Jin-Rong Xu

The genome sequence of the rice blast fungus Magnaporthe grisea

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

The Fusarium graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

Independent Signaling Pathways Regulate Cellular Turgor during Hyperosmotic Stress and Appressorium-Mediated Plant Infection by Magnaporthe grisea

The phytopathogenic fungus Magnaporthe grisea elaborates a specialized infection cell called an appressorium with which it mechanically ruptures the plant cuticle. To generate mechanical force, appressoria produce enormous hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control of cellular turgor, we analyzed the response of M. grisea to hyperosmotic stress. During acute and chronic hyperosmotic stress adaptation, M. grisea accumulates arabitol as its major compatible solute in addition to smaller quantities of glycerol. A mitogen-activated protein kinase–encoding gene OSM1 was isolated from M. grisea and shown to encode a functional homolog of HIGH-OSMOLARITY GLYCEROL1 (HOG1), which encodes a mitogen-activated protein kinase that regulates cellular turgor in yeast. A null mutation of OSM1 was generated in M. grisea by targeted gene replacement, and the resulting mutants were sensitive to osmotic stress and showed morphological defects when grown under hyperosmotic conditions. M. grisea Δosm1 mutants showed a dramatically reduced ability to accumulate arabitol in the mycelium. Surprisingly, glycerol accumulation and turgor generation in appressoria were unaltered by the Δosm1 null mutation, and the mutants were fully pathogenic. This result indicates that independent signal transduction pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection. Consistent with this, exposure of M. grisea appressoria to external hyperosmotic stress induced OSM1-dependent production of arabitol.

Mitogen-Activated Protein Kinase Pathways and Fungal Pathogenesis

In eukaryotic cells, a family of serine/threonine protein kinases known as mitogen-activated protein (MAP) kinases (MAPKs) is involved in the transduction of a variety of extracellular signals and the regulation of different developmental processes. The MAPK is activated by dual phosphorylation of the TXY motif by MAPK kinase (MEK or MAPKK), which is activated in turn by MEK kinase (MEKK or MAPKKK). The sequential activation of the MAPK cascade eventually results in the activation of transcription factors and the expression of specific sets of genes in response to environmental stimuli. In the budding yeast Saccharomyces cerevisiae, five MAPK pathways are known to regulate mating, invasive growth, cell wall integrity, hyperosmoregulation, and ascospore formation (50). In the past decade, MAPKs in various plant and human pathogenic fungi have been characterized. In this review, we will compare their functions in different fungal pathogens with a focus on infection-related morphogenesis and virulence.

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...

Osmoregulation and Fungicide Resistance: the Neurospora crassa os-2 Gene Encodes a HOG1 Mitogen-Activated Protein Kinase Homologue

ABSTRACT Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.

A Genetic Map of Gibberella fujikuroi Mating Population A (Fusarium moniliforme)

We constructed a recombination-based map of the fungal plant pathogen Gibberella fujikuroi mating population A (asexual stage Fusarium moniliforme). The map is based on the segregation of 142 restriction fragment length polymorphism (RFLP) markers, two auxotrophic genes (arg1, nic1), mating type (matA+/matA-), female sterility (ste1), spore-killer (Sk), and a gene governing the production of the mycotoxin fumonisin B1 (fum1) among 121 random ascospore progeny from a single cross. We identified 12 linkage groups corresponding to the 12 chromosome-sized DNAs previously observed in contour-clamped homogeneous electric field (CHEF) gels. Linkage groups and chromosomes were correlated via Southern blots between appropriate RFLP markers and the CHEF gels. Eleven of the 12 chromosomes are meiotically stable, but the 12th (and smallest) is subject to deletions in 3% (4/121) of the progeny. Positive chiasma interference occurred on five of the 12 chromosomes, and nine of the 12 chromosomes averaged more than one crossover per chromosome. The average kb/cM ratio in this cross is approximately 32.

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.

Global gene regulation by Fusarium transcription factors Tri6 and Tri10 reveals adaptations for toxin biosynthesis

Trichothecenes are isoprenoid mycotoxins produced in wheat infected with the filamentous fungus Fusarium graminearum. Some fungal genes for trichothecene biosynthesis (Tri genes) are known to be under control of transcription factors encoded by Tri6 and Tri10. Tri6 and Tri10 deletion mutants were constructed in order to discover additional genes regulated by these factors in planta. Both mutants were greatly reduced in pathogenicity and toxin production and these phenotypes were largely restored by genetic complementation with the wild‐type gene. Transcript levels for over 200 genes were altered ≥u2003twofold for Δtri6 or Δtri10 mutants including nearly all known Tri genes. Also reduced were transcript levels for enzymes in the isoprenoid biosynthetic pathway leading to farnesyl pyrophosphate, the immediate molecular precursor of trichothecenes. DNA sequences 5′ to isoprenoid biosynthetic genes were enriched for the Tri6p DNA binding motif, YNAGGCC, in F.u2003graminearum but not in related species that do not produce trichothecenes. To determine the effect of trichothecene metabolites on gene expression, cultures were treated with trichodiene, the first metabolic intermediate specific to the trichothecene biosynthetic pathway. A total of 153 genes were upregulated by added trichodiene and were significantly enriched for genes likely involved in cellular transport. Differentially regulated genes will be targeted for functional analysis to discover additional factors involved in toxin biosynthesis, toxin resistance and pathogenesis.