In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp1 protein is 520 amino acids long and is comparable to the Ytp1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP (green fluorescent protein) fusion expression results indicate that Mtp1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Deltamtp1 mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.
Fungal Proteins ; genetics ; physiology ; Genes, Fungal ; Magnaporthe ; genetics ; Membrane Proteins ; genetics ; Oryza ; microbiology ; Promoter Regions, Genetic

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.
Autophagy ; genetics ; Gene Expression ; Genes, Fungal ; Host-Pathogen Interactions ; Magnaporthe ; genetics ; growth & development ; pathogenicity ; physiology ; Oryza ; microbiology ; Plant Diseases ; microbiology

The rice blast fungus, Magnaporthe oryzae, is an important pathogen of rice plants. It is well known that genes encoded in the genome have different evolutionary histories that are related to their functions. Phylostratigraphy is a method that correlates the evolutionary origin of genes with evolutionary transitions. Here we applied phylostratigraphy to partition total gene content of M. oryzae into distinct classes (phylostrata), which we designated PS1 to PS7, based on estimation of their emergence time. Genes in individual phylostrata did not show significant biases in their global distribution among seven chromosomes, but at the local level, clustering of genes belonging to the same phylostratum was observed. Our phylostrata-wide analysis of genes revealed that genes in the same phylostratum tend to be similar in many physical and functional characteristics such as gene length and structure, GC contents, codon adaptation index, and level of transcription, which correlates with biological functions in evolutionary context. We also found that a significant proportion of genes in the genome are orphans, for which no orthologs can be detected in the database. Among them, we narrowed down to seven orphan genes having transcriptional and translational evidences, and showed that one of them is implicated in asexual reproduction and virulence, suggesting ongoing evolution in this fungus through lineage-specific genes. Our results provide genomic basis for linking functions of pathogenicity factors and gene emergence time.
Base Composition ; Bias (Epidemiology) ; Child ; Child, Orphaned ; Codon ; Fungi* ; Genome ; Humans ; Magnaporthe ; Methods ; Oryza ; Reproduction, Asexual ; Virulence ; Virulence Factors

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.
Agrobacterium tumefaciens ; genetics ; Databases, Genetic ; Genome, Fungal ; Genomics ; Magnaporthe ; genetics ; pathogenicity ; Mutagenesis, Insertional ; Oryza ; microbiology ; Phenotype ; Plant Diseases ; microbiology ; Transformation, Genetic ; Virulence ; genetics

MGTA1, a putative fungal Zn(II)(2)Cys(6) transcriptional activator-encoding gene, was isolated from rice blast pathogen Magnaporthe grisea, which is homologous to CLTA1 from Colletotrichum lindemuthianum with 51% identity at protein level. MGTA1 cassette contains a 2370 bp open reading frame, consisting of 6 exons, coding a 790 amino acid peptide. MGTA1 gene exists as a single copy in genomes of 7 strains of M. grisea, and is expressed in tip hyphae, conidia, and mature appressoria of strain Guy11.
Amino Acid Sequence ; Gene Expression Profiling ; Gene Expression Regulation, Fungal ; physiology ; Magnaporthe ; genetics ; metabolism ; Molecular Sequence Data ; Oryza ; microbiology ; Sequence Analysis, Protein

The promoter of NAR gene in Magnaporthe grisea was isolated and sequenced. The promoter sequences contained the "TATA" box, the "CAAT" box, and binding sites for fungal regulatory proteins. Programs that predict promoter sequences indicated that promoter sequence lies between locations 430 and 857 of the NAR promoter fragment. GFP expression under the NAR promoter and NAR transcript analysis revealed that this promoter is activated primarily at the mycelial stage in the rice blast fungus and could be used to express native or extrinsic genes in the mycelia of the rice blast fungus.
Base Sequence ; Cloning, Molecular ; Fungal Proteins ; genetics ; Gene Expression ; genetics ; Hyphae ; genetics ; Magnaporthe ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Transcriptional Activation ; genetics

To improve the efficiency of targeted gene replacement (TGR), a dual screen (DS) system with gusA gene as negative selective marker (GUS-DS) was developed in Magnaporthe oryzae. First, we tested the endogenous beta-glucuronidase (GUS) activities of 78 fungal strains. All tested strains were GUS-, only with 3 exceptions. Whereas, after the gusA being introduced in, M. oryzae, Fusarium oxysporum and Colletotrichum lagenarium acquired high GUS activities. The gusA is thus usable as a selective maker in fungal species. With gusA as the negative marker, HPH gene as the positive marker, and the peroxisomal targeting signal receptor genes MGPEX5 and MGPEX7 as 2 instances of target genes, we established the GUS-DS system. After transformation, we collected the transformants from hygromycin B screen media and then tested the GUS activities of them. The GUS- ones were selected as potential mutants and checked in succession by PCR and Southern blotting to identify the true mutants and calculate the efficiency of GUS-DS. As a result, GUS-DS improved the screen efficiency for delta mgpex5 from 65.8% to 90.6%, and for delta mgpex7 from 31.2% to 82.8%. In addition, we established a multiple PCR (M-PCR) method for mutant confirmation. By amplifying the different regions at the targeted locus, M-PCR differentiated the wild type, the ectopic transformants and the mutants effectively and rapidly, and had the same reliability as Southern blotting. In conclusion, GUS-DS and M-PCR are useful tools to improve the efficiency of TGR and would be helpful for fungal genomics.
Escherichia coli ; enzymology ; genetics ; Gene Expression Regulation, Enzymologic ; Genes, Fungal ; Glucuronidase ; genetics ; Magnaporthe ; genetics ; Mutagenesis, Insertional ; methods ; Mutation ; Recombination, Genetic ; Transformation, Genetic

A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a lambdaTriplEx2 vector by SMART cDNA library containing 2.37x10(6) independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M. grisea in GenBank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M. grisea.
Cloning, Molecular ; methods ; DNA, Fungal ; genetics ; Gene Expression Profiling ; methods ; Gene Expression Regulation, Fungal ; Gene Library ; Magnaporthe ; genetics ; Sequence Analysis, DNA ; methods

Appressorium is an infection structure of the phytopathogenic fungus Magnaporthe grisea. Analysis of gene expression profiles of appressorium development provides insight into the molecular basis of pathogenicity and control of this fungal plant disease. A cDNA array representing 2927 unique genes based on a large EST (expressed sequence tag) database of M. grisea strain Y34 was constructed and used to profile the gene expression patterns at mycelium and appressorium maturation stages. Compared with mycelia, 55 up-regulated and 22 down-regulated genes were identified in mature appressoria. Among 77 genes, 16 genes showed no similarity to the genome sequences of M. grisea. A novel homologue of peptidyl-prolyl cis-trans isomerase was found to be expressed at low-level in mature appressoria of M. grisea. The results indicated that the genes such as pyruvate carboxylase, phospholipid metabolism-related protein and glyceraldehyde 3-phosphate dehydrogenase involved in gluconeogenesis, lipid metabolism and glycolysis, showed differential expression in mature appressoria. Furthermore, genes such as PTH11, beta subunit of G protein and SGT1 involved in cell signalling, were expressed differentially in mature appressoria. Northern blot analysis was used to confirm the cDNA array results.
Cell Proliferation ; Fungal Proteins ; metabolism ; Fungal Structures ; metabolism ; Gene Expression Profiling ; methods ; Magnaporthe ; metabolism ; Oligonucleotide Array Sequence Analysis ; methods ; Proteome ; metabolism

Antifungal bacteria for biological control of plant diseases or production of novel antibiotics to plant pathogens were isolated in 1997 from various soils of Ansung, Chunan, Koyang, and Paju in Korea. Sixty-four bacterial strains pre-screened from approximately 1,400 strains were tested on V-8 juice agar against eight plant pathogenic fungi using in vitro bioassay technique for inhibition of mycelial growth. Test pathogens were Alternaria mali, Colletotrichum gloeosporioides, C. orbiculare, Fusarium oxysporum f. sp. cucumerinum, F. oxysporum f. sp. lycopersici, Magnaporthe grisea, Phytophthora capsici, and Rhizoctonia solani. A wide range of antifungal activity of bacterial strains was found against the pathogenic fungi, and strain RC-B77 showed the best antifungal activity. Correlation analysis between inhibition of each fungus and mean inhibition of all eight fungi by 64 bacterial strains revealed that C. gloeosporioides would be best appropriate for detecting bacterial strains producing antibiotics with potential as biocontrol agents for plant pathogens.
Agar ; Alternaria ; Anti-Bacterial Agents ; Bacteria* ; Biological Assay ; Chungcheongnam-do ; Colletotrichum ; Fungi ; Fusarium ; Gyeonggi-do ; Korea ; Magnaporthe ; Mali ; Mass Screening* ; Phytophthora ; Plant Diseases ; Plants* ; Rhizoctonia ; Soil*