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Jinichiro Koga - One of the best experts on this subject based on the ideXlab platform.
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identification of uv induced diterpenes including a new diterpene Phytoalexin phytocassane f from rice leaves by complementary gc ms and lc ms approaches
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Kiyotaka Horie, Jinichiro Koga, Yasuno Inoue, Miki Sakai, Qun Yao, Yosuke Tanimoto, Hiroaki Toshima, Morifumi HasegawaAbstract:Rice Phytoalexins are regarded as one of the most important weapons against pathogenic microorganisms. We attempted to identify novel Phytoalexins and their derivatives using GC/MS and LC/MS analyses. Diterpene derivatives, 9β-pimara-7,15-diene-3β,6β,19-triol, 1, stemar-13-en-2α-ol, 2, and 1α,2α-dihydroxy-ent-12,15-cassadiene-3,11-dione, 3, were isolated from UV-irradiated rice leaves by chromatographic methods. These structures were confirmed by 1D- and 2D-NMR and MS analyses. Interestingly, all three compounds were accumulated following an infection by the rice blast pathogen Magnaporthe oryzae. Compounds 1 and 2 exhibited weak antifungal activity and may be the biosynthetic intermediates of rice Phytoalexins momilactones and oryzalexin S, respectively. Compound 3 exhibited relatively high inhibitory activity against the fungal mycelial growth of M. oryzae to the same extent as the known Phytoalexin phytocassane A. We conclude that 3 is a member of the cassane-type Phytoalexin family and propose the name phytocassane F.
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regulation of a proteinaceous elicitor induced ca2 influx and production of Phytoalexins by a putative voltage gated cation channel ostpc1 in cultured rice cells
Journal of Biological Chemistry, 2012Co-Authors: Haruyasu Hamada, Jinichiro Koga, Kazunori Okada, Takamitsu Kurusu, Eiji Okuma, Hiroshi Nokajima, Masahiro Kiyoduka, Tomoko Koyano, Yoshimi Sugiyama, Hikaru SajiAbstract:Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca2+ followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called Phytoalexins; however, the molecular links and regulatory mechanisms of the Phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca2+, mainly due to a Ca2+ influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca2+-sensitive photoprotein, aequorin, TvX-induced Ca2+ elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid Phytoalexins and the expression of a series of diterpene cyclase genes involved in Phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca2+-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca2+ influx as a plasma membrane Ca2+-permeable channel consequently required for the regulation of Phytoalexin biosynthesis in cultured rice cells.
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Effects of cytokinin on production of diterpenoid Phytoalexins in rice
Journal of Pesticide Science, 2010Co-Authors: Kazunori Okada, Jinichiro Koga, Hideaki Nojiri, Yusuke Jikumaru, Hisakazu YamaneAbstract:The production of Phytoalexins is a well-documented defense response against pathogen infection in rice. Although jasmonic acid has been suggested to be involved in the production of Phytoalexins in both suspension-cultured rice cells and rice leaves, there has been almost no information on other secondary signaling molecules that regulate the production of Phytoalexins in rice. In this study, the production of the major diterpenoid Phytoalexins, momilactones and phytocassanes, was found to be induced by cytokinin treatment in both suspension-cultured rice cells and rice leaves, and also the upregulation of Phytoalexin biosynthetic genes was found to be induced by cytokinin treatment. The roles of cytokinins in the production of diterpenoid Phytoalexins in rice infected with a pathogen are discussed.
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Differential responses of rice to inoculation with wild-type and non-pathogenic mutants of Magnaporthe oryzae
Plant Molecular Biology, 2009Co-Authors: Tomoaki Kato, Jinichiro Koga, Kazunori Okada, Yoko Nishizawa, Yusuke Jikumaru, Shigeru Tanabe, Marie Nishimura, Yuko Ohtake, Takafumi Shimizu, Hisakazu YamaneAbstract:We analyzed the response of rice to Magnaporthe oryzae infection using two mutant strains deficient in Mgb1 and Mst12, which are essential for the development of appresoria and penetration pegs. Both mutants induced the much lower levels of accumulation of Phytoalexins than wild-type, suggesting that the massive production of Phytoalexins requires the fungal invasion of rice cells. Intense accumulation of H_2O_2 in a single whole cell also required fungal penetration. Microarray analysis of rice gene expression revealed mutant-specific gene expression, indicating that signal exchange between rice and M. oryzae commence before fungal penetration of the rice cell. In situ detection of mRNAs for peroxidase and β -1,3-glucanase showed that expression of these genes also occurs after penetration as observed for Phytoalexin production.
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elicitor induced activation of the methylerythritol phosphate pathway toward Phytoalexins biosynthesis in rice
Plant Molecular Biology, 2007Co-Authors: Atsushi Okada, Jinichiro Koga, Kazunori Okada, Hideaki Nojiri, Takafumi Shimizu, Tomohisa Kuzuyama, Naoto Shibuya, Hisakazu YamaneAbstract:Diterpenoid Phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to Phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of Phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in Phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of Phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type Phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of Phytoalexins in infected rice plants.
Hisakazu Yamane - One of the best experts on this subject based on the ideXlab platform.
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overexpression of the bzip transcription factor osbzip79 suppresses the production of diterpenoid Phytoalexin in rice cells
Journal of Plant Physiology, 2015Co-Authors: Koji Miyamoto, Yoko Nishizawa, Eiichi Minami, Hideaki Nojiri, Hisakazu Yamane, Kazunori OkadaAbstract:Phytoalexins are antimicrobial specialised metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are major diterpenoid Phytoalexins in rice that are synthesised from geranylgeranyl diphosphate that is derived from the methylerythritol phosphate (MEP) pathway. We have previously reported that rice cells overexpressing the basic leucine zipper (bZIP) transcription factor OsTGAP1 exhibit a hyperaccumulation of momilactones and phytocassanes, with hyperinductive expression of momilactone and phytocassane biosynthetic genes and MEP pathway genes, upon response to a chitin oligosaccharide elicitor. For a better understanding of OsTGAP1-mediated regulation of diterpenoid Phytoalexin production, we identified OsTGAP1-interacting proteins using yeast two-hybrid screening. Among the OsTGAP1-interacting protein candidates, a TGA factor OsbZIP79 was investigated to verify its physical interaction with OsTGAP1 and involvement in the regulation of Phytoalexin production. An in vitro pull-down assay demonstrated that OsTGAP1 and OsbZIP79 exhibited a heterodimeric as well as a homodimeric interaction. A bimolecular fluorescence complementation analysis also showed the interaction between OsTGAP1 and OsbZIP79 in vivo. Intriguingly, whereas OsbZIP79 transactivation activity was observed in a transient reporter assay, the overexpression of OsbZIP79 resulted in suppression of the elicitor-inducible expression of diterpenoid Phytoalexin biosynthetic genes, and thus caused a decrease in the accumulation of Phytoalexin in rice cells. These results suggest that OsbZIP79 functions as a negative regulator of Phytoalexin production triggered by a chitin oligosaccharide elicitor in rice cells, although it remains open under which conditions OsbZIP79 can work with OsTGAP1.
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Biosynthesis of Phytoalexins and regulatory mechanisms of it in rice.
Bioscience biotechnology and biochemistry, 2013Co-Authors: Hisakazu YamaneAbstract:We performed extensive functional characterization of diterpenoid Phytoalexin biosynthetic genes in rice, and found that the genes for the biosynthesis of the major diterpenoid Phytoalexins, phytocassanes and momilactones, are clustered on chromosomes 2 and 4, and that their expression is coordinately induced in rice cells after elicitation. Isopentenyl diphosphate, an early precursor of diterpenoid Phytoalexins, was found to be synthesized through the plastidic methylerythritol phosphate pathway. We also found that chitin elicitor receptor kinase OsCERK1 and a mitogen-activated protein kinase cascade, the OsMKK4-OsMPK6 cascade, play essential roles in the elicitor-induced production of diterpenoid Phytoalexins. In addition, a basic leucine zipper transcription factor, OsTGAP1, was identified as a key regulator of the coordinated expression of the clustered genes and the methylerythritol phosphate pathway genes. Naringenin 7-O-methyltransferase (OsNOMT) was also identified as a key enzyme in the biosynthe...
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Effects of cytokinin on production of diterpenoid Phytoalexins in rice
Journal of Pesticide Science, 2010Co-Authors: Kazunori Okada, Jinichiro Koga, Hideaki Nojiri, Yusuke Jikumaru, Hisakazu YamaneAbstract:The production of Phytoalexins is a well-documented defense response against pathogen infection in rice. Although jasmonic acid has been suggested to be involved in the production of Phytoalexins in both suspension-cultured rice cells and rice leaves, there has been almost no information on other secondary signaling molecules that regulate the production of Phytoalexins in rice. In this study, the production of the major diterpenoid Phytoalexins, momilactones and phytocassanes, was found to be induced by cytokinin treatment in both suspension-cultured rice cells and rice leaves, and also the upregulation of Phytoalexin biosynthetic genes was found to be induced by cytokinin treatment. The roles of cytokinins in the production of diterpenoid Phytoalexins in rice infected with a pathogen are discussed.
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Differential responses of rice to inoculation with wild-type and non-pathogenic mutants of Magnaporthe oryzae
Plant Molecular Biology, 2009Co-Authors: Tomoaki Kato, Jinichiro Koga, Kazunori Okada, Yoko Nishizawa, Yusuke Jikumaru, Shigeru Tanabe, Marie Nishimura, Yuko Ohtake, Takafumi Shimizu, Hisakazu YamaneAbstract:We analyzed the response of rice to Magnaporthe oryzae infection using two mutant strains deficient in Mgb1 and Mst12, which are essential for the development of appresoria and penetration pegs. Both mutants induced the much lower levels of accumulation of Phytoalexins than wild-type, suggesting that the massive production of Phytoalexins requires the fungal invasion of rice cells. Intense accumulation of H_2O_2 in a single whole cell also required fungal penetration. Microarray analysis of rice gene expression revealed mutant-specific gene expression, indicating that signal exchange between rice and M. oryzae commence before fungal penetration of the rice cell. In situ detection of mRNAs for peroxidase and β -1,3-glucanase showed that expression of these genes also occurs after penetration as observed for Phytoalexin production.
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elicitor induced activation of the methylerythritol phosphate pathway toward Phytoalexins biosynthesis in rice
Plant Molecular Biology, 2007Co-Authors: Atsushi Okada, Jinichiro Koga, Kazunori Okada, Hideaki Nojiri, Takafumi Shimizu, Tomohisa Kuzuyama, Naoto Shibuya, Hisakazu YamaneAbstract:Diterpenoid Phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to Phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of Phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in Phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of Phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type Phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of Phytoalexins in infected rice plants.
Soledade M C Pedras - One of the best experts on this subject based on the ideXlab platform.
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biotransformation of rutabaga Phytoalexins by the fungus alternaria brassicicola unveiling the first hybrid metabolite derived from a Phytoalexin and a fungal polyketide
Bioorganic & Medicinal Chemistry, 2017Co-Authors: Soledade M C Pedras, Abbas AbdoliAbstract:The biotransformations of the rutabaga Phytoalexins rutalexin, brassicanate A, isalexin and rapalexin A by the plant pathogenic fungus Alternaria brassicicola are reported. While the biotransformations of rutalexin, brassicanate A, and isalexin are fast, rapalexin A is resistant to fungal transformation. Unexpectedly, biotransformation of rutalexin yields a hybrid metabolite named rutapyrone, derived from rutalexin metabolism and phomapyrone G, a fungal metabolite produced by A. brassicicola. These fungal transformations are detoxification reactions likely carried out by different enzymes. The discovery of rapalexin A resistance to detoxification suggests that this Phytoalexin in combination with additional Phytoalexins could protect crucifers against this pathogen. Phytoalexins resistant to degradation by A. brassicicola are expected to provide the producing plants with higher disease resistance levels.
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the phytopathogenic fungus alternaria brassicicola phytotoxin production and Phytoalexin elicitation
Phytochemistry, 2009Co-Authors: Soledade M C Pedras, Paulos B Chumala, Wei Jin, Mohammed S Islam, Dominic W HauckAbstract:Abstract The metabolites and phytotoxins produced by the phytopathogenic fungus Alternaria brassicicola (Schwein.) Wiltshire, as well as the Phytoalexins induced in host plants, were investigated. Brassicicolin A emerged as the most selective phytotoxic metabolite produced in liquid cultures of A. brassicicola and spirobrassinin as the major Phytoalexin produced in infected leaves of Brassica juncea (whole plants). In detached infected leaves of B. juncea , the main component was N′ -acetyl-3-indolylmethanamine, the product of detoxification of the Phytoalexin brassinin by A. brassicicola . In addition, the structure elucidation of three hitherto unknown metabolites having a fusicoccane skeleton was carried out and the antifungal activity of several plant defenses against A. brassicicola was determined.
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cloning purification and characterisation of brassinin glucosyltransferase a Phytoalexin detoxifying enzyme from the plant pathogen sclerotinia sclerotiorum
Fungal Genetics and Biology, 2009Co-Authors: Adrienne Sexton, Soledade M C Pedras, Zoran Minic, Anton J Cozijnsen, Barbara J HowlettAbstract:Abstract The plant-pathogenic fungus Sclerotinia sclerotiorum can detoxify cruciferous Phytoalexins such as brassinin via glucosylation. Here we describe a multifaceted approach including genome mining, transcriptional induction, Phytoalexin quantification, protein expression and enzyme purification that led to identification of a S. sclerotiorum glucosyltransferase that detoxifies brassinin. Transcription of this gene, denoted as brassinin glucosyltransferase 1 (SsBGT1), was induced significantly in response to the cruciferous Phytoalexins camalexin, cyclobrassinin, brassilexin, brassinin and 3-phenylindole, a camalexin analogue. This gene was also up-regulated during infection of Brassica napus leaves. Levels of brassinin decreased significantly between 48 and 72 h post-inoculation, with a concomitant increase in levels of 1-β- d -glucopyranosylbrassinin, the product of the reaction catalysed by SsBGT1. These findings strongly implicate the involvement of this gene during infection of B. napus. This gene was cloned and expressed in Saccharomyces cerevisiae. The purified recombinant enzyme was able to glucosylate brassinin and two other Phytoalexins, albeit much less effectively. This is the first report of a fungal gene involved in detoxification of plant defence molecules via glucosylation.
Philippe Jeandet - One of the best experts on this subject based on the ideXlab platform.
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Structure, Chemical Analysis, Biosynthesis, Metabolism, Molecular Engineering and Biological Functions of Phytoalexins
MDPI - Multidisciplinary Digital Publishing Institute, 2018Co-Authors: Philippe JeandetAbstract:Ever since the concept of Phytoalexins was proposed by Müller and Borger in 1940, these compounds have attracted considerable attention due to the central role they play in the defense mechanisms of various plants. Besides displaying antifungal activity in numerous plant–pathogen interactions, Phytoalexins have been implicated in human health and disease as antioxidant, anticancer and cardioprotective agents as well as being supposed to act positively in neurodegenerative illnesses. More than 25 years after the work of Siemann and Creasy which established a relationship between the concentration of the Phytoalexin resveratrol in wine and the beneficial effects of wine consumption on health, the relevant literature on Phytoalexins and their role in health and disease has increased tremendously. Knowledge on Phytoalexins relies on fields as diverse as organic synthesis, analytical chemistry, plant molecular pathology, biocontrol, biochemistry and various aspects of biomedicine and biotechnology. It is almost impossible to review all of these aspects and, therefore, an attempt is made in the present book to illustrate some of them with a particular emphasis on the induction mechanisms of Phytoalexin biosynthesis, methods for their analysis in complex matrices, fungal metabolism and Phytoalexin bioactivity. This book will serve as a resource for teachers, researchers and students concerned with the study of Phytoalexins
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Modulation of Phytoalexin Biosynthesis in Engineered Plants for Disease Resistance
International journal of molecular sciences, 2013Co-Authors: Philippe Jeandet, Christophe Clément, Eric Courot, Sylvain CordelierAbstract:Phytoalexins are antimicrobial substances of low molecular weight produced by plants in response to infection or stress, which form part of their active defense mechanisms. Starting in the 1950’s, research on Phytoalexins has begun with biochemistry and bio-organic chemistry, resulting in the determination of their structure, their biological activity as well as mechanisms of their synthesis and their catabolism by microorganisms. Elucidation of the biosynthesis of numerous Phytoalexins has permitted the use of molecular biology tools for the exploration of the genes encoding enzymes of their synthesis pathways and their regulators. Genetic manipulation of Phytoalexins has been investigated to increase the disease resistance of plants. The first example of a disease resistance resulting from foreign Phytoalexin expression in a novel plant has concerned a Phytoalexin from grapevine which was transferred to tobacco. Transformations were then operated to investigate the potential of other Phytoalexin biosynthetic genes to confer resistance to pathogens. Unexpectedly, engineering Phytoalexins for disease resistance in plants seem to have been limited to exploiting only a few Phytoalexin biosynthetic genes, especially those encoding stilbenes and some isoflavonoids. Research has rather focused on indirect approaches which allow modulation of the accumulation of Phytoalexin employing transcriptional regulators or components of upstream regulatory pathways. Genetic approaches using gain- or less-of functions in Phytoalexin engineering together with modulation of Phytoalexin accumulation through molecular engineering of plant hormones and defense-related marker and elicitor genes have been reviewed.
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chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew
Phytopathology, 2006Co-Authors: Aziz Aziz, Philippe Jeandet, Patricia Trotelaziz, Michel Couderchet, Laurent Dhuicq, Guy VernetAbstract:Aziz, A., Trotel-Aziz, P., Dhuicq, L., Jeandet, P., Couderchet, M., and Vernet, G. 2006. Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew. Phytopathology 96:1188-1194. Chitosan (CHN), a deacetylated derivative of chitin, was shown to be efficient in promoting plant defense reactions. CHN oligomers of different molecular weight (MW) and degree of acetylation (DA) triggered an accumulation of Phytoalexins, trans- and cis-resveratrol and their derivatives e-viniferin and piceid, in grapevine leaves. Highest Phytoalexin production was achieved within 48 h of incubation with CHN at 200 µg/ml with an MW of 1,500 and a DA of 20% (CHN1.5/20), while oligomers with greater MW were less efficient, indicating that a specific MW threshold could be required for Phytoalexin response. Treatment of grapevine leaves by highly active CHN1.5/20 also led to marked induction of chitinase and β-1,3-glucanase activities. CHN1.5/20 applied together with copper sulfate (CuSO4) strongly induced Phytoalexin accumulation. CuSO4 alone, especially at low concentrations also elicited a substantial production of Phytoalexins in grapevine leaves. Evidence is also provided that CHN1.5/20 significantly reduced the infection of grapevine leaves by Botrytis cinerea and Plasmopara viticola, and in combination with CuSO4 conferred protection against both pathogens.
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Phytoalexins from the Vitaceae: Biosynthesis, Phytoalexin Gene Expression in Transgenic Plants, Antifungal Activity, and Metabolism
Journal of Agricultural and Food Chemistry, 2002Co-Authors: Philippe Jeandet, Anne-céline Douillet-breuil, S. Debord, M. Sbaghi, Roger Bessis, Marielle AdrianAbstract:Resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defense reactions comprising both constitutive and inducible barriers. In grapevine, the most frequently observed and best characterized defense mechanisms are the accumulation of Phytoalexins and the synthesis of PR-proteins. Particular attention has been given here to stilbene Phytoalexins produced by Vitaceae, specifically, their pathway of biosynthesis (including stilbene Phytoalexin gene transfer experiments to other plants) and their biological activity together with fungal metabolism.
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hplc analysis of grapevine Phytoalexins coupling photodiode array detection and fluorometry
Analytical Chemistry, 1997Co-Authors: Philippe Jeandet, S. Debord, Marielle Adrian, Anne Celine Breuil, Leslie A Weston, Philippe Meunier, And Gabrielle Maume, Roger BessisAbstract:A reversed-phase HPLC method useful for the analysis of the grapevine Phytoalexins resveratrol, its β-d-glucoside, e-viniferin, and pterostilbene in leaf extracts was developed by coupling diode array detection and fluorometry. Phytoalexins were extracted from UV-irradiated grapevine leaves with methanol/water (80:20) and prepurified on C18 solid phase extraction cartridges. Separation by HPLC was achieved using a C18 column and a gradient elution with acetonitrile and water (from 10 to 85% acetonitrile). Analyses of grapevine leaf extracts were performed by injecting the equivalent of 1 mg of leaf fresh weight. Recovery was nearly 100% for the three stilbenes resveratrol, pterostilbene, and e-viniferin (ranging from 100.2 to 104.8%), and replicate analyses gave coefficients of variation of 0.6−2.5%. Identification of each Phytoalexin was accomplished by line spectral comparisons with known standards, and e-viniferin was further characterized by MS and GC/MS. Simultaneously, stilbenes were detected by flu...
Kazunori Okada - One of the best experts on this subject based on the ideXlab platform.
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overexpression of the bzip transcription factor osbzip79 suppresses the production of diterpenoid Phytoalexin in rice cells
Journal of Plant Physiology, 2015Co-Authors: Koji Miyamoto, Yoko Nishizawa, Eiichi Minami, Hideaki Nojiri, Hisakazu Yamane, Kazunori OkadaAbstract:Phytoalexins are antimicrobial specialised metabolites that are produced by plants in response to pathogen attack. Momilactones and phytocassanes are major diterpenoid Phytoalexins in rice that are synthesised from geranylgeranyl diphosphate that is derived from the methylerythritol phosphate (MEP) pathway. We have previously reported that rice cells overexpressing the basic leucine zipper (bZIP) transcription factor OsTGAP1 exhibit a hyperaccumulation of momilactones and phytocassanes, with hyperinductive expression of momilactone and phytocassane biosynthetic genes and MEP pathway genes, upon response to a chitin oligosaccharide elicitor. For a better understanding of OsTGAP1-mediated regulation of diterpenoid Phytoalexin production, we identified OsTGAP1-interacting proteins using yeast two-hybrid screening. Among the OsTGAP1-interacting protein candidates, a TGA factor OsbZIP79 was investigated to verify its physical interaction with OsTGAP1 and involvement in the regulation of Phytoalexin production. An in vitro pull-down assay demonstrated that OsTGAP1 and OsbZIP79 exhibited a heterodimeric as well as a homodimeric interaction. A bimolecular fluorescence complementation analysis also showed the interaction between OsTGAP1 and OsbZIP79 in vivo. Intriguingly, whereas OsbZIP79 transactivation activity was observed in a transient reporter assay, the overexpression of OsbZIP79 resulted in suppression of the elicitor-inducible expression of diterpenoid Phytoalexin biosynthetic genes, and thus caused a decrease in the accumulation of Phytoalexin in rice cells. These results suggest that OsbZIP79 functions as a negative regulator of Phytoalexin production triggered by a chitin oligosaccharide elicitor in rice cells, although it remains open under which conditions OsbZIP79 can work with OsTGAP1.
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biosynthesis elicitation and roles of monocot terpenoid Phytoalexins
Plant Journal, 2014Co-Authors: Eric A Schmelz, Alisa Huffaker, James Sims, Shawn A Christensen, Kazunori Okada, Reuben J PetersAbstract:Summary A long-standing goal in plant research is to optimize the protective function of biochemical agents that impede pest and pathogen attack. Nearly 40 years ago, pathogen-inducible diterpenoid production was described in rice, and these compounds were shown to function as antimicrobial Phytoalexins. Using rice and maize as examples, we discuss recent advances in the discovery, biosynthesis, elicitation and functional characterization of monocot terpenoid Phytoalexins. The recent expansion of known terpenoid Phytoalexins now includes not only the labdane-related diterpenoid superfamily but also casbane-type diterpenoids and β-macrocarpene-derived sequiterpenoids. Biochemical approaches have been used to pair pathway precursors and end products with cognate biosynthetic genes. The number of predicted terpenoid Phytoalexins is expanding through advances in cereal genome annotation and terpene synthase characterization that likewise enable discoveries outside the Poaceae. At the cellular level, conclusive evidence now exists for multiple plant receptors of fungal-derived chitin elicitors, phosphorylation of membrane-associated signaling complexes, activation of mitogen-activated protein kinase, involvement of phytohormone signals, and the existence of transcription factors that mediate the expression of Phytoalexin biosynthetic genes and subsequent accumulation of pathway end products. Elicited production of terpenoid Phytoalexins exhibit additional biological functions, including root exudate-mediated allelopathy and insect antifeedant activity. Such findings have encouraged consideration of additional interactions that blur traditionally discrete Phytoalexin classifications. The establishment of mutant collections and increasing ease of genetic transformation assists critical examination of further biological roles. Future research directions include examination of terpenoid Phytoalexin precursors and end products as potential signals mediating plant physiological processes.
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regulation of a proteinaceous elicitor induced ca2 influx and production of Phytoalexins by a putative voltage gated cation channel ostpc1 in cultured rice cells
Journal of Biological Chemistry, 2012Co-Authors: Haruyasu Hamada, Jinichiro Koga, Kazunori Okada, Takamitsu Kurusu, Eiji Okuma, Hiroshi Nokajima, Masahiro Kiyoduka, Tomoko Koyano, Yoshimi Sugiyama, Hikaru SajiAbstract:Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca2+ followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called Phytoalexins; however, the molecular links and regulatory mechanisms of the Phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca2+, mainly due to a Ca2+ influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca2+-sensitive photoprotein, aequorin, TvX-induced Ca2+ elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid Phytoalexins and the expression of a series of diterpene cyclase genes involved in Phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca2+-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca2+ influx as a plasma membrane Ca2+-permeable channel consequently required for the regulation of Phytoalexin biosynthesis in cultured rice cells.
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Effects of cytokinin on production of diterpenoid Phytoalexins in rice
Journal of Pesticide Science, 2010Co-Authors: Kazunori Okada, Jinichiro Koga, Hideaki Nojiri, Yusuke Jikumaru, Hisakazu YamaneAbstract:The production of Phytoalexins is a well-documented defense response against pathogen infection in rice. Although jasmonic acid has been suggested to be involved in the production of Phytoalexins in both suspension-cultured rice cells and rice leaves, there has been almost no information on other secondary signaling molecules that regulate the production of Phytoalexins in rice. In this study, the production of the major diterpenoid Phytoalexins, momilactones and phytocassanes, was found to be induced by cytokinin treatment in both suspension-cultured rice cells and rice leaves, and also the upregulation of Phytoalexin biosynthetic genes was found to be induced by cytokinin treatment. The roles of cytokinins in the production of diterpenoid Phytoalexins in rice infected with a pathogen are discussed.
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Differential responses of rice to inoculation with wild-type and non-pathogenic mutants of Magnaporthe oryzae
Plant Molecular Biology, 2009Co-Authors: Tomoaki Kato, Jinichiro Koga, Kazunori Okada, Yoko Nishizawa, Yusuke Jikumaru, Shigeru Tanabe, Marie Nishimura, Yuko Ohtake, Takafumi Shimizu, Hisakazu YamaneAbstract:We analyzed the response of rice to Magnaporthe oryzae infection using two mutant strains deficient in Mgb1 and Mst12, which are essential for the development of appresoria and penetration pegs. Both mutants induced the much lower levels of accumulation of Phytoalexins than wild-type, suggesting that the massive production of Phytoalexins requires the fungal invasion of rice cells. Intense accumulation of H_2O_2 in a single whole cell also required fungal penetration. Microarray analysis of rice gene expression revealed mutant-specific gene expression, indicating that signal exchange between rice and M. oryzae commence before fungal penetration of the rice cell. In situ detection of mRNAs for peroxidase and β -1,3-glucanase showed that expression of these genes also occurs after penetration as observed for Phytoalexin production.