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Characterization of the Pinus massoniana transcriptional response to Bursaphelenchus xylophilus infection using suppression subtractive hybridization.

ABSTRACT: Pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, is the most destructive diseases of pine and poses a threat of serious economic losses worldwide. Although several of the mechanisms involved in disease progression have been discovered, the molecular response of Pinus massoniana to PWN infection has not been explored. We constructed four subtractive suppression hybridization cDNA libraries by taking time-course samples from PWN-inoculated Masson pine trees. One-hundred forty-four significantly differentially expressed sequence tags (ESTs) were identified, and 124 high-quality sequences with transcriptional features were selected for gene ontology (GO) and individual gene analyses. There were marked differences in the types of transcripts, as well as in the timing and levels of transcript expression in the pine trees following PWN inoculation. Genes involved in signal transduction, transcription and translation and secondary metabolism were highly expressed after 24 h and 72 h, while stress response genes were highly expressed only after 72 h. Certain transcripts responding to PWN infection were discriminative; pathogenesis and cell wall-related genes were more abundant, while detoxification or redox process-related genes were less abundant. This study provides new insights into the molecular mechanisms that control the biochemical and physiological responses of pine trees to PWN infection, particularly during the initial stage of infection.

SUBMITTER: Xu L

PROVIDER: S-EPMC3709736 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Characterization of the Pinus massoniana transcriptional response to Bursaphelenchus xylophilus infection using suppression subtractive hybridization.

Xu Liang L Liu Zhen-Yu ZY Zhang Kai K Lu Quan Q Liang Jun J Zhang Xing-Yao XY

International journal of molecular sciences 20130528 6

Pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, is the most destructive diseases of pine and poses a threat of serious economic losses worldwide. Although several of the mechanisms involved in disease progression have been discovered, the molecular response of Pinus massoniana to PWN infection has not been explored. We constructed four subtractive suppression hybridization cDNA libraries by taking time-course samples from PWN-inoculated Masson pine trees. ...[more]

PMID: 23759987

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Project description:BACKGROUND: Pine wilt disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which threatens pine forests and forest ecosystems worldwide and causes serious economic losses. In the 40 years since the pathogen was identified, the physiological changes occurring as the disease progresses have been characterized using anatomical and biochemical methods, and resistant trees have been selected via breeding programs. However, no studies have assessed the molecular genetics, e.g. transcriptional changes, associated with infection-induced physiological changes in resistant or susceptible trees. RESULTS: We constructed seven subtractive suppression hybridization (SSH) cDNA libraries using time-course sampling of trees inoculated with pine wood nematode at 1, 3, or 7 days post-inoculation (dpi) in susceptible trees and at 1, 3, 7, or 14 dpi in resistant trees. A total of 3,299 sequences was obtained from these cDNA libraries, including from 138 to 315 non-redundant sequences in susceptible SSH libraries and from 351 to 435 in resistant SSH libraries. Using Gene Ontology hierarchy, those non-redundant sequences were classified into 15 subcategories of the biological process Gene Ontology category and 17 subcategories of the molecular function category. The transcriptional components revealed by the Gene Ontology classification clearly differed between resistant and susceptible libraries. Some transcripts were discriminative: expression of antimicrobial peptide and putative pathogenesis-related genes (e.g., PR-1b, 2, 3, 4, 5, 6) was much higher in susceptible trees than in resistant trees at every time point, whereas expression of PR-9, PR-10, and cell wall-related genes (e.g., for hydroxyproline-rich glycoprotein precursor and extensin) was higher in resistant trees than in susceptible trees at 7 and 14 dpi. CONCLUSIONS: Following inoculation with pine wood nematode, there were marked differences between resistant and susceptible trees in transcript diversity and the timing and level of transcripts expressed in common; in particular, expression of stress response and defense genes differed. This study provided new insight into the differences in the physiological changes between resistant and susceptible trees that have been observed in anatomical and biochemical studies.

Project description:BackgroundPine trees challenged by Bursaphelenchus xylophilus invasion produce phytoalexins to combat this nematode. Nevertheless, the phytoalexins of Asian pine trees are ineffective against B. xylophilus. The anti-phytoalexin genes of B. xylophilus disable almost all Asian pine phytoalexins, which has allowed B. xylophilus to 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.ResultsHere, we described anti-phytoalexin genes in B. xylophilus using transcriptomic and bioinformatics analyses. The genes that were induced by both Pinus massoniana and 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 gene Bx-cathepsin W was cloned and characterized. The results of the RNAi assessment indicated that the knockdown of Bx-cathepsin W reduced the survival rates of B. xylophilus under carvone or P. massoniana stress. The correlation between Bx-cathepsin W and the susceptibility of pines showed that Bx-cathepsin W might help improve the anti-phytotoxin ability of B. xylophilus.ConclusionsThe results indicated that the anti-phytoalexin gene Bx-cathepsin W supported the survival of B. xylophilus under P. massoniana phytoalexin stress. The cDNA library sequencing, differentially expressed gene identification, and WGCNA algorithm analysis provided insight at a systemic level into the gene regulation of B. xylophilus in response to the immune reaction of P. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.

Project description:IntroductionPine wilt disease (PWD) is responsible for extensive economic and ecological damage to Pinus spp. forests and plantations worldwide. PWD is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and transmitted into pine trees by a vector insect, the Japanese pine sawyer (JPS, Monochamus alternatus). Host infection by PWN will attract JPS to spawn, which leads to the co-existence of PWN and JPS within the host tree, an essential precondition for PWD outbreaks. Through the action of their metabolites, microbes can manipulate the co-existence of PWN and JPS, but our understanding on how key microorganisms engage in this process remains limited, which severely hinders the exploration and utilization of promising microbial resources in the prevention and control of PWD.MethodsIn this study we investigated how the PWN-associated fungus Aspergillus promotes the co-existence of PWN and JPS in the host trees (Pinus massoniana) via its secondary metabolite, sterigmatocystin (ST), by taking a multi-omics approach (phenomics, transcriptomics, microbiome, and metabolomics).ResultsWe found that Aspergillus was able to promote PWN invasion and pathogenicity by increasing ST biosynthesis in the host plant, mainly by suppressing the accumulation of ROS (reactive oxygen species) in plant tissues that could counter PWN. Further, ST accumulation triggered the biosynthesis of VOC (volatile organic compounds) that attracts JPS and drives the coexistence of PWN and JPS in the host plant, thereby encouraging the local transmission of PWD. Meanwhile, we show that application of an Aspergillus inhibitor (chiricanine A treatment) results in the absence of Aspergillus and decreases the in vivo ST amount, thereby sharply restricting the PWN development in host. This further proved that Aspergillus is vital and sufficient for promoting PWD transmission.DiscussionAltogether, these results document, for the first time, how the function of Aspergillus and its metabolite ST is involved in the entire PWD transmission chain, in addition to providing a novel and long-term effective nematicide for better PWD control in the field.

Dataset Information

Characterization of the Pinus massoniana transcriptional response to Bursaphelenchus xylophilus infection using suppression subtractive hybridization.

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Characterization of the Pinus massoniana transcriptional response to Bursaphelenchus xylophilus infection using suppression subtractive hybridization.

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