Moss Pathogenesis-Related-10 Protein Enhances Resistance to Pythium irregulare in Physcomitrella patens and Arabidopsis thaliana.
ABSTRACT: Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR)-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR-10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants.
Project description:Plants have developed complex defense mechanisms to cope with microbial pathogens. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are perceived by pattern recognition receptors (PRRs), leading to the activation of defense. While substantial progress has been made in understanding the activation of plant defense by PAMPs and DAMPs recognition in tracheophytes, far less information exists on related processes in early divergent plants like mosses. The aim of this study was to identify genes that were induced in P. patens in response to elicitors of Pectobacterium carotovorum subsp. carotovorum, using a cDNA suppression subtractive hybridization (SSH) method. A total of 239 unigenes were identified, including genes involved in defense responses related to the shikimate, phenylpropanoid, and oxylipin pathways. The expression levels of selected genes related to these pathways were analyzed using quantitative RT-PCR, confirming their rapid induction by P.c. carotovorum derived elicitors. In addition, P. patens induced cell wall reinforcement after elicitor treatment by incorporation of phenolic compounds, callose deposition, and elevated expression of Dirigent-like encoding genes. Small molecule defense markers and phytohormones such as cinnamic acid, 12-oxo-phytodienoic acid, and auxin levels all increased in elicitor-treated moss tissues. In contrast, salicylic acid levels decreased while abscisic acid levels remained unchanged. P. patens reporter lines harboring an auxin-inducible promoter fused to ?-glucuronidase revealed GUS activity in protonemal and gametophores tissues treated with elicitors of P.c. carotovorum, consistent with a localized activation of auxin signaling. These results indicate that P. patens activates the shikimate, phenylpropanoid, oxylipins, and auxin pathways upon treatment with P.c. carotovorum derived elicitors.
Project description:The moss Physcomitrella patens is a suitable model plant to analyze the activation of defense mechanisms after pathogen assault. In this study, we show that Colletotrichum gloeosporioides isolated from symptomatic citrus fruit infects P. patens and cause disease symptoms evidenced by browning and maceration of tissues. After C. gloeosporioides infection, P. patens reinforces the cell wall by the incorporation of phenolic compounds and induces the expression of a Dirigent-protein-like encoding gene that could lead to the formation of lignin-like polymers. C. gloeosporioides-inoculated protonemal cells show cytoplasmic collapse, browning of chloroplasts and modifications of the cell wall. Chloroplasts relocate in cells of infected tissues toward the initially infected C. gloeosporioides cells. P. patens also induces the expression of the defense genes PAL and CHS after fungal colonization. P. patens reporter lines harboring the auxin-inducible promoter from soybean (GmGH3) fused to ?-glucuronidase revealed an auxin response in protonemal tissues, cauloids and leaves of C. gloeosporioides-infected moss tissues, indicating the activation of auxin signaling. Thus, P. patens is an interesting plant to gain insight into defense mechanisms that have evolved in primitive land plants to cope with microbial pathogens.
Project description:The moss Physcomitrella patens is an evolutionarily basal model system suitable for the analysis of plant defence responses activated after pathogen assault. Upon infection with the necrotroph Botrytis cinerea, several defence mechanisms are induced in P. patens, including the fortification of the plant cell wall by the incorporation of phenolic compounds and the induced expression of related genes. Botrytis cinerea infection also activates the accumulation of reactive oxygen species and cell death with hallmarks of programmed cell death in moss tissues. Salicylic acid (SA) levels also increase after fungal infection, and treatment with SA enhances transcript accumulation of the defence gene phenylalanine ammonia-lyase (PAL) in P. patens colonies. The expression levels of the genes involved in 12-oxo-phytodienoic acid (OPDA) synthesis, including lipoxygenase (LOX) and allene oxide synthase (AOS), increase in P. patens gametophytes after pathogen assault, together with a rise in free linolenic acid and OPDA concentrations. However, jasmonic acid (JA) could not be detected in healthy or infected tissues of this plant. Our results suggest that, although conserved defence signals, such as SA and OPDA, are synthesized and are probably involved in the defence response of P. patens against B. cinerea infection, JA production appears to be missing. Interestingly, P. patens responds to OPDA and methyl jasmonate by reducing moss colony growth and rhizoid length, suggesting that jasmonate perception is present in mosses. Thus, P. patens can provide clues with regard to the evolution of different defence pathways in plants, including signalling and perception of OPDA and jasmonates in nonflowering and flowering plants.
Project description:Mosses are ecologically important plants also used for greening, gardening, and decorative purposes. Knowledge of the microbial flora associated with mosses is expected to be important for control and preservation of global and local environments. However, the moss-associated microbial flora is often poorly known. Moss-associated fungi and bacteria may promote plant growth and pest control, but they may be alternative hosts for pathogens of vascular plants. In this study, the fungus Sclerotinia delphinii was identified for the first time as a pathogen that causes severe damage to Sunagoke moss (Racomitrium japonicum). This moss is used for greening roofs and walls of buildings in urban environments owing to its notable tolerance of environmental stresses. Inoculation with the S. delphinii strain SR1 of the mono- and dicotyledonous seed plants Hordeum vulgare, Brassica rapa var. pekinensis, Lactuca sativa, and Spinacia oleracea, in addition to the liverwort Marchantia polymorpha and the moss Physcomitrella patens, showed that the fungus has a wide host range. Colonization with SR1 progressed more rapidly in non-vascular than in vascular plant species. Studies with P. patens under controlled conditions showed that SR1 secreted a fluid during colonization. Treatment with the secretion induced production of reactive oxygen species in the moss. Endogenous peroxidase partially inhibited SR1 colonization of P. patens. A bacterial isolate, most likely Bacillus amyloliquefaciens, that coexists with R. japonicum was antagonistic to SR1 growth. Taken together, the present results suggest that fungal colonization of mosses may be prevented by a peroxidase secreted by the moss and an antagonistic bacterium coexisting in the moss habitat. The findings suggest that there is potential to apply biological control measures for protection of mosses against fungal pathogens.
Project description:Wounding is a serious environmental stress in plants. Oxylipins such as jasmonic acid play an important role in defense against wounding. Mechanisms to adapt to wounding have been investigated in vascular plants; however, those mechanisms in nonvascular plants remain elusive. To examine the response to wounding in Physcomitrella patens, a model moss, a proteomic analysis of wounded P. patens was conducted. Proteomic analysis showed that wounding increased the abundance of proteins related to protein synthesis, amino acid metabolism, protein folding, photosystem, glycolysis, and energy synthesis. 12-Oxo-phytodienoic acid (OPDA) was induced by wounding and inhibited growth. Therefore, OPDA is considered a signaling molecule in this plant. Proteomic analysis of a P. patens mutant in which the PpAOS1 and PpAOS2 genes, which are involved in OPDA biosynthesis, are disrupted showed accumulation of proteins involved in protein synthesis in response to wounding in a similar way to the wild-type plant. In contrast, the fold-changes of the proteins in the wild-type plant were significantly different from those in the aos mutant. This study suggests that PpAOS gene expression enhances photosynthesis and effective energy utilization in response to wounding in P. patens.
Project description:BACKGROUND: The endogenous circadian clock allows the organism to synchronize processes both to daily and seasonal changes. In plants, many metabolic processes such as photosynthesis, as well as photoperiodic responses, are under the control of a circadian clock. Comparative studies with the moss Physcomitrella patens provide the opportunity to study many aspects of land plant evolution. Here we present a comparative overview of clock-associated components and the circadian network in the moss P. patens. RESULTS: The moss P. patens has a set of conserved circadian core components that share genetic relationship and gene expression patterns with clock genes of vascular plants. These genes include Myb-like transcription factors PpCCA1a and PpCCA1b, pseudo-response regulators PpPRR1-4, and regulatory elements PpELF3, PpLUX and possibly PpELF4. However, the moss lacks homologs of AtTOC1, AtGI and the AtZTL-family of genes, which can be found in all vascular plants studied here. These three genes constitute essential components of two of the three integrated feed-back loops in the current model of the Arabidopsis circadian clock mechanism. Consequently, our results suggest instead a single loop circadian clock in the moss. Possibly as a result of this, temperature compensation of core clock gene expression appears to be decreased in P. patens. CONCLUSIONS: This study is the first comparative overview of the circadian clock mechanism in a basal land plant, the moss P. patens. Our results indicate that the moss clock mechanism may represent an ancestral state in contrast to the more complex and partly duplicated structure of subsequent land plants. These findings may provide insights into the understanding of the evolution of circadian network topology.
Project description:Anthracnose caused by the hemibiotroph fungus <i>Colletotrichum gloeosporioides</i> is a devastating plant disease with an extensive impact on plant productivity. The process of colonization and disease progression of <i>C. gloeosporioides</i> has been studied in a number of angiosperm crops. To better understand the evolution of the plant response to pathogens, the study of this complex interaction has been extended to bryophytes. The model moss <i>Physcomitrium patens</i> Hedw. B&S (former <i>Physcomitrella patens</i>) is sensitive to known bacterial and fungal phytopathogens, including <i>C. gloeosporioides</i>, which cause infection and cell death. <i>P. patens</i> responses to these microorganisms resemble that of the angiosperms. However, the molecular events during the interaction of <i>P. patens</i> and <i>C. gloeosporioides</i> have not been explored. In this work, we present a comprehensive approach using microscopy, phenomics and RNA-seq analysis to explore the defense response of <i>P. patens</i> to <i>C. gloeosporioides</i>. Microscopy analysis showed that appressoria are already formed at 24 h after inoculation (hai) and tissue colonization and cell death occur at 24 hai and is massive at 48 hai. Consequently, the phenomics analysis showed progressing browning of moss tissues and impaired photosynthesis from 24 to 48 hai. The transcriptomic analysis revealed that more than 1200 <i>P. patens</i> genes were differentially expressed in response to <i>Colletotrichum</i> infection. The analysis of differentially expressed gene function showed that the <i>C. gloeosporioides</i> infection led to a transcription reprogramming in <i>P. patens</i> that upregulated the genes related to pathogen recognition, secondary metabolism, cell wall reinforcement and regulation of gene expression. In accordance with the observed phenomics results, some photosynthesis and chloroplast-related genes were repressed, indicating that, under attack, <i>P. patens</i> changes its transcription from primary metabolism to defend itself from the pathogen.
Project description:The mosses and flowering plants diverged >400 million years ago. The mosses have haploid-dominant life cycles, whereas the flowering plants are diploid-dominant. The common ancestors of land plants have been inferred to be haploid-dominant, suggesting that genes used in the diploid body of flowering plants were recruited from the genes used in the haploid body of the ancestors during the evolution of land plants. To assess this evolutionary hypothesis, we constructed an EST library of the moss Physcomitrella patens, and compared the moss transcriptome to the genome of Arabidopsis thaliana. We constructed full-length enriched cDNA libraries from auxin-treated, cytokinin-treated, and untreated gametophytes of P. patens, and sequenced both ends of >40,000 clones. These data, together with the mRNA sequences in the public databases, were assembled into 15,883 putative transcripts. Sequence comparisons of A. thaliana and P. patens showed that at least 66% of the A. thaliana genes had homologues in P. patens. Comparison of the P. patens putative transcripts with all known proteins, revealed 9,907 putative transcripts with high levels of similarity to vascular plant genes, and 850 putative transcripts with high levels of similarity to other organisms. The haploid transcriptome of P. patens appears to be quite similar to the A. thaliana genome, supporting the evolutionary hypothesis. Our study also revealed that a number of genes are moss specific and were lost in the flowering plant lineage.
Project description:Glucosinolates are defense-related secondary metabolites found in Brassicaceae. When Brassicaceae come under attack, glucosinolates are hydrolyzed into different forms of glucosinolate hydrolysis products (GHPs). Among the GHPs, isothiocyanates are the most comprehensively characterized defensive compounds, whereas the functional study of nitriles, another group of GHP, is still limited. Therefore, this study investigates whether 3-butenenitrile (3BN), a nitrile, can trigger the signaling pathways involved in the regulation of defense responses in Arabidopsis thaliana against biotic stresses. Briefly, the methodology is divided into three stages, (i) evaluate the physiological and biochemical effects of exogenous 3BN treatment on Arabidopsis, (ii) determine the metabolites involved in 3BN-mediated defense responses in Arabidopsis, and (iii) assess whether a 3BN treatment can enhance the disease tolerance of Arabidopsis against necrotrophic pathogens. As a result, a 2.5 mM 3BN treatment caused lesion formation in Arabidopsis Columbia (Col-0) plants, a process found to be modulated by nitric oxide (NO). Metabolite profiling revealed an increased production of soluble sugars, Krebs cycle associated carboxylic acids and amino acids in Arabidopsis upon a 2.5 mM 3BN treatment, presumably via NO action. Primary metabolites such as sugars and amino acids are known to be crucial components in modulating plant defense responses. Furthermore, exposure to 2.0 mM 3BN treatment began to increase the production of salicylic acid (SA) and jasmonic acid (JA) phytohormones in Arabidopsis Col-0 plants in the absence of lesion formation. The production of SA and JA in nitrate reductase loss-of function mutant (nia1nia2) plants was also induced by the 3BN treatments, with a greater induction for JA. The SA concentration in nia1nia2 plants was lower than in Col-0 plants, confirming the previously reported role of NO in controlling SA production in Arabidopsis. A 2.0 mM 3BN treatment prior to pathogen assays effectively alleviated the leaf lesion symptom of Arabidopsis Col-0 plants caused by Pectobacterium carotovorum ssp. carotovorum and Botrytis cinerea and reduced the pathogen growth on leaves. The findings of this study demonstrate that 3BN can elicit defense response pathways in Arabidopsis, which potentially involves a coordinated crosstalk between NO and phytohormone signaling.
Project description:Triterpenoids produced by plants play important roles in the protection against biotic stress. Roots of Arabidopsis thaliana produce different triterpenoids, which include the tricyclic triterpene diol, arabidiol. In a degradation reaction induced by infection with the oomycete pathogen, Pythium irregulare, arabidiol is cleaved to the 11-carbon volatile homoterpene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), and the 19-carbon ketone, apo-arabidiol. The arabidiol pathway and its volatile breakdown product DMNT have been implicated in the defense against P. irregulare infection. Here we show that the non-volatile breakdown product apo-arabidiol is further converted to the acetylated derivative ?-14-acetyl-apo-arabidiol via a presumed epimerization and subsequent acetylation reaction. ?-14-acetyl-apo-arabidiol and the detected intermediates in the derivatization pathway are partially exuded from the root indicating possible defensive activities of these molecules in the rhizosphere. The conversion steps of apo-arabidiol vary among different Arabidopsis accessions and are present in only rudimentary form in the close relative Arabidopsis lyrata, which supports an intra- and inter-specific modularity in triterpenoid metabolism.