Sesterterpene sulfates as isocitrate lyase inhibitors from tropical sponge Hippospongia sp

Two sesterterpene sulfates (1-2) were isolated from tropical sponge Hippospongia sp. and their inhibitory activities against isocitrate lyase (ICL) from the rice blast fungus Mgnaporthe grisea were evaluated. Compound 3 was obtained by hydrolysis of compound 1. Compounds 1 and 3 were found to be potent ICL inhibitors, which inhibited appressorium formation and C(2) carbon utilization in M. grisea. Our results suggest that ICL plays crucial role in appressorium formation of M. grisea and is a new target for the protection of rice blast disease.     

Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea

We describe the isolation and characterization of ICL1 from the rice blast fungus Magnaporthe grisea, a gene that encodes isocitrate lyase, one of the principal enzymes of the glyoxylate cycle. ICL1 shows elevated expression during development of infection structures and cuticle penetration, and a targeted gene replacement showed that the gene is required for full virulence by M. grisea. In particular, we found that the prepenetration stage of development, before entry into plant tissue, is affected by loss of the glyoxylate cycle. There is a delay in germination, infection-related development and cuticle penetration in Delta icl1 mutants. Recent reports have shown the importance of the glyoxylate cycle in the virulence of the human pathogenic fungus Candida albicans and the bacterial pathogen Mycobacterium tuberculosis. Our results indicate that the glyoxylate cycle is also important in this plant pathogenic fungus, demonstrating the widespread utility of the pathway in microbial pathogenesis.     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.     

Overexpression of a rice diacylglycerol kinase gene OsBIDK1 enhances disease resistance in transgenic tobacco

A rice diacylglycerol kinase (DGK) gene, OsBIDK1, which encodes a 499-amino acid protein, was cloned and characterized. OsBIDK1 contains a conserved DGK domain, consisting of a diacylglycerol kinase catalytic subdomain and a diacylglycerol kinase accessory subdomain. Expression of OsBIDK1 in rice seedlings was induced by treatment with benzothiadiazole (BTH), a chemical activator of the plant defense response, and by infection with Magnaporthe grisea, causal agent of blast disease. In BTH-treated rice seedlings, expression of OsBIDK1 was induced earlier and at a higher level than in water-treated control seedlings after inoculation with M. grisea. Transgenic tobacco plants that constitutively express the OsBIDK1 gene were generated and disease resistance assays showed that overexpression of OsBIDK1 in transgenic tobacco plants resulted in enhanced resistance against infection by tobacco mosaic virus and Phytophthora parasitica var. nicotianae. These results suggest that OsBIDK1 may play a role in disease resistance responses.     

转拟南芥AtNPR1基因增强水稻对水稻白叶枯病和稻瘟病的抗性

AtNPR1基因是拟南芥系统获得抗性的一个重要调节基因,在拟南芥中过量表达AtNPR1基因能使拟南芥对细菌和真菌的抗性同时增强.为了研究在水稻中过量表达AtNPR1基因对水稻抗病性的影响,将该基因转入到广西主栽籼稻恢复系品种桂99中.经PCR验证得到了79株转基因植株,DNA斑点杂交表明ATNPR1基因已经整合到桂99染色体DNA中.Northern杂交和RT-PCR分析表明,AtNPR1基因在桂99中已经表达;同时还检测了转基因植株对水稻白叶枯病和稻瘟病的抗性,结果表明转基因植株对该两种病害的抗性均显著增强.     

Gene-Expression Patterns and Levels of Jasmonic Acid in Rice Treated with the Resistance Inducer 2,6-Dichloroisonicotinic Acid

Acquired disease resistance can be induced in rice (Oryza sativa) by a number of synthetic or natural compounds, but the molecular mechanisms behind the phenomenon are poorly understood. One of the synthetic inducers of resistance, 2,6-dichloroisonicotinic acid (INA), efficiently protected rice leaves from infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr. A comparison of gene-expression patterns in plants treated with INA versus plants inoculated with the compatible pathogen M. grisea or the incompatible pathogen Pseudomonas syringae pv syringae revealed only a marginal overlap: 6 gene products, including pathogenesis-related proteins (PR1-PR9), accumulated in both INA-treated and pathogen-attacked leaves, whereas 26 other gene products accumulated only in INA-treated or only in pathogen-attacked leaves. Lipoxygenase enzyme activity and levels of nonconjugated jasmonic acid (JA) were enhanced in leaves of plants treated with a high dose of INA (100 ppm). Exogenously applied JA enhanced the gene induction and plant protection caused by lower doses of INA (0.1 to 10 ppm) that by themselves did not give rise to enhanced levels of endogenous (-)-JA. These data suggest that INA, aside from activating a pathogen-induced signaling pathway, also induces events that are not related to pathogenesis. JA acts as an enhancer of both types of INA-induced reactions in rice.     

Overexpression of Bax inhibitor suppresses the fungal elicitor-induced cell death in rice (Oryza sativa L) cells

Treatment of suspension-cultured cells of rice (Oryza sativa L.) with cell wall extract of rice blast fungus (Magnaporthe grisea) elicits a rapid generation of H2O2, alkalinization of culture medium, and eventual cell death. To elucidate genes involved in these processes, we exploited SAGE (Serial Analysis of Gene Expression) technique for the molecular analysis of cell death in suspension-cultured cells treated with the elicitor. Among the downregulated genes in the elicitor-treated cells, a BI-1 gene coding for Bax inhibitor was identified. Transgenic rice cells overexpressing Arabidopsis BI-1 gene showed sustainable cell survival when challenged with M. grisea elicitor. Thus, the plant Bax inhibitor plays a functional role in regulating cell death in the rice cell culture system.     

Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of omega-3 fatty acid desaturases

Linolenic acid (18:3) is the most abundant fatty acid in plant membrane lipids and is a source for various oxidized metabolites, called oxylipins. 18:3 and oxylipins play important roles in the induction of defense responses to pathogen infection and wound stress in Arabidopsis. However, in rice, endogenous roles for 18:3 and oxylipins in disease resistance have not been confirmed. We generated 18:3-deficient transgenic rice plants (F78Ri) with co-suppression of two omega-3 fatty acid desaturases, OsFAD7 and OsFAD8. that synthesize 18:3. The F78Ri plants showed enhanced resistance to the phytopathogenic fungus Magnaporthe grisea. A typical 18:3-derived oxylipin, jasmonic acid (JA), acts as a signaling molecule in defense responses to fungal infection in Arabidopsis. However, in F78Ri plants, the expression of JA-responsive pathogenesis-related genes, PBZ1 and PR1b, was induced after inoculation with M. grisea, although the JA-mediated wound response was suppressed. Furthermore, the application of JA methyl ester had no significant effect on the enhanced resistance in F78Ri plants. Taken together, our results indicate that, although suppression of fatty acid desaturases involves the concerted action of varied oxylipins via diverse metabolic pathways, 18:3 or 18:3-derived oxylipins, except for JA, may contribute to signaling on defense responses of rice to M. grisea infection.     

稻瘟病菌致病性的分子遗传学研究进展

由稻瘟病菌引起的稻瘟病是水稻生产上危害最为严重的真菌病害,对世界粮食生产造成巨大损失。稻瘟病菌成功侵染寄主包括分生孢子萌发、附着胞形成、侵染钉分化和侵染性菌丝扩展等一系列错综复杂的过程,其中每一环节都是由特定基因控制的。稻瘟病菌与水稻的互作符合经典的基因对基因学说,二者的不亲和互作是无毒基因与抗病基因相互作用的结果。近几十年来,世界各国的科学家对稻瘟病菌致病性的生物学及其遗传的分子机制进行了深入的研究。文章就稻瘟病菌致病性的分子遗传学及其遗传变异机制的研究进行了综述,同时对功能基因的研究方法进行了总结。     

<Emphasis Type="Italic">OsBIMK1</Emphasis>, a rice MAP kinase gene involved in disease resistance responses

The activation of mitogen-activated protein kinases (MAPKs) has been previously implicated in signal transduction during plant responses to pathogen attack as well as to various environmental stresses. We have isolated from rice a new MAPK cDNA, OsBIMK1 ( O ryza s ativa L. BTH-induced MAPK 1), which encodes a 369-amino-acid protein with moderate to high nucleotide sequence similarity to previously reported plant MAPK genes. OsBIMK1 contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY. We analyzed in detail the expression of OsBIMK1 upon treatment with various chemical and biological inducers of resistance responses in rice and in both incompatible and compatible interactions between rice and Magnaporthe grisea. Expression of OsBIMK1 was activated rapidly upon treatment with benzothiadiazole (BTH) as well as with dichloroisonicotinic acid, probenazole, jasmonic acid and its methyl ester, Pseudomonas syringae pv. syringae, or wounding. Expression of OsBIMK1 was induced rapidly during the first 36 h after inoculation with M. grisea in BTH-treated rice seedlings and in an incompatible interaction between M. grisea and a blast-resistant rice genotype. BTH treatment induced a systemic activation of OsBIMK1 expression. These results suggest that OsBIMK1 plays an important role in rice disease resistance.     

MGOS: A resource for studying Magnaporthe grisea and Oryza sativa interactions

The MGOS (Magnaporthe grisea Oryza sativa) web-based database contains data from Oryza sativa and Magnaporthe grisea interaction experiments in which M. grisea is the fungal pathogen that causes the rice blast disease. In order to study the interactions, a consortium of fungal and rice geneticists was formed to construct a comprehensive set of experiments that would elucidate information about the gene expression of both rice and M. grisea during the infection cycle. These experiments included constructing and sequencing cDNA and robust long-serial analysis gene expression libraries from both host and pathogen during different stages of infection in both resistant and susceptible interactions, generating >50,000 M. grisea mutants and applying them to susceptible rice strains to test for pathogenicity, and constructing a dual O. sativa-M. grisea microarray. MGOS was developed as a central web-based repository for all the experimental data along with the rice and M. grisea genomic sequence. Community-based annotation is available for the M. grisea genes to aid in the study of the interactions.     

Effect of a Photo-synthetic Inhibitor on Tryptamine Pathway-mediated Sekiguchi Lesion Formation in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

A lesion-mimic mutant of rice (cv. Sekiguchi-asahi) showed enhanced resistance to Magnaporthe grisea infection, thereby inducing Sekiguchi lesion ( sl ) formation and tryptamine accumulation under light. Both Sekiguchi lesion formation and tryptamine accumulation in leaves infected with M. grisea were inhibited by pretreatment with the photosynthetic inhibitor, 3-(3, 4-Dichlorophenyl)-1,1-dimethylurea (DCMU), which suppressed the gene expression of tryptophan decarboxylase ( TDC ), monoamine oxidase activity, H₂O₂ generation and DNA fragmentation. Catalase activity was inhibited by M. grisea infection under light, but magnitude of the inhibition was reduced in leaves pretreated with DCMU. Furthermore, tryptophan accumulated in M. grisea- infected leaves under light but not in DCMU-pretreated ones. Interestingly, such DCMU inhibition was reduced in the presence of tryptophan. Our studies suggest that chloroplasts function as the inhibitor of anti-oxidant system such as catalase activity and the supplier of a precursor of tryptamine and tryptophan in the sl mutant infected with M. grisea .     

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.     

Wheat puroindolines enhance fungal disease resistance in transgenic rice

Antimicrobial peptides play a role in the immune systems of animals and plants by limiting pathogen infection and growth. The puroindolines, endosperm-specific proteins involved in wheat seed hardness, are small proteins reported to have in vitro antimicrobial properties. Rice, the most widely used cereal crop worldwide, normally does not contain puroindolines. Transgenic rice plants that constitutively express the puroindoline genes pinA and/or pinB throughout the plants were produced. PIN extracts of leaves from the transgenic plants reduced in vitro growth of Magnaporthe grisea and Rhizoctonia solani, two major fungal pathogens of rice, by 35 to 50%. Transgenic rice expressing pinA and/or pinB showed significantly increased tolerance to M. grisea (rice blast), with a 29 to 54% reduction in symptoms, and R. solani (sheath blight), with an 11 to 22% reduction in symptoms. Puroindolines are effective in vivo in antifungal proteins and could be valuable new tools in the control of a wide range of fungal pathogens of crop plants.     

Disruption of the alternative oxidase gene in Magnaporthe grisea and its impact on host infection

Plants and numerous fungi including Magnaporthe grisea protect mitochondria from interference by respiration inhibitors by expressing alternative oxidase, the enzymatic core of alternative respiration. The alternative oxidase gene AOXMg of M. grisea was disrupted. Several lines of evidence suggested that the disruption of AOXMg was sufficient to completely curb the expression of alternative respiration. In the infection of barley leaves, several AOXMg-minus and, thus, alternative respiration-deficient mutants of M. grisea retained their pathogenicity without significant impairment of virulence. However, differences between the wild-type strain and an AOXMg-minus mutant were apparent under oxidative stress conditions generated by the treatment of infected barley leaves with the commercial respiration inhibitor azoxystrobin. Symptom development was effectively suppressed on leaves infected with the alternative respiration-deficient mutant, while lesions on leaves infected with the wild-type strain continued to develop at much higher inhibitor doses. However, respective lesions rarely developed to the stage of full maturity. The results did not conform to a previous model implying that expression of alternative respiration is silenced during pathogenesis by the presence of constitutive plant antioxidants. Rather, alternative respiration provided protection from azoxystrobin during both saprophytic and infectious stages of the pathogen. The nature of similar oxidative stress conditions in the ecology of M. grisea remains an open question.