John E. Hamer

MPG1 Encodes a Fungal Hydrophobin Involved in Surface Interactions during Infection-Related Development of Magnaporthe grisea.

The rice blast fungus expresses a pathogenicity gene, MPG1, during appressorium formation, disease symptom development, and conidiation. The MPG1 gene sequence predicts a small protein belonging to a family of fungal proteins designated hydrophobins. Using random ascospore analysis and genetic complementation, we showed that MPG1 is necessary for infection-related development of Magnaporthe grisea on rice leaves and for full pathogenicity toward susceptible rice cultivars. The protein product of MPG1 appears to interact with hydrophobic surfaces, where it may act as a developmental sensor for appressorium formation. Ultrastructural studies revealed that MPG1 directs formation of a rodlet layer on conidia composed of interwoven ~5-nm rodlets, which contributes to their surface hydrophobicity. Using combined genetic and biochemical approaches, we identified a 15-kD secreted protein with characteristics that establish it as a class I hydrophobin. The protein is able to form detergent-insoluble high molecular mass complexes, is soluble in trifluoroacetic acid, and exhibits mobility shifts after treatment with performic acid. The production of this protein is directed by MPG1.

An ATP‐driven efflux pump is a novel pathogenicity factor in rice blast disease

Cells tolerate exposure to cytotoxic compounds through the action of ATP‐driven efflux pumps belonging to the ATP‐binding cassette (ABC) superfamily of membrane transporters. Phytopathogenic fungi encounter toxic environments during plant invasion as a result of the plant defense response. Here we demonstrate the requirement for an ABC transporter during host infection by the fungal plant pathogen Magnaporthe grisea. The ABC1 gene was identified in an insertional mutagenesis screen for pathogenicity mutants. The ABC1 insertional mutant and a gene‐replacement mutant arrest growth and die shortly after penetrating either rice or barley epidermal cells. The ABC1‐encoded protein is similar to yeast ABC transporters implicated in multidrug resistance, and ABC1 gene transcripts are inducible by toxic drugs and a rice phytoalexin. However, abc1 mutants are not hypersensitive to antifungal compounds. The non‐pathogenic, insertional mutation in ABC1 occurs in the promoter region and dramatically reduces transcript induction by metabolic poisons. These data strongly suggest that M.grisea requires the up‐regulation of specific ABC transporters for pathogenesis; most likely to protect itself against plant defense mechanisms.

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 CPKA gene of Magnaporthe grisea is essential for appressorial penetration

The rice blast fungus Magnaporthe grisea uses appressoria to penetrate into plant cells. Appressorium formation occurs following conidial germination on hydrophobic surfaces and may involve a cyclic AMP (cAMP)-dependent signaling mechanism. Recently, gene replacement mutants of CPKA, a gene encoding a proposed catalytic subunit of cAMP-dependent protein kinase A, were shown to be defective in appressorium formation, cAMP responsiveness, and lesion formation (T. K. Mitchell and R. A. Dean, Plant Cell, 7:1869–1878, 1995). Here we report a detailed phenotypic characterization of three cpkA mutants. cpkA mutants are dramatically reduced in pathogenicity toward healthy plants. However, the reduced pathogenicity does not appear to be due to a loss of appressorium formation. cpkA mutants are delayed in appressorium formation but form appressoria at the same level as wild-type strains over a 24-h period. Appressoria formed by cpkA mutants are fully melanized but are smaller than wild type and are defective in pen...

DNA Fingerprinting with a Dispersed Repeated Sequence Resolves Pathotype Diversity in the Rice Blast Fungus.

The poor definition of pathotype variation in the rice blast fungus has historically handicapped strategies for reducing blast disease damage to the worlds rice crop. We have employed a probe for a dispersed repeated DNA sequence called MGR [Hamer et al. (1989). Proc. Natl. Acad. Sci. USA 86, 9981-9985] to construct genotype-specific, EcoRl restriction fragment length profiles (MGR-DNA fingerprints) from United States field isolates of this fungus. By using a blind-test design, we demonstrated that MGR-DNA fingerprints distinguished the major pathotypes in the United States, accurately identified the pathotypes of isolates collected over a 30-year period, and defined the organization of clonal lineages within and among pathotype groups. These results resolved a lingering controversy regarding rice blast pathotype stability and illustrated new opportunities for tracking the population dynamics and evolution of this important crop pathogen.

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.

Characterization of a PR-10 Pathogenesis-Related Gene Family Induced in Rice During Infection with Magnaporthe grisea

A partial cDNA with homology to the PR-10 class of pathogenesis-related proteins was used to screen a rice genomic library. One 16-kb genomic clone contained three genes with PR-10 similarity. These genes, RPR10a, RPR10b, and RPR10c, were arranged in tandem and separated by approximately 2.5 kb. RPR10a cDNA was obtained by reverse transcription-polymerase chain reaction, and sequence analysis revealed that RPR10a and RPR10b encode predicted proteins of 158 and 160 amino acids, respectively, and share 71% amino acid identity. RPR10c appears to be a nonfunctional pseudogene. Gene-specific probes were used to study transcript accumulations of the three RPR10 genes in rice plants following inoculation with Magnaporthe grisea. RPR10a transcripts were induced from a low basal level within 12 h after inoculation and showed a second higher level induction at 48 h, which continued throughout the 144 h it was examined. In addition, RPR10a was induced strongly by salicylic and jasmonic acid applications to rice plants. Transcripts of RPR10b also were enhanced by M. grisea, but were not strongly visible until 48 h after inoculation. Tissue prints of M. grisea-infected rice leaves when the RPR10a-specific probe was used indicate that RPR10a is expressed most strongly in a localized fashion in response to the pathogen.

Cellular Localization and Role of Kinase Activity of PMK1 in Magnaporthe grisea

ABSTRACT 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.

The Aspergillus nidulans sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity.

Cytokinesis (septation) in the fungus Aspergillus nidulans occurs through the formation of a transient actin ring at the incipient division site. Temperature‐sensitive mutations in the sepA gene prevent septation and cause defects in the maintenance of cellular polarity, without affecting growth and nuclear division. The sepA gene encodes a member of the growing family of FH1/2 proteins, which appear to have roles in morphogenesis and cytokinesis in organisms such as yeast and Drosophila. Results from temperature shift and immunofluorescence microscopy experiments strongly suggest that sepA function requires a preceding mitosis and that sepA acts prior to actin ring formation. Deletion mutants of sepA exhibit temperature‐sensitive growth and severe delays in septation at the permissive temperature, indicating that expression of another gene may compensate for the loss of sepA. Conidiophores formed by sepA mutants exhibit abnormal branching of the stalk and vesicle. These results suggest that sepA interacts with the actin cytoskeleton to promote formation of the actin ring during cytokinesis and that sepA is also required for maintenance of cellular polarity during hyphal growth and asexual morphogenesis.

Regulatory Genes Controlling MPG1 Expression and Pathogenicity in the Rice Blast Fungus Magnaporthe grisea.

MPG1, a pathogenicity gene of the rice blast fungus Magnaporthe grisea, is expressed during pathogenesis and in axenic culture during nitrogen or glucose limitation. We initiated a search for regulatory mutations that would impair nitrogen metabolism, MPG1 gene expression, and pathogenicity. First, we developed a pair of laboratory strains that were highly fertile and pathogenic toward barley. Using a combinatorial genetic screen, we identified mutants that failed to utilize a wide range of nitrogen sources (e.g., nitrate or amino acids) and then tested the effect of these mutations on pathogenicity. We identified five mutants and designated them Nr- (for nitrogen regulation defective). We show that two of these mutations define two genes, designated NPR1 and NPR2 (for nitrogen pathogenicity regulation), that are essential for pathogenicity and the utilization of many nitrogen sources. These genes are nonallelic to the major nitrogen regulatory gene in M. grisea and are required for expression of the pathogenicity gene MPG1. We propose that NPR1 and NPR2 are major regulators of pathogenicity in M. grisea and may be novel regulators of nitrogen metabolism in fungi.