Many fungal pathogens invade plants using specialized infection structures called appressoria that differentiate from the tips of fungal hyphae contacting the plant surface. We demonstrate a role for a MAP kinase that is essential for appressorium formation and infectious growth in Magnaporthe grisea, the fungal pathogen responsible for rice blast disease. The PMK1 gene of M. grisea is homologous to the Saccharomyces cererisiae MAP kinases FUS3/KSS1, and a GST-Pmkl fusion protein has kinase activity in vitro, pink1 mutants of M. grisea fail to form appressoria and fail to grow invasively in rice plants, pink1 mutants are still responsive to cAMP for early stages of appressorium formation, which suggests Pmkl acts downstream of a cAMP signal for infection structure formation. PMK1 is nonessential for vegetative growth and sexual and asexual reproduction in culture. Surprisingly, when expressed behind the GALl promoter in yeast, PMK1 can rescue the mating defect in a fus3 kssl double mutant. These results demonstrate that PMK1 is part of a highly conserved MAP kinase signal transduction pathway that acts cooperatively with a cAMP signaling pathway for fungal pathogenesis.

Differential cDNA cloning was used to identify genes expressed during infectious growth of the fungal pathogen Magnaporthe grisea in its host, the rice plant. We characterized one of these genes, MPG1, in detail. Using a nove1 assay to determine the proportion of fungal biomass present in the plant, we determined that the MPGl transcript was 60-fold more abundant during growth in the plant than in culture. Mpgl mutants have a reduced ability to cause disease symptoms that appears to result from an impaired ability to undergo appressorium formation. MPGl mRNA was highly abundant very early in plant infection concomitant with appressorium formation and was also abundant at the time of symptom development. The MPGl mRNA was also expressed during conidiation and i n mycelial cultures starved for nitrogen or carbon. MPGl potentially encodes a small, secreted, cysteine-rich, moderately hydrophobic protein with the characteristics of a fungal hydrophobin. Consistent with the role of the MPGl gene product as a hydrophobin, Mpgl mutants show an basily wettable" phenotype. Our results suggest that hydrophobins may have a role in the elaboration of infective structures by fungi and may fulfill other functions in fungal phytopathogenesis.

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1-1⌬ mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1-1⌬ mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1-1⌬ mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.The plant cell wall provides such an effective barrier that many microorganisms can infect plants only through wounds or natural openings in the wall (1). A variety of fungal pathogens have evolved a complex morphogenetic program to sense specific components and properties of the plant cell surface and to differentiate specialized infectious cells, called appressoria, for penetrating the plant cell wall (2). Appressoria are produced by a wide range of taxonomically diverse fungal and nonfungal species (e.g., Oomycetes), suggesting that appressorium differentiation is a widespread microbial adaptation that assists in plant cell-wall penetration.The rice blast fungus, Magnaporthe grisea, infects most of the economically important cereal crops, particularly rice (3). Dispersed fungal spores (conidia) attach tightly to the plant surface, and under high moisture conditions, they germinate and differentiate a dome-shaped appressorium (4-6). The appressorium attaches tightly to the plant surface, and over several hours, turgor pressures within the appressorium reach 3-8 MPa, the highest measured turgor pressures for any cell (7,8). The fact that appressoria are capable of denting and penetrating inert hydrophobic surfaces (4) suggests that turgor pressure is the major force used to drive a thin penetration hypha through the plant cell wall.Turgor within the M. grisea appressorium is generated through a dramatic rise in intracellular glycerol levels (9). The efflux of glycerol from within the appressorium is prevented partially by a wall layer rich in polyketide-derived melanin.Mutations or chemicals that block the formation of the melanin wall layer allow the rapid efflux of glycerol, impair the maintenance of appressorium turgor, and prevent penetration (7, 9). Other cell-wall or membra...

We constructed a chimeric plasmid carrying a complete copy of the trifunctional trpC gene from the Ascomycete fungus Aspergillus nidulans. This plasmid, designated pHY201, replicates in Escherichia coli, where it confers resistance to ampicillin and chloramphenicol and complements thpC mutants lacking phosphoribosylanthranilate isomerase activity. We used pHY201 to transform an A. nidulans trpCstrain to trpC' at frequencies of >20 stable transformants per ,ug of DNA. Southern blot analysis of DNA from transformants showed that pHY201 DNA had integrated into the A. nidulans chromosomes in a majority of cases. Most of the integration events appeared to occur at the site of the tipC-allele of the recipient strain. In several instances, we succeeded in recovering pHY201, or derivatives thereof, from A. nidulans transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of E. coli.The Ascomycete fungus Aspergillus nidulans has been used extensively for the study of eukaryotic gene structure, organization, and regulation (1-4) because features of its life cycle make it particularly amenable to biochemical and genetic analysis. A. nidulans has been especially valuable for investigating the genetic and molecular processes controlling fungal cell differentiation (5-9). In contrast to the situation with its close relatives Saccharomyces cerevisiae (yeast) and Neurospora crassa, however, investigations of A. nidulans have not been facilitated by the availability of a tractable DNA-mediated transformation system.We recently isolated the trifunctional trpC gene from A. nidulans (10) for use as a selective marker in transformation experiments with this organism. Here we report construction of a chimeric plasmid, designated pHY201, consisting of a complete wild-type copy of the Aspergillus trpC gene inserted into the unique Sal I site of pBR329 (11). We find that pHY201 DNA, either in circular or linear form, transforms a trpC-A. nidulans strain to trpC+ at frequencies of >20 stable transformants per pg of DNA. The transforming DNA becomes integrated into the host genome, frequently at the site of the resident gene. In several instances, we were able to recover pHY201 in native or altered form from Aspergillus transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of Escherichia coli to ampicillin resistance. Thus, pHY201 has properties that suggest that it may serve as a valuable prototype for the development of more sophisticated Aspergillus cloning vectors. (15) of E. coli MC1066 by using a recombinant phage library formed between A. nidulans nuclear DNA and X Charon 4 (9, 10). We determined that a 4.1-kilobase Xho I restriction fragment present in a phage designated XAn trpC12 contained the entire trpC gene plus about 0.4 kilobase of 3' and 5' flanking sequences (unpublished results). This fragment was recloned into the unique Sal I site of pBR329 (11) to form pHY201 (Fig. 1). MATERIALS AND METHODS MaterialsIsolation and Labeli...

Differential cDNA cloning was used to identify genes expressed during infectious growth of the fungal pathogen Mag-naporthe grisea in its host, the rice plant. We characterized one of these genes, MPG1, in detail. Using a nove1 assay to determine the proportion of fungal biomass present in the plant, we determined that the MPGl transcript was 60-fold more abundant during growth in the plant than in culture. Mpgl mutants have a reduced ability to cause disease symptoms that appears to result from an impaired ability to undergo appressorium formation. MPGl mRNA was highly abundant very early in plant infection concomitant with appressorium formation and was also abundant at the time of symptom development. The MPGl mRNA was also expressed during conidiation and i n mycelial cultures starved for nitrogen or carbon. MPGl potentially encodes a small, secreted, cysteine-rich, moderately hydrophobic protein with the characteristics of a fungal hydrophobin. Consistent with the role of the MPGl gene product as a hydrophobin, Mpgl mutants show an basily wettable" phenotype. Our results suggest that hydrophobins may have a role in the elaboration of infective structures by fungi and may fulfill other functions in fungal phytopathogenesis.