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.     

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.     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

The mitogen-activated protein kinase gene MAF1 is essential for the early differentiation phase of appressorium formation in Colletotrichum lagenarium

Colletotrichum lagenarium, the causal agent of cucumber anthracnose, invades host plants by forming a specialized infection structure called an appressorium. In this fungus, the mitogen-activated protein kinase (MAPK) gene CMK1 is involved in several steps of the infection process, including appressorium formation. In this study, the goal was to investigate roles of other MAPKs in C. lagenarium. The MAPK gene MAF1, related to Saccharomyces cerevisiae MPK1 and Magnaporthe grisea MPS1, was isolated and functionally characterized. The maf1 gene replacement mutants grew normally, but there was a significant reduction in conidiation and fungal pathogenicity. The M. grisea mps1 mutant forms appressoria, but conidia of the C. lagenarium maf1 mutants produced elongated germ tubes without appressoria on both host plant and glass, on which the wild type forms appressoria, suggesting that MAF1 has an essential role in appressorium formation on inductive surfaces. On a nutrient agar, wild-type conidia produced elongated germ tubes without appressoria. The morphological phenotype of the wild type on the nutrient agar was similar to that of the maf1 mutants on inductive surfaces, suggesting repression of the MAF1-mediated appressorium differentiation on the nutrient agar. The cmk1 mutants failed to form normal appressoria but produced swollen, appressorium-like structures on inductive surfaces, which is morphologically different from the maf1 mutants. These findings suggest that MAF1 is required for the early differentiation phase of appressorium formation, whereas CMK1 is involved in the maturation of appressoria.     

Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea

A mitogen-activated protein (MAP) kinase gene, PMK1, is known to regulate appressorium formation and infectious hyphal growth in the rice blast fungus Magnaporthe grisea. In this study, we constructed a green fluorescent protein gene-PMK1 fusion (GFP-PMK1) to examine the expression and localization of PMK1 in M. grisea during infection-related morphogenesis. The GFP-PMK1 fusion encoded a functional protein that complemented the defect of the pmk1 deletion mutant in appressorium formation and plant infection. Although a weak GFP signal was detectable in vegetative hyphae, conidia, and germ tubes, the expression of GFP-Pmk1 was increased in appressoria and developing conidia. Nuclear localization of GFP-Pmk1 proteins was observed in a certain percentage of appressoria. A kinase-inactive allele and a nonphosphorylatable allele of PMK1 were constructed by site-directed mutagenesis. Expression of these mutant PMK1 alleles did not complement the pmk1 deletion mutant. These data confirm that kinase activity and activation of PMK1 by the upstream MAP kinase kinase are required for appressorium formation and plant infection in M. grisea. When overexpressed with the RP27 promoter in the wild-type strain, both the kinase-inactive and nonphosphorylatable PMK1 fusion proteins caused abnormal germ tube branching. Overexpression of these PMK1 mutant alleles may interfere with the function of native PMK1 during appressorium formation.     

PDE1 encodes a P-type ATPase involved in appressorium-mediated plant infection by the rice blast fungus Magnaporthe grisea

Plant infection by the rice blast fungus Magnaporthe grisea is brought about by the action of specialized infection cells called appressoria. These infection cells generate enormous turgor pressure, which is translated into an invasive force that allows a narrow penetration hypha to breach the plant cuticle. The Magnaporthe pde1 mutant was identified previously by restriction enzyme-mediated DNA integration mutagenesis and is impaired in its ability to elaborate penetration hyphae. Here we report that the pde1 mutation is the result of an insertion into the promoter of a P-type ATPase-encoding gene. Targeted gene disruption confirmed the role of PDE1 in penetration hypha development and pathogenicity but highlighted potential differences in PDE1 regulation in different Magnaporthe strains. The predicted PDE1 gene product was most similar to members of the aminophospholipid translocase group of P-type ATPases and was shown to be a functional homolog of the yeast ATPase gene ATC8. Spatial expression studies showed that PDE1 is expressed in germinating conidia and developing appressoria. These findings implicate the action of aminophospholipid translocases in the development of penetration hyphae and the proliferation of the fungus beyond colonization of the first epidermal cell.     

The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea

In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.     

Mutations at the smo genetic locus affect the shape of diverse cell types in the rice blast fungus

Teflon film surfaces are highly conducive to the formation of infection structures (appressoria) in the plant pathogenic fungus, Magnaporthe grisea. We have utilized Teflon films to screen and select for mutants of M. grisea that are defective in appressorium formation. This approach and several others yielded a group of 14 mutants with a similar phenotype. All the mutant strains make abnormally shaped conidia and appressoria. When two mutant strains are crossed, abnormally shaped asci are formed. Ascus shape is normal when a mutant strain is crossed with a wild-type strain. Despite dramatic alterations in cell shape these strains otherwise grow, form conidia, undergo meiosis, and infect plants normally. This mutant phenotype, which we have termed Smo(-), for abnormal spore morphology, segregates in simple Mendelian fashion in crosses with wild-type strains. Some ascospore lethality is associated with smo mutations. In genetic crosses between mutants, smo mutations fail to recombine and do not demonstrate complementation of the abnormal ascus shape phenotype. We conclude that the smo mutations are alleles of a single genetic locus and are recessive with regard to the the ascus shape defect. Mutations at the SMO locus also permit germinating M. grisea conidia to differentiate appressoria on surfaces that are not normally conducive to infection structure formation. A number of spontaneous smo mutations have been recovered. The frequent occurrence of this mutation suggests that the SMO locus may be highly mutable.     

Saccharomyces cerevisiae SSD1 orthologues are essential for host infection by the ascomycete plant pathogens Colletotrichum lagenarium and Magnaporthe grisea

Fungal plant pathogens have evolved diverse strategies to overcome the multilayered plant defence responses that confront them upon host invasion. Here we show that pathogenicity of the cucumber anthracnose fungus, Colletotrichum lagenarium, and the rice blast fungus, Magnaporthe grisea, requires a gene orthologous to Saccharomyces cerevisiae SSD1, a regulator of cell wall assembly. Screening for C. lagenarium insertional mutants deficient in pathogenicity led to the identification of ClaSSD1. Following targeted gene replacement, appressoria of classd1 mutants retained the potential for penetration but were unable to penetrate into host epidermal cells. Transmission electron microscopy suggested that appressorial penetration by classd1 mutants was restricted by plant cell wall-associated defence responses, which were observed less frequently with the wild-type strain. Interestingly, on non-host onion epidermis classd1 mutants induced papilla formation faster and more abundantly than the wild type. Similarly, colonization of rice leaves by M. grisea was severely reduced after deletion of the orthologous MgSSD1 gene and attempted infection by the mutants was accompanied by the accumulation of reactive oxygen species within the host cell. These results suggest that appropriate assembly of the fungal cell wall as regulated by SSD1 allows these pathogens to establish infection by avoiding the induction of host defence responses.     

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.     

Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure

Very little is known about how cellular osmosensors monitor changes in osmolarity of the environment. Here, we report that in yeast, Sln1 osmosensor histidine kinase monitors changes in turgor pressures. Reductions in turgor caused by either hyperosmotic stress, nystatin, or removal of cell wall activate MAPK Hog1 specifically through the SLN1 branch, but not through the SHO1 branch of the high osmolarity glycerol pathway. The integrity of the periplasmic region of Sln1 was essential for its sensor function. We found that activity of the plant histidine kinase cytokinin response 1 (Cre1) is also regulated by changes in turgor pressure, in a manner identical to that of Sln1, in the presence of cytokinin. We propose that Sln1 and Cre1 are turgor sensors, and that similar turgor-sensing mechanisms might regulate hyperosmotic stress responses both in yeast and plants.     

The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea

Trimeric G-proteins transmit extracellular signals to various downstream effectors (e.g. MAP kinases) in eukaryotes. In the rice blast fungus Magnaporthe grisea, the Pmk1 MAP kinase is essential for appressorium formation and infectious growth. The pmk1 deletion mutant fails to form appressoria but still responds to exogenous cAMP for tip deformation. Since gene disruption mutants of three Galpha subunits still form appressoria and are phenotypically different from pmk1 mutants, it is likely that the Pmk1 pathway is activated by Gbeta in M. grisea. In this study, we isolated and characterized the MGB1 gene that encodes the G subunit in M. grisea. Mutants disrupted in MGB1 were reduced in conidiation. Conidia from mgb1 mutants were defective in appressorium formation and failed to penetrate or grow invasively on rice leaves. Exogenous cAMP induced appressorium formation in mgb1 mutants, but these appressoria were abnormal in shape and could not penetrate. The intracellular cAMP level was reduced in mgb1 mutants and the defects in conidiation and hyphal growth were partially suppressed with 1 mM cAMP. Transformants expressing multiple copies of MGB1 were able to form appressoria on hydrophilic surfaces. Our results suggest that MGB1 may be involved in the cAMP signalling for regulating conidiation, surface recognition and appressorium formation. The Pmk1 pathway may be the downstream target of MGB1 for regulating penetration and infectious hyphae growth in M. grisea.     

Functional analysis of lipid metabolism in Magnaporthe grisea reveals a requirement for peroxisomal fatty acid beta-oxidation during appressorium-mediated plant infection

The rice blast fungus Magnaporthe grisea infects plants by means of specialized infection structures known as appressoria. Turgor generated in the appressorium provides the invasive force that allows the fungus to breach the leaf cuticle with a narrow-penetration hypha gaining entry to the underlying epidermal cell. Appressorium maturation in M. grisea involves mass transfer of lipid bodies to the developing appressorium, coupled to autophagic cell death in the conidium and rapid lipolysis at the onset of appressorial turgor generation. Here, we report identification of the principal components of lipid metabolism in M. grisea based on genome sequence analysis. We show that deletion of any of the eight putative intracellular triacylglycerol lipase-encoding genes from the fungus is insufficient to prevent plant infection, highlighting the complexity and redundancy associated with appressorial lipolysis. In contrast, we demonstrate that a peroxisomally located multifunctional, fatty acid beta-oxidation enzyme is critical to appressorium physiology, and blocking peroxisomal biogenesis prevents plant infection. Taken together, our results indicate that, although triacylglycerol breakdown in the appressorium involves the concerted action of several lipases, fatty acid metabolism and consequent generation of acetyl CoA are necessary for M. grisea to complete its prepenetration phase of development and enter the host plant.     

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.     

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.     

Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea.

cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA, do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea, we have identified strains in which a null mutation in the adenylate cyclase gene (MAC1) has an unstable phenotype such that the bypass suppressors of the Mac1(-) phenotype (sum) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation (sum1-99) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA (SUM1), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA, encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis.     

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.