRNA targeting Bx-tlp-1 (dsTLP1) infections, reflecting the decreased pathogenicity of B. xylophilus and the delayed disease progression in pine trees. And the results of micro-CT showed that the degree of cavitation for the trees inoculated with Bx-TLP-1 (0.3811 mm3) was greater than that for the trees inoculated with dsTLP1 PWNs (0.1204 mm3) on day 15 after inoculation. Results from this study indicated that B. xylophilus Bx-tlp-1 gene may induce the upregulated expression of related genes encoding enzymes in the terpene synthesis pathway of P. massoniana, resulting in the accumulation of terpenes, which also provided an insight to investigate the B. xylophilus pathogenicity in the future.
... [39], though no highly similar sequence (e-value < 1e -10 ) was found in all organisms. Meng et al. (2020) [40] reported that Bx-TH2 is similar to the Hall's panicgrass Panicum hallii thaumatin-like protein (XP_025808117.1), and it shares 52.05% identity with this protein, with over 85% query coverage. In plants, thaumatin is induced by a variety of phytopathogens via salicylic acid [33,34]. ...
... [39], though no highly similar sequence (e-value < 1e -10 ) was found in all organisms. Meng et al. (2020) [40] reported that Bx-TH2 is similar to the Hall's panicgrass Panicum hallii thaumatin-like protein (XP_025808117.1), and it shares 52.05% identity with this protein, with over 85% query coverage. In plants, thaumatin is induced by a variety of phytopathogens via salicylic acid [33,34]. ...
... We previously reported that Bx-CPI is most similar to a cystatin-like peptidase inhibitor in the herbaceous plant Medicago truncatula [18]. However, Meng et al. (2020) [40] reported that Bx-CPI is more similar to the CP inhibitor five-like protein of the common sunflower Helianthus annuus (XP_022033148.1), which shares 32.5% identity with this protein, with over 62% query coverage based on a BLAST analysis of the current database. Although the phylogenetic analysis showed that Bx-CPI clustered in the same clade with an unnamed Enterobius vermicularis (Oxyuridae) protein, no other nematode proteins were clustered to the same group [40]. ...
Pine wilt disease (PWD) is an infectious disease of pines that typically kills affected trees. The causal pathogen of PWD is the pine wood nematode (PWN), Bursaphelenchus xylophilus. Understanding of the disease has advanced in recent years through the use of a highly sensitive proteomics procedure and whole genome sequence analysis; in combination, these approaches have enabled identification of proteins secreted by PWNs. However, the roles of these proteins during the onset of parasitism have not yet been elucidated. In this study, we used a leaf-disk assay based on transient overexpression in Nicotiana benthamiana to allow functional screening of 10 candidate pathogenic proteins secreted by PWNs. These proteins were selected based on previous secretome and RNA-seq analyses. We found that five molecules induced significant cell death in tobacco plants relative to a GFP-only control. Three of these proteins (Bx-TH1, Bx-TH2, and Bx-CPI) may have a role in molecular mimicry and likely make important contributions to inducing hypersensitive responses in host plants.
One of the most damaging pathogens of pinewood is the pinewood nematode (PWN), Bursaphelenchus xylophilus , which could cause an adverse effect on the ecosystems of forests and the commerce of timber. Therefore, it is crucial to realize rapid and accurate B. xylophilus detection. In this work, an accelerated strand exchange amplification method (ASEA) was established to detect B. xylophilus for the first time. By integrating with fast nucleic acid extraction, the whole detection procedure could be finished within 30 min, dramatically shortened the detection time. The ASEA method exhibited high specificity toward B. xylophilus and the detection limit for B. xylophilus plasmid DNA was as low as 1.0 × 10 ⁰ copies/μL. Furthermore, the ASEA approach also exhibited accurate detection for B. xylophilus when applied to actual pinewood samples, meeting the demand of B. xylophilus detection in realistic scenario. We believe the ASEA method has significant potential for B. xylophilus detection, and it will be helpful for controlling forest pest and quarantine regulations.
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Pine wood nematode, Bursaphelenchus xylophilus, is a worldwide pest of pine trees, spreading at an alarming rate and with great ecological adaptability. In the process of causing disease, the nematode causes metabolic disorders and changes in the endophytic microbial community of the pine tree. However, the changes at the pine nidus during early nematode invasion have not been well studied, especially the differential metabolites, in Pinus massoniana, the main host of B. xylophilus in China. In this study, we analyzed the endophytic bacterial and fungal communities associated with healthy and B. xylophilus-caused wilted pine trees. The results show that 1333 bacterial OTUs and 502 fungal OTUs were annotated from P. massoniana stem samples. The abundance of bacterial communities in pine trees varies more following infection by B. xylophilus, but the abundance changes of fungal communities are less visible. There were significant differences in endophytic microbial diversity between wilted and healthy P. massoniana. In wilted pine trees, Actinobacteria and Bacteroidia were differential indicators of bacterial communities, whereas, in healthy pine trees, Rhizobiales in the Proteobacteria phylum were the major markers of bacterial communities. Meanwhile, the differential markers of fungal communities in healthy pines are Malasseziales, Tremellales, Sordariales, and Fusarium, whereas Pleosporaceae is the key marker of fungal communities in wilted pines. Our study examines the effect of changes in the endophytic microbial community on the health of pine trees that may be caused by B. xylophilus infection. In parallel, a non-targeted metabolomic study based on liquid mass spectrometry (LC-MS) technology was conducted on pine trees inoculated with pine nematodes and healthy pine trees with a view to identifying key compounds affecting early pine lesions. Ultimately, 307 distinctly different metabolites were identified. Among them, the riboflavin metabolic pathway in pine trees may play a key role in the early pathogenesis of pine wood nematode disease.
Pinewood releases ethanol and other volatile compounds after Bursaphelenchus xylophilus infection. In the current study, we examined the influence of different ethanol concentrations on B. xylophilus reproduction. Low-concentrations of ethanol (8.5, 17, and 34 mM) increased egg production in B. xylophilus, whereas higher-concentrations (156 and 312 mM) reduced egg production. Transcriptome analysis was conducted to explore the molecular response of a low concentration of ethanol on the nematodes. The results suggest that the nematodes use ethanol as an energy source, which may promote survival. Ethanol induced changes in the expression of genes involved in the biosynthesis and metabolism of fatty acids and amino acids. Furthermore, ethanol promoted the expression of detoxification-related, cell wall-degrading, and reproduction-related genes. Such responses might contribute to the pathogenicity of B. xylophilus.
