Rice blast fungus (Magnaporthe oryzae) infects Arabidopsis via a mechanism distinct from that required for the infection of rice

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice (Oryza sativa) blast. Although M. oryzae as a whole infects a wide variety of monocotyledonous hosts, no dicotyledonous plant has been reported as a host. We found that two rice pathogenic strains of M. oryzae, KJ201 and 70-15, interacted differentially with 16 ecotypes of Arabidopsis (Arabidopsis thaliana). Strain KJ201 infected all ecotypes with varying degrees of virulence, whereas strain 70-15 caused no symptoms in certain ecotypes. In highly susceptible ecotypes, small chlorotic lesions appeared on infected leaves within 3 d after inoculation and subsequently expanded across the affected leaves. The fungus produced spores in susceptible ecotypes but not in resistant ecotypes. Fungal cultures recovered from necrotic lesions caused the same symptoms in healthy plants, satisfying Koch's postulates. Histochemical analyses showed that infection by the fungus caused an accumulation of reactive oxygen species and eventual cell death. Similar to the infection process in rice, the fungus differentiated to form appressorium and directly penetrated the leaf surface in Arabidopsis. However, the pathogenic mechanism in Arabidopsis appears distinct from that in rice; three fungal genes essential for pathogenicity in rice played only limited roles in causing disease symptoms in Arabidopsis, and the fungus seems to colonize Arabidopsis as a necrotroph through the secretion of phytotoxic compounds, including 9,12-octadecadienoic acid. Expression of PR-1 and PDF1.2 was induced in response to infection by the fungus, suggesting the activation of salicylic acid- and jasmonic acid/ethylene-dependent signaling pathways. However, the roles of these signaling pathways in defense against M. oryzae remain unclear. In combination with the wealth of genetic and genomic resources available for M. oryzae, this newly established pathosystem allows comparison of the molecular and cellular mechanisms underlying pathogenesis and host defense in two well-studied model plants.     

Molecular cloning and differential expression of an aldehyde dehydrogenase gene in rice leaves in response to infection by blast fungus

Aldehyde dehydrogenase (ALDH) superfamily is a group of enzymes metabolizing endogenous and exogenous aldehydes. Using differential display RT-PCR and cDNA library screening, a full-length aldehyde dehydrogenase cDNA (ALDH7B7) was isolated from rice leaves infected by incompatible race of blast fungus Magnaporthe grisea. The deduced amino acid sequence consists of 509 amino acid residues and shares 74∼81% identity with those of ALDH7Bs from other plants. ALDH7B7 expression was induced by blast fungus infection, ultraviolet, mechanical wound in rice leaves and was not detected in untreated rice organs. This gene has also been found to be inducible after exogenous phytohormones application, such as salicylic acid, methyl ester of jasmonic acid and abscisic acid. The function of ALDH7B7 in the interaction process between blast fungus and rice is discussed.     

Structures of Conversion Products Formed from 18β-Glycyrrhetinic Acid by Streptomyces sp. G-20

Dynamic profiles of the rate of O₂ generation from press-injured and inoculated rice leaf slices, versus the time after inoculation, discriminated between the incompatible and compatible combination of blast fungus races with a cultivar. The application of sodium saccharin to rice seedlings via the root system for 6 days changed the compatible to incompatible profile. Even after press- injury and inoculation with the compatible conidia, the leaf application of sodium saccharin enhanced superoxide generation. The application of N-methylsaccharin in a similar manner, however, did not enhance the superoxide generation. Inoculation of press-injured leaves with incompatible conidia in the presence of an aqueous diffusate of the germinating compatible conidia changed the incompatible to compatible profile. The application to press-injured of concanavalin A or a lyophylized preparation from 5 m ammonia extracts of rice leaf homogenate prior to stimulating with a resistance-inducing factor (RIF) from the fungus also enhanced the superoxide generation. The RIF, either from the incompatible or compatible race, gave a quite similar profile of activation upon the generation of the superoxide anion.     

Cloning and Characterization of a Probenazole-Inducible Gene for an Intracellular Pathogenesis-Related Protein in Rice

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) inducesdisease resistance in rice against rice blast fungus. To investigatethe molecular mechanism of probenazole-induced resistance, weisolated and characterized a cDNA clone of a probenazole-induciblegene in rice, which encoded a protein designated PBZ1. Sequenceanalysis revealed that significant homology at the amino acidlevel exists between the predicted PBZ1 protein and intracellularpathogenesis-related (IPR) proteins. Accumulation of PBZ1 mRNAwas not induced by wounding, but markedly induced by inoculationwith rice blast fungus. In addition, it was induced sooner byinoculation with rice blast fungus. In addition, it was inducedsooner by inoculation with an incompatible race than that witha compatible race. On the other hand, when the accumulationof the PBZ1 mRNA was examined after treatment with probenazole-relatedcompounds, it was not fully correlated with anti-rice blastactivity. However, it was induced after treatement with N-cyanomethyl-2-chloro-isonicotinamide(NCI), which belongs to another group of compounds known toinduce disease resistance. Thus, although the accumulation ofthe PBZ1 mRNA was not fully correlated with anti-rice blastactivity, our findings suggest that the PBZ1 gene has an importantfunction during the disease resistance response in rice. (Received June 19, 1995; Accepted October 13, 1995)     

Possible role of phytocassane,rice phytoalexin,in disease resistance of rice against the blast fungus Magnaporthe grisea

In addition to momilactone, phytocassanes A through E (diterpene phytoalexins) were detected in rice leaves in fields suffering from rice blast. Furthermore, phytocassane accumulation was most abundant at the edges of necrotic lesions, indicating that the phytoalexins prevent subsequent spread of the fungus from the infected site. In pot experiments the pattern of phytocassane accumulation in rice leaves in an incompatible interaction (infection with an avirulent race of Magnaporthe grisea) was more rapidly induced than in a compatible interaction (infection with a virulent race of M. grisea).     

Galactolipid depletion in blast fungus‐infected rice leaves

This research focuses on galactolipid depletion in blast fungus‐infected rice leaves. Two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), from rice leaves were isolated and purified. The chemical structure of MGDG was identified as 1,2‐dilinolenyl‐3‐O‐β‐d ‐galactopyranosyl‐sn‐glycerol, and that of DGDG as 1,2‐dilinolenyl‐3‐O‐[α‐d ‐galactopyranosyl‐(1→6)‐O‐β‐d ‐galactopyranosyl]‐sn‐glycerol. Both the MGDG and DGDG content in the incompatible blast fungus race‐infected leaves decreased more than those in the compatible blast fungus race‐infected leaves during the infection process. Active oxygen species had the ability to peroxygenate and de‐esterify MGDG or DGDG in vitro, suggesting that active oxygen species play an important role in galactolipid depletion during the process of rice blast fungus invasion. Other possible functions of rice galactolipids during disease resistance are also discussed.     

Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungus Magnaporthe oryzae (Mo), was expressed in Pichia pastoris. Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an α-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that Mo-MnLOX illustrates a partly unique variation of the structural theme of FeLOX.     

Molecular Cloning of Early-expressed cDNAs from Rice in Response to Infection by Rice Blast Fungus Magnaporthe grisea

Compatible and incompatible reactions in rice plants (Oryza sativa L. cv. Shenxianggen No.4) were resulted from inoculation with two different virulent races of rice blast fungus (Magnaporthe grisea (Hebert) Barr), and thus an effective infecting system was established between rice plants and the rice blast pathogen. Two cDNA clones that showed induced and temporal patterns in expression in the very early stage in response to infection of the fungus were obtained from the plants by use of differential display. Of the two cDNA clones, Fastresp-a was induced to express in both compatible and incompatible interactions although it was expressed earlier in the former reaction. The second one, Fastresp-b, was only expressed in incompatible interaction. Southern blot analysis of the rice genomic DNA indicated that both of the two clones were from genome of the plant. No significant homology to the two genes was found from the rice gene database. This suggested that they were novel genes in rice and may play important roles in rice resistant response to infection of rice blast fungus.     

受稻瘟病原真菌侵染的水稻早期表达基因的cDNA克隆

以亲和性与非亲和性两个稻瘟病原真菌小种（Ｍａｇｎａｐｏｒｔｈｅ ｇｒｉｓｅａ（Ｈｅｂｅｒｔ）Ｂａｒｒ）感染同一水稻品种（Ｏｒｙｚａｓａｔｉｖａ Ｌ．ｃｖ．Ｓｈｅｎｘｉａｎｇｇｅｎｇ Ｎｏ．４）的植株产生明显不同的致病和抗病反应,由此建立了有效的感染系统。应用差异显示技术获得两个在侵染早期具有诱导表达特征的ｃＤＮＡ克隆,其中一个同时在致病和抗病反应中进行早期诱导表达,但在抗病反应中的诱导相对早于其在     

Soybean Lipoxygenase-1 Oxidizes 3Z-Nonenal : A Route to 4S-Hydroperoxy-2E-Nonenal and Related Products

In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O₂ uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z,11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer.     

Molecular cloning and differential expression of an γ-aminobutyrate transaminase gene, OsGABA-T, in rice (Oryza sativa) leaves infected with blast fungus

γ-Aminobutyrate transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde. Using differential display PCR and cDNA library screening, a full-length GABA-T cDNA (OsGABA-T) was isolated from rice (Oryza sativa) leaves infected with an incompatible race of Magnaporthe grisea. The deduced amino acid sequence comprises 483 amino acid residues and shares 85–69% identity with GABA-T sequences from other plants. OsGABA-T expression is induced by blast fungus infection, mechanical wounding and ultraviolet radiation in rice leaves and is not detected in normal rice organs. This gene is also induced by defense signal molecules such as salicylic acid and abscisic acid, but not by jasmonic acid. Our data suggest that OsGABA-T (GABA shunt) may play a role in restricting the levels of cell death during the host–pathogen interaction.     

Purification, Characterization, and Immunological Properties of NADH-Dependent Glutamate Synthase from Rice Cell Cultures

To obtain a monospecific antibody against NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), the enzyme was purified to homogeneity from cultured rice cells (Oryza sativa) with column chromatography using Butyl Toyopearl 650M, Sephacryl S-300, Blue Sepharose CL-6B, and Butyl Toyopearl 650S. The specific activity at the final stage of the purification was 9.8 micromoles of glutamate formed per minute per milligram of protein. The yield was 6.1% and purification was 815-fold. Analysis by denaturing gel electrophoresis revealed a single polypeptide with an apparent molecular weight of 196,000, similar to the value of 194,000 estimated for the native protein. Apparent K_m values for l-glutamine, 2-oxoglutarate, and NADH were 811, 76, and 3.0 micromolar, respectively. Neither NADPH nor l-asparagine substituted for NADH and l-glutamine, respectively. The enzyme had its absorption maxima at 273, 373, and 440 nanometers with a shoulder at 475 nanometers, suggesting that the rice NADH-GOGAT is a flavoprotein. Monospecific antibody raised against NADH-GOGAT purified from the rice cells was obtained as the first instance for the enzyme in higher plants. Immunological analyses showed that the antibody for rice cell NADH-GOGAT reacted with only the enzyme in extracts from the cells. The anti-NADH-GOGAT antibody did not recognize the ferredoxin-GOGAT purified from rice leaves, and likewise the anti-rice leaf ferredoxin-GOGAT antibody did not react with the NADH-GOGAT purified from the cultured rice cells.     

Changes in Lipoxygenase Components of Rice Seedlings during Germination

Changes in lipoxygenase (LOX) activity were followed duringthe germination of rice seeds. The enzyme activity of 3-day-oldseedlings was 20 times higher than that of ungerminated seeds.Sixty per cent of the increased activity was found in shoots.The increase in LOX activity was mainly due to an increase inlipoxygenase-2 (LOX-2), a minor component in ungerminated seeds;this increase was inhibited by cycloheximide. LOX-2 was isolatedfrom the 3-day-old seedlings and compared for its enzymologicalproperties with rice lipoxygenase-3 (LOX-3), a major componentin ungerminated seeds. Both LOX-2 and LOX-3 were stable at pH5 to 8, but LOX-2 was more heatstable than LOX-3. Apparent K_mvalues of LOX-2 and LOX-3 for linoleic acid were 170 and 59µM, and those for linolenic acid were 5,300 and 88 µM,respectively. Both LOXs were inhibited by some metal ions andantioxidants. (Received February 5, 1986; Accepted May 9, 1986)     

Identification of oxylipins with antifungal activity by LC–MS/MS from the supernatant of Pseudomonas 42A2

In microorganisms hydroxy fatty acids are produced from the biotransformation of unsaturated fatty acids. Such compounds belong to a class of oxylipins which are reported to perform a variety of biological functions such as anti-inflammatory or cytotoxic activity. These compounds have been found in rice and timothy plants after being infected by specific fungus. When grown in submerged culture with linoleic acid, Pseudomonas 42A2 accumulated in the supernatant several hydroxy fatty acids. In this work LC–MS/MS has been used to elucidate the structure of the components form the organic extract: 9-hydroxy-10,12-octadecadienoic acid; 13-hydroxy-9,11-octadecadienoic acid; 7,10-dihydroxy-8E-octadecenoic acid; 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid. Antimicrobial activity against several pathogenic fungal strains is presented: MIC (μg/mL) Verticillium dhaliae, 32; Macrophonia phaesolina, 32; Arthroderma uncinatum, 32; Trycophyton mentagrophytes, 64.     

Effect of endocannabinoids on soybean lipoxygenase-1 activity

Endocannabinoids appear to be involved in a variety of physiological processes. Lipoxygenase activity has been known to be affected by unsaturated fatty acids or phenolic compounds. In this study, we examined whether endocannabinoids containing both N-acyl group and phenolic group can affect the activity of soybean lipoxygenase (LOX)-1, similar to mammalian 15-lipoxygenase in physicochemical properties. First, N-arachidonoyl dopamine and N-oleoyl dopamine were found to inhibit soybean LOX-1-catalyzed oxygenation of linoleic acid in a non-competitive manner with a K_i value of 3.7 μM and 6.2 μM, respectively. Meanwhile, other endocannabinoids failed to show a remarkable inhibition of soybean LOX-1. Separately, N-arachidonoyl dopamine and N-arachidonoyl serotonin were observed to inactivate soybean LOX-1 with K_in value of 27 μM and 24 μM, respectively, and k₃ value of 0.12 min⁻¹ and 0.35 min⁻¹, respectively. Furthermore, such an inactivation was enhanced by ascorbic acid, but suppressed by 13(S)-hydroperoxy-9,11-octadecadienoic acid. Taken together, it is proposed that endocannabinoids containing polyunsaturated acyl moiety and phenolic group may be efficient for the inhibition as well as inactivation of 15-lipoxygenase.     

Interaction Transcriptome Analysis Identifies Magnaporthe oryzae BAS1-4 as Biotrophy-Associated Secreted Proteins in Rice Blast Disease

Biotrophic invasive hyphae (IH) of the blast fungus Magnaporthe oryzae secrete effectors to alter host defenses and cellular processes as they successively invade living rice (Oryza sativa) cells. However, few blast effectors have been identified. Indeed, understanding fungal and rice genes contributing to biotrophic invasion has been difficult because so few plant cells have encountered IH at the earliest infection stages. We developed a robust procedure for isolating infected-rice sheath RNAs in which ∼20% of the RNA originated from IH in first-invaded cells. We analyzed these IH RNAs relative to control mycelial RNAs using M. oryzae oligoarrays. With a 10-fold differential expression threshold, we identified known effector PWL2 and 58 candidate effectors. Four of these candidates were confirmed to be fungal biotrophy-associated secreted (BAS) proteins. Fluorescently labeled BAS proteins were secreted into rice cells in distinct patterns in compatible, but not in incompatible, interactions. BAS1 and BAS2 proteins preferentially accumulated in biotrophic interfacial complexes along with known avirulence effectors, BAS3 showed additional localization near cell wall crossing points, and BAS4 uniformly outlined growing IH. Analysis of the same infected-tissue RNAs with rice oligoarrays identified putative effector-induced rice susceptibility genes, which are highly enriched for sensor-transduction components rather than typically identified defense response genes.