Showing papers on "Phytoalexin published in 2012"

Simultaneous RNA-seq analysis of a mixed transcriptome of rice and blast fungus interaction.

Abstract: A filamentous fungus, Magnaporthe oryzae, is a causal agent of rice blast disease, which is one of the most serious diseases affecting cultivated rice, Oryza sativa. However, the molecular mechanisms underlying both rice defense and fungal attack are not yet fully understood. Extensive past studies have characterized many infection-responsive genes in the pathogen and host plant, separately. To understand the plant-pathogen interaction comprehensively, it is valuable to monitor the gene expression profiles of both interacting organisms simultaneously in the same infected plant tissue. Although the host-pathogen interaction during the initial infection stage is important for the establishment of infection, the detection of fungal gene expression in infected leaves at the stage has been difficult because very few numbers of fungal cells are present. Using the emerging RNA-Seq technique, which has a wide dynamic range for expression analyses, we analyzed the mixed transcriptome of rice and blast fungus in infected leaves at 24 hours post-inoculation, which is the point when the primary infection hyphae penetrate leaf epidermal cells. We demonstrated that our method detected the gene expression of both the host plant and pathogen simultaneously in the same infected leaf blades in natural infection conditions without any artificial treatments. The upregulation of 240 fungal transcripts encoding putative secreted proteins was observed, suggesting that these candidates of fungal effector genes may play important roles in initial infection processes. The upregulation of transcripts encoding glycosyl hydrolases, cutinases and LysM domain-containing proteins were observed in the blast fungus, whereas pathogenesis-related and phytoalexin biosynthetic genes were upregulated in rice. Furthermore, more drastic changes in expression were observed in the incompatible interactions compared with the compatible ones in both rice and blast fungus at this stage. Our mixed transcriptome analysis is useful for the simultaneous elucidation of the tactics of host plant defense and pathogen attack.

The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance

Abstract: Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na(+)/K(+) ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels.

A convenient method for simultaneous quantification of multiple phytohormones and metabolites: application in study of rice-bacterium interaction

Abstract: Simultaneous analysis of multiple functional-related phytohormones and their metabolites will improve our understanding of interactions among different hormones in the same biologic process. A method was developed for simultaneous quantification of multiple phytohormones, abscisic acid, indole-3-acetic acid (IAA), jasmonic acid (JA), and salicylic acid, hormone conjugates, IAA-aspartic acid, JA-isoleucine, and methyl JA, and phytoalexins, momilactone A, naringenin, and sakuranetin. This method combines a convenient procedure for preparing filtrated crude extracted samples and a sensitive quantification assay using ultra fast liquid chromatography-electrospray ionization tandem mass spectrometry (UFLC-ESI-MS). With this method, we determined the dynamic profiles of defense-related phytohormones, hormone metabolites, and phytoalexins in the interaction of rice with Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, one of the most devastating diseases of rice worldwide. This UFLC-ESI-MS method is convenient, sensitive, reliable, and inexpensive for quantification of multiple phytohormones and metabolites compared to current methods. The results obtained by application of this method in studying rice-bacterial interaction provide a basis for understanding the molecular mechanisms of rice defense responses.

Dissecting Phosphite-Induced Priming in Arabidopsis Infected with Hyaloperonospora arabidopsidis

Abstract: Phosphite (Phi), a phloem-mobile oxyanion of phosphorous acid (H3PO3), protects plants against diseases caused by oomycetes. Its mode of action is unclear, as evidence indicates both direct antibiotic effects on pathogens as well as inhibition through enhanced plant defense responses, and its target(s) in the plants is unknown. Here, we demonstrate that the biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) exhibits an unusual biphasic dose-dependent response to Phi after inoculation of Arabidopsis (Arabidopsis thaliana), with characteristics of indirect activity at low doses (10 mm or less) and direct inhibition at high doses (50 mm or greater). The effect of low doses of Phi on Hpa infection was nullified in salicylic acid (SA)-defective plants (sid2-1, NahG) and in a mutant impaired in SA signaling (npr1-1). Compromised jasmonate (jar1-1) and ethylene (ein2-1) signaling or abscisic acid (aba1-5) biosynthesis, reactive oxygen generation (atrbohD), or accumulation of the phytoalexins camalexin (pad3-1) and scopoletin (f6′h1-1) did not affect Phi activity. Low doses of Phi primed the accumulation of SA and Pathogenesis-Related protein1 transcripts and mobilized two essential components of basal resistance, Enhanced Disease Susceptibility1 and Phytoalexin Deficient4, following pathogen challenge. Compared with inoculated, Phi-untreated plants, the gene expression, accumulation, and phosphorylation of the mitogen-activated protein kinase MPK4, a negative regulator of SA-dependent defenses, were reduced in plants treated with low doses of Phi. We propose that Phi negatively regulates MPK4, thus priming SA-dependent defense responses following Hpa infection.

Role of glutathione in plant signaling under biotic stress.

Abstract: Glutathione (GSH) is a non-protein thiol compound which has been repeatedly reported to play an important role in plant responses during biotic stresses. However, our knowledge of glutathione-related molecular mechanisms underlying plant defense responses still remains limited. We first discovered that the Arabidopsis thaliana phytoalexin deficient 2-1 (pad2-1) mutant was linked to glutathione deficiency since the mutation was identified in the GSH1 gene encoding the first enzyme of glutathione biosynthesis: Glutamate Cysteine Ligase (GCL). Interestingly, this glutathione-deficient mutant pad2-1 also displays a high susceptibility to a wide range of invaders. We recently reported that the glutathione deficiency in pad2-1 is directly related to a low content of GCL protein. In parallel, we highlighted that the altered redox potential in pad2-1 upregulates the oxidative-stress marker genes GR1, GSTF6 and RbohD during infection with the hemibiotrophic oomycete Phytophthora brassicae. Moreover, the impairment of early signaling events such as plasma membrane depolarization, production of nitric oxide and reactive oxygen species also correlates with the reduced hypersensitive response (HR) observed during P. brassicae infection. Concerning the impaired salicylic acid (SA)-dependent pathway in pad2-1, our results indicated that transcripts of IsoChorismate Synthase1 (ICS1, a main enzyme of SA biosynthesis) do not accumulate in response to pathogen. In this review, we integrate previous knowledge and recent discoveries about pad2-1 to better understand the involvement of glutathione in the pad2-1 pleiotropic phenotype observed during biotic stresses.

Accumulation of salicylic acid, jasmonic acid and phytoalexins in rice, Oryza sativa, infested by the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae)

Abstract: In order to clarify the mechanism of induced resistance to blast disease in rice, Oryza sativa, that had been previously infested by the white-backed planthopper, Sogatella furcifera Horvath, we first investigated the accumulation of salicylic acid (SA) and jasmonic acid (JA) in rice plants infested by the planthopper. The results confirmed that infestation of S. furcifera strongly stimulates the production of SA and JA in rice. These results indicate that both salicylate- and jasmonate-mediated pathways (SA and JA pathways), which are involved in the general defense system in plants, were activated in rice infested by S. furcifera. Further results confirmed that S. furcifera infestation induces accumulation of a major rice diterpenoid phytoalexin, momilactone A, and a flavonoid phytoalexin, sakuranetin, which are well known as antimicrobial chemicals, particularly in blast disease caused by the blast fungus, Magnaporthe oryzae B. Couch. All these results strongly suggest the following hypothetical mechanism of induced-resistance to M. oryzae in rice infested by S. furcifera. First, S. furcifera releases some elicitor-active compounds, which might be produced in the salivary glands, into the rice plant during feeding. Next, the defense signal systems, SA- and JA-mediated pathways, are activated by the elicitor. Finally, phytoalexins are induced in rice as antimicrobial compounds mainly through activation of the JA-mediated pathway.

Effect of Aspergillus oryzae-challenged germination on soybean isoflavone content and antioxidant activity.

Abstract: Application of microbial stress to soybean during germination induces the accumulation of phytoalexins, which have many health benefits. In this study, the effects of stress induced by Aspergillus oryzae on the phytochemical composition of germinating soybeans were investigated, and their radical scavenging activity was compared with those of ungerminated (US) and germinated (GS) soybeans. Additionally, the antioxidant activity of coumestrol, a soybean phytoalexin, against hydrogen peroxide-induced reactive oxygen species (ROS) was investigated in HepG2 cells. A. oryzae exposure significantly decreased the total isoflavone content and induced coumestrol and glyceollin I. A. oryzae-challenged germinated soybeans exhibited the highest radical scavenging activity (IC(50) = 0.55 mg/mL) as compared to US and GS. Coumestrol exhibited significantly higher radical scavenging activity than daidzein and genistein. Furthermore, coumestrol significantly prevented hydrogen peroxide-induced ROS production and lipid peroxidation and inhibited decreases in cell viability, intracellular glutathione (GSH) levels, and superoxide dismutase (SOD) activity. These results indicate that using food-grade A. oryzae to elicit the biosynthesis of phytoalexins alters the secondary metabolite profiles of the soybeans and offers enhanced bioactivity of soybean as a functional food ingredient.

Influence of ATP-Binding Cassette Transporters in Root Exudation of Phytoalexins, Signals, and in Disease Resistance.

Abstract: The roots of plants secrete compounds as a way to exchange information with organisms living in the soil. Here, we report the involvement of seven root-expressed ATP-binding cassette (ABC) transporters corresponding to both full and half-size molecules (Atabcg36, Atabcg37, Atabcc5, Atabcf1, Atabcf3, Atnap5 and Atath10) in root exudation processes using Arabidopsis thaliana. Root exuded phytochemicals were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS), and it was determined that some of the root exudates from the corresponding ABC transporter mutants were significantly different compared to the wild type. For example, Atabcg37 and Atabcc5 secreted higher levels of the phytoalexin camalexin, and Atabcg36 secreted higher levels of organic acids, specifically salicylic acid (SA). Furthermore, we analyzed the root tissue metabolites of these seven ABC transporter mutants and found that the levels of SA, quercetin and kaempferol glucosides were higher in Atabcg36, which was correlated with higher expression levels of defense genes in the root tissues compared with the wild type. We did not observe significant changes in the root exudates of the half-size transporters except for Atabcf1 that showed lower levels of few organic acids. In summary, full size transporters are involved in root secretion of phytochemicals.

The potential bioproduction of the pharmaceutical agent sakuranetin, a flavonoid phytoalexin in rice

Abstract: Sakuranetin, the major flavonoid phytoalexin in rice, can be induced by ultraviolet (UV) irradiation, treatment with CuCl2 or jasmonic acid (JA), or phytopathogenic infection. In addition to sakuranetin’s biological significance on disease resistance in rice, its broad bioactivities have recently been described. Results from these studies have shown that sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin, a precursor of sakuranetin, by naringenin 7-O-methyltransferase (NOMT), but the relevant gene has not yet been identified in rice. Recently, we identified the OsNOMT gene, which is involved in the final step of sakuranetin biosynthesis in rice. In previous studies, OsNOMT was purified to apparent homogeneity from UV-treated wild-type rice leaves; however, the purified protein, termed OsCOMT1, exhibited caffeic acid 3-O-methyltransferase (COMT) activity, but not NOMT activity. Based on the analysis of an oscomt1 T-DNA ...

Ectopic expression of Hrf1 enhances bacterial resistance via regulation of diterpene phytoalexins, silicon and reactive oxygen species burst in rice.

Abstract: Harpin proteins as elicitor derived from plant gram negative bacteria such as Xanthomonas oryzae pv. oryzae (Xoo), Erwinia amylovora induce disease resistance in plants by activating multiple defense responses. However, it is unclear whether phytoalexin production and ROS burst are involved in the disease resistance conferred by the expression of the harpinXoo protein in rice. In this article, ectopic expression of hrf1 in rice enhanced resistance to bacterial blight. Accompanying with the activation of genes related to the phytoalexin biosynthesis pathway in hrf1-transformed rice, phytoalexins quickly and consistently accumulated concurrent with the limitation of bacterial growth rate. Moreover, the hrf1-transformed rice showed an increased ability for ROS scavenging and decreased hydrogen peroxide (H2O2) concentration. Furthermore, the localization and relative quantification of silicon deposition in rice leaves was detected by scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometer (EDS). Finally, the transcript levels of defense response genes increased in transformed rice. These results show a correlation between Xoo resistance and phytoalexin production, H2O2, silicon deposition and defense gene expression in hrf1-transformed rice. These data are significant because they provide evidence for a better understanding the role of defense responses in the incompatible interaction between bacterial disease and hrf1-transformed plants. These data also supply an opportunity for generating nonspecific resistance to pathogens.

In silico study of interaction between rice proteins enhanced disease susceptibility 1 and phytoalexin deficient 4, the regulators of salicylic acid signalling pathway.

Abstract: Enhanced disease susceptibility 1 (EDS1), a plant-specific protein has homology with the eukaryotic lipase in their N-terminal halves and a unique domain at its C-termini. EDS1 is known to be an important regulator of biotic stress and an essential component of basal immunity. EDS1 interacts with its positive co-regulator phytoalexin deficient 4 (PAD4), resulting in mobilization of the salicylic acid defence pathway. Limited information regarding this interaction in rice is available. To study this interaction, a model of EDS1 and PAD4 proteins from rice was generated and validated with Accelrys DS software version 3.1 using bioinformatics interface. The in silico docking between the two proteins showed a significant protein–protein interaction between rice EDS1 and PAD4, suggesting that they form a dimeric protein complex, which, similar to that in Arabidopsis, is perhaps also important for triggering the salicylic acid signalling pathway in plants.

Endogenous hydrogen peroxide is a key factor in the yeast extract-induced activation of biphenyl biosynthesis in cell cultures of Sorbus aucuparia

Abstract: Biphenyls are unique phytoalexins produced by plants belonging to Pyrinae, a subtribe of the economically important Rosaceae family. The formation of aucuparin, a well-known biphenyl, is induced by yeast extract (YE) in cell cultures of Sorbus aucuparia. However, the molecular mechanism underlying YE-induced activation of biphenyl biosynthesis remains unknown. Here we demonstrate that the addition of YE to the cell cultures results in a burst of reactive oxygen species (ROS; H2O2 and O2 −), followed by transcriptional activation of the biphenyl synthase 1 gene (BIS1) encoding the key enzyme of the biphenyl biosynthetic pathway and aucuparin accumulation. Pretreatment of the cell cultures with ROS scavenger dihydrolipoic acid and NADPH oxidase-specific inhibitor diphenylene iodonium abolished all of the above YE-induced biological events. However, when the cell cultures was pretreated with superoxide dismutase specific inhibitor N,N-diethyldithiocarbamic acid, although O2 − continued to be generated, the H2O2 accumulation, BIS1 expression and aucuparin production were blocked. Interestingly, exogenous supply of H2O2 in the range of 0.05–10 mM failed to induce aucuparin accumulation. These results indicate that endogenous generation of H2O2 rather than that of O2 − is a key factor in YE-induced accumulation of biphenyl phytoalexins in cell cultures of S. aucuparia.

Glycerolipids and the plant cuticle contribute to plant immunity

Abstract: The conserved metabolites, oleic acid (18:1), a major monounsaturated fatty acid (FA), and glycerol-3-phosphate (G3P) are obligatory precursors of glycerolipid biosynthesis in plants. In Arabidopsis, the SSI2-encoded SACPD is the major isoform that contributes to 18:1 biosynthesis. Signaling induced upon reduction in oleic acid (18:1) levels not only upregulates salicylic acid (SA)-mediated responses but also inhibits jasmonic acid (JA)- inducible defenses. I examined the transcription profile of ssi2 plants and identified two transcription factors, WRKY50 and WRKY51. Although the ssi2 wrky50 and ssi2 wrky51 plants were constitutively upregulated in SA-derived signaling, they were restored in JAdependent defense signaling. Not only did these plants show JA-inducible PDF1.2 expression, but they were also restored for basal resistance to the necrotrophic pathogen, Botrytis cinerea. Overall, my results show that the WRKY50 and WRKY51 proteins mediate both SA- and low 18:1-dependent repression of JA signaling in Arabidopsis plants. My studies also show that cellular G3P levels are important for plant defense to necrotrophic pathogens. I showed that G3P levels are induced in Arabidopsis in response to the necrotrophic fungal pathogen B. cinerea. G3P-dependant induction of basal defense is not via the activities of other defense-related hormones such as SA, JA or the phytoalexin camalexin. Arabidopsis mutants unable to accumulate G3P (gly1, gli1) showed enhanced susceptibility to B. cinerea. Previous studies in our lab identified acyl-carrier protein 4 (ACP4), a component of FA and lipid biosynthesis, as an important regulator of plant systemic immunity. ACP4 mutant plants were defective in systemic acquired resistance (SAR) because they contained a defective cuticle. I further investigated the role of the plant cuticle in SAR by studying the involvement of long-chain acyl-CoA synthetases (LACS), a gene family involved in long-chain FA and cuticle biosynthesis, in SAR. In all, eight lacs mutants (lacs1, lacs2, lacs3, lacs4, lacs6, lacs7, lacs8, lacs9) were isolated and characterized. Six mutants were compromised in SAR. Together, my studies show that the various LACS isoforms contribute differentially to both cuticle formation and systemic immunity in Arabidopsis.

Effects of Methyl Jasmonate Treatment on Levels of Nitric Oxide and Hydrogen Peroxide and Phytoalexin Synthesis in Postharvest Grape Berries

Abstract: In order to reveal the mechanisms of methyl jasmonat(eMeJA)inhibiting postharvest decay in grape berries,‘Kyoho’grape berries were pretreated with 10 μmol · L-1 MeJA vapor for 6 h and then stored at 1 ℃ for 28 days. The fruit decay incidence,weight loss rate,levels of endogenous signaling molecules nitric oxide(NO)and hydrogen peroxide(H2O2),activities of phytoalexin synthesis related enzymes and individual phytoalexin compounds contents were determined at 7-day intervals during the storage. The results exhibited that the MeJA treatment could significantly inhibit the increase in decay incidence and weight loss rate,promote endogenous NO release and H2O2 content during the early stage of the storage,and simultaneously induce the increase in activities of phenylalanine-ammonia-lyase(PAL), cinnamate 4-hydroxylase(C4H),4-coumarate coenzyme A ligase(4-CL)and resveratrol synthase(RS)associated with phytoalexin synthesis and the contents of phytoalexins including resveratrol and viniferin in grape berries during the storage. Therefore,we speculate that MeJA might play an important role in signal transduction in grape cells,which regulated the downstream signaling molecules NO and H2O2 levels to enhance activities of enzymes related to phytoalexin synthesis,resulting in promoting phytoalexin accumulation and furthermore improving disease resistance in grape berries.

Metabolism of phytoalexins and analogs, and inhibitors of brassinin detoxification in Leptosphaeria maculans

Abstract: ................................................................................. ii Acknowledgements......................................................................iv Dedication.................................................................................v Table of contents........................................................................vi List of figures........................................................................ ...ix List of tables.............................................................................xiv List of schemes...........................................................................xv List of abbreviations.....................................................................xix Chapter 1: Introduction ...................................................................................................... 1 1.1 General objectives .................................................................................................... 1 1.2 Cruciferous plants and fungal pathogens ............................................................... 2 1.3 Secondary metabolites of cruciferous plants and blackleg fungi in attack and defense 3 1.3.1 Fungal metabolites .............................................................................................................. 3 1.3.2 Cruciferous plant metabolites .............................................................................................. 6 1.4 Biotransformation of cruciferous phytoalexins and related structures by phytopathogenic fungi ......................................................................................................... 10 1.4.1 Syntheses of phytoalexin-related structures ...................................................................... 10 1.4.2 Biotransformations in fungal cultures ............................................................................... 12 1.4.3 Enzymes and inhibitors of brassinin transformation by Leptosphaeria maculans ............ 22 1.5 Crop protectants against pathogenic fungi .......................................................... 27 1.5.1 Inhibitors of fungal respiration .......................................................................................... 29 1.5.1.1 Complex I inhibitors ................................................................................................ 30 1.5.1.2 Complex II inhibitors ............................................................................................... 31 1.5.1.3 Complex III inhibitors ............................................................................................. 34 1.5.2 Inhibitors of ergosterol biosynthesis ................................................................................. 38 1.5.2.1 Inhibitors of demethylase (DMI) ............................................................................. 40 1.5.2.2 Inhibitors of squalene epoxidase .............................................................................. 41 1.5.2.3 Inhibitors of ∆14 reductase and ∆8 ∆7 isomerase ................................................. 42