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.     

A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea

Many fungal pathogens invade plants by means of specialized infection structures called appressoria. In the rice (Oryza sativa) blast fungus Magnaporthe grisea, the pathogenicity mitogen-activated protein (MAP) kinase1 (PMK1) kinase is essential for appressorium formation and invasive growth. In this study, we functionally characterized the MST7 and MST11 genes of M. grisea that are homologous with the yeast MAP kinase kinase STE7 and MAP kinase kinase kinase STE11. Similar to the pmk1 mutant, the mst7 and mst11 deletion mutants were nonpathogenic and failed to form appressoria. When a dominant MST7 allele with S212D and T216E mutations was introduced into the mst7 or mst11 mutant, appressorium formation was restored in the resulting transformants. PMK1 phosphorylation also was detected in the vegetative hyphae and appressoria of transformants expressing the MST7(S212D T216E) allele. However, appressoria formed by these transformants failed to penetrate and infect rice leaves, indicating that constitutively active MST7 only partially rescued the defects of the mst7 and mst11 mutants. The intracellular cAMP level was reduced in transformants expressing the MST7(S212D T216E) allele. We also generated MST11 mutant alleles with the sterile alpha motif (SAM) and Ras-association (RA) domains deleted. Phenotype characterizations of the resulting transformants indicate that the SAM domain but not the RA domain is essential for the function of MST11. These data indicate that MST11, MST7, and PMK1 function as a MAP kinase cascade regulating infection-related morphogenesis in M. grisea. Although no direct interaction was detected between PMK1 and MST7 or MST11 in yeast two-hybrid assays, a homolog of yeast STE50 in M. grisea directly interacted with both MST7 and MST11 and may function as the adaptor protein for the MST11-MST7-PMK1 cascade.     

Ferricrocin synthesis in Magnaporthe grisea and its role in pathogenicity in rice

Iron is an essential element for the growth of nearly all organisms. In order to overcome the problem of its low bioavailability, microorganisms (including fungi) secrete siderophores, high-affinity iron chelators. As the acquisition of iron is also a key step in infection processes, siderophores have been considered as potential virulence factors in several host–pathogen interactions. Most fungi produce siderophores of the hydroxamate-type, which are synthesized by non-ribosomal peptide synthetases (NRPSs). Magnaporthe grisea , the causal agent of rice blast disease, produces ferricrocin as intracellular storage siderophore and excretes coprogens. In the M. grisea genome we identified SSM1 , an NRPS gene, and a gene encoding an l -ornithine N5-monooxygenase ( OMO1 ) that is clustered with SSM1 and responsible for catalysing the first step in siderophore biosynthesis, the N⁵ hydroxylation of ornithine. Disruption of SSM1 confirmed that the gene encodes ferricrocin synthetase. Pathogenicity of these mutants towards rice was reduced, suggesting a role of this siderophore in pathogenicity of M. grisea .     

Two PAK kinase genes, CHM1 and MST20, have distinct functions in Magnaporthe grisea

In the rice blast fungus Magnaporthe grisea, the Pmk1 mitogen-activated protein (MAP) kinase is essential for appressorium formation and infectious growth. PMK1 is homologous to yeast Fus3 and Kss1 MAP kinases that are known to be regulated by the Ste20 PAK kinase for activating the pheromone response and filamentation pathways. In this study, we isolated and characterized two PAK genes, CHM1 and MST20, in M. grisea. Mutants disrupted in MST20 were reduced in aerial hyphae growth and conidiation, but normal in growth rate, appressorium formation, penetration, and plant infection. In chm1 deletion mutants, growth, conidiation, and appressorium formation were reduced significantly. Even though appressoria formed by chm1 mutants were defective in penetration, chm1 mutants were able to grow invasively on rice leaves and colonize through wounds. The chm1 mutants were altered in conidiogenesis and produced conidia with abnormal morphology. Hyphae of chm1 mutants had normal septation, but the length of hyphal compartments was reduced. On nutritionally poor oatmeal agar, chm1 mutants were unstable and produced sectors that differed from original chm1 mutants in growth rate, conidiation, or colony morphology. However, none of the monoconidial cultures derived from these spontaneous sectors were normal in appressorial penetration and fungal pathogenesis. These data suggest that MST20 is dispensable for plant infection in M. grisea, but CHM1 plays a critical role in appressorium formation and penetration. Both mst20 and chm1 deletion mutants were phenotypically different from the pmk1 mutant that is defective in appressorium formation and infectious hyphae growth. It is likely that MST20 and CHM1 individually play no critical role in activating the PMK1 MAP kinase pathway during appressorium formation and infectious hyphae growth. However, CHM1 appears to be essential for appressorial penetration and CHM1 and MST20 may have redundant functions in M. grisea.     

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.     

MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea

Magnaporthe grisea produces an infection structure called an appressorium, which is used to breach the plant cuticle by mechanical force. Appressoria generate hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control and biochemical mechanism for turgor generation, we assayed glycerol biosynthetic enzymes during appressorium development, and the movement of storage reserves was monitored in developmental mutants. Enzymatic activities for glycerol generation from carbohydrate sources were present in appressoria but did not increase during development. In contrast, triacylglycerol lipase activity increased during appressorium maturation. Rapid glycogen degradation occurred during conidial germination, followed by accumulation in incipient appressoria and dissolution before turgor generation. Lipid droplets also moved to the incipient appressorium and coalesced into a central vacuole before degrading at the onset of turgor generation. Glycogen and lipid mobilization did not occur in a Deltapmk1 mutant, which lacked the mitogen-activated protein kinase (MAPK) required for appressorium differentiation, and was retarded markedly in a DeltacpkA mutant, which lacks the catalytic subunit of cAMP-dependent protein kinase A (PKA). Glycogen and lipid degradation were very rapid in a Deltamac1 sum1-99 mutant, which carries a mutation in the regulatory subunit of PKA, occurring before appressorium morphogenesis was complete. Mass transfer of storage carbohydrate and lipid reserves to the appressorium therefore occurs under control of the PMK1 MAPK pathway. Turgor generation then proceeds by compartmentalization and rapid degradation of lipid and glycogen reserves under control of the CPKA/SUM1-encoded PKA holoenzyme.     

水稻受稻瘟菌侵染后发病初期的细胞学反应

采用致病性不同的3个稻瘟菌株接种水稻IR64品种。结果显示：在抗性反应中,病菌接种后水稻细胞中形成原生质颗粒,逐渐向病菌侵入部位聚集;原生质浓缩及沉积的形成、细胞坏死与病菌侵染菌丝的受抑制在时间和空间上是一致的。在中度抗性反应中,原生质颗粒化的时间延迟。在感病反应中,尚未观察到水稻细胞质浓缩现象。病菌侵染后寄主细胞在蓝光激发下的自发荧光表明,在寄主细胞中有多酚类物质的产生和积累。抗性反应中稻瘟菌接种后多酚类物质产生早,荧光范围大而强;中度抗性反应中,多酚类物质产生迟,荧光范围小而弱;而在感病反应中,难以观察到寄主细胞的自发荧光。胼胝质、磷脂酶Dγ(phospholipase Dγ,PLDγ)产生和积累的趋势与多酚类积累的情况大致是相似的。不同互作类型寄主细胞中多酚类物质、胼胝质和PLDγ产生和积累的差异表明,这些物质在水稻的抗病性中起了重要作用。     

Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection

Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses. In Arabidopsis, 20 MAPKs have been identified and divided into four major groups. In rice, a monocot model and economically important cereal crop, only five MAPKs were characterized, including three related to the host defense response. In this study, we have identified 17 members of the rice MAPK gene (OsMPK) family through an in silico search of rice genome databases. Based on the phylogenetic analysis and pairwise comparison of Arabidopsis and rice MAPKs, we propose that MAPKs can be divided into six groups. Interestingly, the rice genome contains many more MAPKs with the TDY phosphorylation site (11 members) than with the TEY motif (six members). In contrast, the Arabidopsis genome contains more MAPKs with the TEY motif (12 members) than with the TDY motif (eight members). Upon inoculation with the blast fungus (Magnaporthe grisea), nine of 17 OsMPK genes were found to be induced at the mRNA level during either early, late, or both stages of infection. Four of the M. grisea-induced OsMPK genes were associated with host-cell death in the lesion-mimic rice mutant, and eight of them were differentially induced in response to defense signal molecules such as jasmonic acid, salicylic acid, abscisic acid, and ethylene. The genome-wide expression analysis suggests that about half of the rice MAPK genes are associated with pathogen infection and host defense response.     

稻瘟菌Ⅰ型烯醇化酶基因全长cDNA的电子克隆

利用电子克隆技术从稻瘟菌中克隆到一个新的Ⅰ型烯醇化酶全长cDNA,暂命为MgEno-1。MgEno-1全长1571核薯酸,其预测的ORF为1317核苷酸,共编码438个氨基酸。起始密码子ATG位于第53位,终止密码子TAA位于第1369位。序列分析表明该烯醇化酶与丝状真菌中已报道的其它烯醇化酶高度同源,且长度一致,这暗示烯醇化酶基因进化上高度保守,甚至有可能像18SrRNA一样可作为进化尺度。这将是第一个用电子克隆技术从稻瘟菌中克隆到的基因。     

Biomechanical model for appressorial design in Magnaporthe grisea

The fungus Magnaporthe grisea, commonly referred to as the rice blast fungus, is responsible for destroying from 10% to 30% of the world's rice crop each year. The fungus attaches to the rice leaf and forms a dome-shaped structure, the appressorium, in which enormous pressures are generated that are used to blast a penetration peg through the rice cell walls and infect the plant. We develop a model of the appressorial design in terms of a bioelastic shell that can explain the shape of the appressorium, and its ability to maintain that shape under the enormous increases in turgor pressure that can occur during the penetration phase.     

Regions of microsynteny in Magnaporthe grisea and Neurospora crassa.

A bacterial artificial chromosome (BAC) clone containing 110,467 bp of genomic DNA from Magnaporthe grisea was sequenced, annotated, and compared to the genomes of Neurospora crassa, Candida albicans, and Saccharomyces cerevisiae. Twenty-six open reading frames (ORFs), involved in multiple biochemical pathways, were identified in the BAC sequence. A region of 53 kb, containing 18 of the 26 ORFs, was found to be syntenic to a portion of the N. crassa genome. Subregions of complete colinearity as well as interrupted colinearity were present. No synteny was evident with either C. albicans or S. cerevisiae. The identification of syntenic regions containing highly conserved genes across two genera that have been evolutionarily separated for approximately 200 million years elicits many biological questions as to the function and identity of these genes.     

Molecular structure of rDNA repeat unit in Magnaporthe grisea

The 8-kb repeat unit of M. grisea rRNA-encoding DNA (rDNA) was cloned as three subclones, pM50, pM21, and pM86. Nucleotide sequencing of these subclones uncovered the structure of an rDNA repeat unit similar to those of other ascomycetes. The intergenic spacer (IGS) of the rDNA cistron contained a repetitive (R) region, which was rich in two kinds of short tandemly repeated elements.     

Cellular localization of MAGI-1 caspase cleavage products and their role in apoptosis

MAGI-1 is a membrane-associated guanylate kinase (MAGUK) protein present at adherent and tight junctions, where it acts as a structural and signaling scaffold. During apoptosis, MAGI-1 is cleaved by caspases at Asp761 into N- and C-terminal cleavage products, allowing further dismantling of the cell junctions, one of the key features of apoptosis. Here, we investigated the cellular distribution and possible proapototic role of MAGI-1 caspase cleavage products. Full-length MAGI-1 exhibited submembrane localization, while the N-terminal caspase cleavage product of MAGI-1 is translocated to the cytosol and the C-terminal caspase cleavage product accumulates in the nucleus. When overexpressed in MDCK cells, both N- and C-terminal MAGI-1 caspase cleavage products exhibited minor proapoptotic activity, although their role in apoptosis is probably more passive.     

A novel gene MGA1 is required for appressorium formation in Magnaporthe grisea

Insertional mutagenesis is an effective way to study the infection mechanism of fungal pathogens. In an attempt to identify the genes involved in appressorium formation from Magnaporthe grisea, we carried out Agrobacterium tumefaciens mediated transformation (ATMT) of the fungus. Analysis of the region flanking the T-DNA integration site in one of the appressorium mutants showed insertion in a gene coding a 78 amino acid protein (MGA1), showing no significant homology to any of the known proteins. The mutant mga1 caused neither foliar nor root infection. Complementation of the mutated gene with the full length wild type gene restored appressorium formation as well as rice infection demonstrating the involvement of this gene in pathogenicity of M. grisea. In an indirect immunolocalisation assay, the MGA1 expression was seen predominantly in germ tube and appressoria. The mutant was impaired in glycogen and lipid mobilization required for appressorium formation. The glycerol content in the mycelia of the mutant under hyperosmotic stress conditions was less as compared to wild type and was thus unable to tolerate the hyperosmotic stress induced by sorbitol. We hypothesize that MGA1 plays a crucial role in signal transduction leading to the metabolism of glycogen and lipids, which is a part of appressorium differentiation process.     

Disruption of a Magnaporthe grisea cutinase gene.

Summary Using a one-step strategy to disrupt CUT1, a gene for cutinase, cut1^– mutants were generated in two strains of Magnaporthe grisea. One strain, pathogenic on weeping lovegrass and barley and containing the arg3–12 mutation, was transformed with a disruption vector in which the Aspergillus nidulans ArgB⁺ gene was inserted into CUT1. Prototrophic transformants were screened by Southern hybridization, and 3 of 53 tested contained a disrupted CUT1 gene (cut1 : : ArgB⁺). A second strain, pathogenic on rice, was transformed with a disruption vector in which a gene for hyg B resistance was inserted into CUT1. Two of the 57 transformants screened by Southern hybridization contained a disrupted CUT1 gene (cut1:. Hyg). CUT1 mRNA was not detectable in transformants that contained a disrupted gene. Transformants with a disrupted CUT1 gene failed to produce a cutin-inducible esterase that is normally detected by activity staining on non-denaturing polyacrylamide gels. Enzyme activity, measured either with tritiated cutin or with p-nitrophenyl butyrate as a substrate, was reduced but not eliminated in strains with a disrupted CUT1 gene. The infection efficiency of the cut1^– disruption transformants was equal to that of the parent strains on all three host plants. Lesions produced by these mutants had an appearance and a sporulation rate similar to those produced by the parent strains. We conclude that the M. grisea CUT1 gene is not required for pathogenicity.     

Molecular characterization of a cystathionine beta-synthase gene,CBS1, in Magnaporthe grisea

CBS1 from Magnaporthe grisea is a structural and functional homolog of the cystathionine β-synthase (CBS) gene from Saccharomyces cerevisiae. Our studies indicated that M. grisea can utilize homocysteine and methionine through a CBS-independent pathway. The results also revealed responses of M. grisea to homocysteine that are reminiscent of human homocystinuria.     

Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.