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.     

Mnh6, a nonhistone protein, is required for fungal development and pathogenicity of Magnaporthe grisea

Mnh6, a nonhistone protein containing an HMG1 box, was isolated from the rice blast fungus, Magnaporthe grisea. In the current study, we utilized an MNH6-deletion mutant to investigate the role of Mnh6 in the disease cycle of M. grisea. The Deltamnh6 mutant exhibited pleiotropic effects on fungal morphogenesis, including reduction in mycelial growth, conidiation, appressorium development, plant penetration, and infectious growth in host cells. Furthermore, Deltamnh6 mutant had greatly reduced pathogenicity on barley and rice compared to the wild-type. The reintroduction of an intact copy of MNH6 into the Deltamnh6 mutant restored morphological features and pathogenicity, suggesting that Mnh6 is required for fungal development, effective pathogenicity, and completion of the disease cycle of M. grisea.     

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     

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.     

Detection of Magnaporthe grisea in infested rice seeds using polymerase chain reaction

AIM: To develop a diagnostic assay based on polymerase chain reaction for the detection of Magnaporthe grisea from infested rice seeds. METHODS AND RESULTS: Primers were designed based on the nucleotide sequence of the mif 23, an infection-specific gene of M. grisea. The primers amplified target DNA from genetically and geographically diverse isolates of the pathogen. The lowest concentration of template DNA that led to amplification was 20 rhog. No PCR product was detected when DNA from other fungi was used, indicating the specificity of the primers. With this PCR based seed assay, M. grisea was detected in rice seedlots with infestation rates as low as 0.2%. CONCLUSION: The PCR detection of M. grisea is simple, rapid, specific, sensitive and suitable for the routine detection of the pathogen in infested seeds. SIGNIFICANCE AND IMPACT OF THE STUDY: Introduction of the blast fungus into new areas where it has not been previously recorded could be avoided by the detection of infested seedlots. A PCR-based seed assay could facilitate risk assessment of naturally infested rice seeds; help design management programs and optimize fungicide use.     

Infection-related development in the rice blast fungus Magnaporthe grisea

Recent developments have been made in the identification of signal transduction pathways and gene products involved in the infection-related development of the rice blast fungus, Magnaporthe grisea. It has been established that cAMP-dependent and MAP kinase-mediated signaling are both critical for appressorium morphogenesis and function. These signaling pathways may act downstream of hydrophobin-mediated surface sensing by the growing germ tube. Several genes have been identified that are required for invasive growth of M. grisea including genes that allow adaptation of fungal metabolism to growth within plant tissues.     

Effects of neolignans from the stem bark of Magnolia obovata on plant pathogenic fungi

Aims:  To characterize antifungal principles from the methanol extract of Magnolia obovata and to evaluate their antifungal activities against various plant pathogenic fungi.
Methods and Results:  Four neolignans were isolated from stem bark of M. obovata as antifungal principles and identified as magnolol, honokiol, 4-methoxyhonokiol and obovatol. In mycelial growth inhibition assay, both magnolol and honokiol displayed more potent antifungal activity than 4-methoxyhonokiol and obovatol. Both magnolol and honokiol showed similar in vivo antifungal spectrum against seven plant diseases tested; both compounds effectively suppressed the development of rice blast, tomato late blight, wheat leaf rust and red pepper anthracnose. 4-Methoxyhonokiol and obovatol were highly active to only rice blast and wheat leaf rust respectively.
Conclusions:  The extract of M. obovata and four neolignans had potent in vivo antifungal activities against plant pathogenic fungi.
Significance and Impact of the Study:  Neolignans from Magnolia spp. can be used and suggested as a novel antifungal lead compound for the development of new fungicide and directly as a natural fungicide for the control of plant diseases such as rice blast and wheat leaf rust.     

Antifungal activity of rice Pex5p, a receptor for peroxisomal matrix proteins

We have purified a novel antifungal protein from blast fungus (Magnaporthe grisea)-treated rice leaves using consecutive chromatographies on CM-Sepharose ion-change, Affi-gel blue, and HPLC gel filtration columns. We determined the N-terminal peptide sequence of the purified protein and subjected it to the NCBI/BLAST database and found the protein to be a partial fragment of the peroxisomal receptor protein in rice (OsPex5p). After cloning two cDNAs encoding OsPEX5L and OsPEX5S genes that are splice variants of OsPEX5 from a rice leaf cDNA library, we investigated their antifungal properties. The recombinant proteins were expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the OsPEX5L gene was induced by diverse external stresses such as rice blast fungus, fungal elicitor, and other signaling molecules including H(2)O(2), abscisic acid, jasmonic acid, and salicylic acid. These results suggest that the peroxisomal receptor protein, OsPex5p, plays a critical role in the rice defense system against diverse external stresses including fungal pathogenic attack.     

MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization

Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re-introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infection-related fungal development, and is required for pathogenicity of M. grisea.     

Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.     

RAR1, ROR1, and the actin cytoskeleton contribute to basal resistance to Magnaporthe grisea in barley

The fungus Magnaporthe grisea, the causal agent of rice blast disease, is a major pathogen of rice and is capable of producing epidemics on other cultivated cereals, including barley (Hordeum vulgare). We explored the requirements for basal resistance of barley against a compatible M. grisea isolate using both genetic and chemical approaches. Mutants of the RAR1 gene required for the function of major resistance gene-mediated resistance and mutants of the ROR1 and ROR2 genes required for full expression of cell-wall-penetration resistance against powdery mildew pathogens were examined for macroscopic and microscopic alterations in M. grisea growth and symptoms. RAR1 contributed to resistance in epidermis and mesophyll at different stages of fungal infection dependent on the MLO/mlo-5 status. Whereas no ROR2 effect was detected, ROR1 was found to contribute to cell-wall-penetration resistance, at least in the epidermis. Application of the actin agonist cytochalasin E promoted cell wall penetration by M. grisea in a dose-dependent manner, demonstrating an involvement of the actin cytoskeleton in penetration resistance.     

Cellular differentiation and host invasion by the rice blast fungus Magnaporthe grisea

This review describes current advances in understanding the biology of plant infection by the rice blast fungus Magnaporthe grisea. Development of the specialized infection structure, the appressorium, in M. grisea has recently been shown to be controlled by cell cycle progression and initiation of autophagic, programmed cell death in the fungal spore. Re-cycling of the contents of the fungal spore and peroxisomal fatty acid beta-oxidation are therefore important processes for appressorium function. Following entry to the host plant, new evidence suggests that M. grisea grows biotrophically within rice cells, bounded by the plant plasmalemma, and the fungus moves from cell-to-cell by means of plasmodesmata. Biotrophic proliferation of the fungus is likely to require secretion of effector proteins and suppression of host defences. Consistent with this, a component of the polarized exocytosis machinery of M. grisea is necessary for pathogenicity and also for induction of host defences in an incompatible interaction. Large-scale insertional mutagenesis is now allowing the rapid analysis of gene function in M. grisea, heralding a new approach to the study of this important fungal pathogen.     

转基因改良水稻抗稻瘟病的策略及其进展

稻瘟病是由子囊菌引起的广泛发生在世界各水稻产区的主要真菌病害。由于病原菌致病性的高度分化,使得对稻瘟病很难控制和防治。长期实践证明,培育抗病品种是稻瘟病抗病育种的主要目标。随着基因工程的发展,利用转基因技术导入外源基因改良稻瘟病抗性已成为一条新途径。现有研究表明,通过某些抗病基因、抗真菌蛋白基因、杀菌肽基因的克隆和转育,可以培育出获得对稻瘟病广谱抗性的水稻品种(系)。     

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).     

Ascherxanthone B from Aschersonia luteola, a new antifungal compound active against rice blast pathogen Magnaporthe grisea

Aims:  Fungicide resistance now exists in the rice blast fungus, Magnaporthe grisea , necessitating the need for new active agents. Fungi isolated from habitats in Thailand were screened with reference to this problem.
Methods and Results:  A new, reliable in vitro screening system based on a microdilution plate format was set up using a virulent strain of M. grisea THL 16. Culture broth extracts from approximately 800 fungal strains were investigated, one of these, Aschersonia luteola BCC 8774, was found to produce an active fungicidal compound, ascherxanthone B, with an IC₉₀ value of 0·58 μg ml⁻¹ (0·95 μmol l⁻¹). An in vivo study of anti-blast efficacy of ascherxanthone B showed a positive effect in disease reduction.
Conclusions:  Previous report has shown that a species of Aschersonia produces ascherxanthone A. Research on the species, A. luteola BCC 8774, led to the discovery of related novel metabolite, ascherxanthone B with fungicidal properties.
Significance and Impact of the Study:  Current methods of rice blast control seem to fail leading to increase in crop losses. Our discovery of the anti-blast activity shown by ascherxanthone B is the first step in the development of a potentially novel fungicide.     

Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis

We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea.     

A fungal metallothionein is required for pathogenicity of Magnaporthe grisea

The causal agent of rice blast disease, the ascomycete fungus Magnaporthe grisea, infects rice (Oryza sativa) plants by means of specialized infection structures called appressoria, which are formed on the leaf surface and mechanically rupture the cuticle. We have identified a gene, Magnaporthe metallothionein 1 (MMT1), which is highly expressed throughout growth and development by M. grisea and encodes an unusual 22-amino acid metallothionein-like protein containing only six Cys residues. The MMT1-encoded protein shows a very high affinity for zinc and can act as a powerful antioxidant. Targeted gene disruption of MMT1 produced mutants that show accelerated hyphal growth rates and poor sporulation but had no effect on metal tolerance. Mmt1 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. Mmt1 appears to be distributed in the inner side of the cell wall of the fungus. These findings indicate that Mmt1-like metallothioneins may play a novel role in fungal cell wall biochemistry that is required for fungal virulence.     

Antifungal activity against plant pathogenic fungi of chaetoviridins isolated from Chaetomium globosum

The Chaetomium globosum strain F0142, which was isolated from barnyard grass, showed potent disease control efficacy against rice blast (Magnaporthe grisea) and wheat leaf rust (Puccinia recondita). Two antifungal substances were purified from broth from this organism and identified as chaetoviridins A and B. Chaetoviridin A exhibited higher antifungal activity than chaetoviridin B against plant pathogenic fungi both in vitro and in vivo. Treatment with chaetoviridin A at 62.5 microg/mL suppressed the development of rice blast and wheat leaf rust by over 80%. The molecule also exhibited moderate control of tomato late blight, resulting in 50% control following the application of 125 microg/mL chaetoviridin A.     

2-epi-botcinin A and 3-O-acetylbotcineric acid from Botrytis cinerea

Two metabolites, 2-epi-botcinin A and 3-O-acetylbotcineric acid, were isolated from Botrytis cinerea (AEM211). The former compound was new, and the latter was known but structurally revised by us. In a test for antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease, 2-epi-botcinin A was 8 times less active than botcinin A (MIC 100 microM), and the MIC value for 3-O-acetylbotcineric acid being 100 microM.