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Genome-wide functional analysis reveals that infection-associated fungal…

- - - - necessary to develop a high
        throughput method for gene functional analysis
      - the deletion of genes encoding components of the
        nonhomologous DNA end-joining pathway
        
        to generate
        fungal strains with enhanced frequencies of homologous recombination
      - deleted the ku70-encoding gene of M. oryzae and tested the resulting strain for the frequency of targeted gene replacement.
        
        deletion of the BUF1 gene, which encodes tri-hydroxy-naphthalene reductase, an enzyme required for melanin biosynthesis
      - Buf1 mutants have a buff color compared to the olive green/gray color of wild-type M. oryzae cultures
      - Homologous gene replacement occurred at
        a frequency of 80% (n 100) in the ku70 mutant background.
        
        Compares to the highly variable frequency of gene replacement
        in M. oryzae
        
        locus-dependent and ranges from 1% to 25%
        
        Growth rate, sporulation, and pathogenicity of the ku70
        mutant were found to be unaltered by the mutation
        
        the mutant was selected as an entry strain for rapid evaluation of the
        function of new genes in M. oryzae
  - - - modified at its C terminus by addition of phosphatidylethanolamine, t
    - - it was functional
        within M. oryzae
        
        a reliable marker for analysis of the
        cellular pattern of autophagy
    - - GFP-MoATG8-labeled autophagosomes
        accumulated in conidia during germination
      - decreased in number during the onset of conidial cell death and
        appressorium maturation
    - - Autophagosome number within developing appressoria increased during appressorium maturation and intense autophagic activity
    - - To investigate whether autophagy is specifically associated with appressorium development in M. oryzae
        
        GFP-MoATG8 in a pmk1 MAP kinase
        mutant was expressed
        
        it does not elaborate appressoria and is consequently nonpathogenic
        
        pmk1 mutant, GFP-MoATG8-labeled autophagosomes were present in the conidium during germination but in significantly smaller numbers
        
        Conidia of the pmk1 mutant remained intact throughout germination and germ tube elongation
        
        conidial programmed cell death does not occur
  - - - 23 autophagy-related genes
        
        autophagy
        (MoATG1, MoATG13, MoATG17),
        
        nucleation (MoATG6)
        
        phagophore and autophagosome expansion (MoATG3, MoATG4,MoATG5, MoATG7, MoATG8, MoATG10, MoATG12, and MoATG16)
        
        recycling (MoATG2, MoATG9, MoATG15, and MoATG18)
      - To test the role of each gene in plant infection by M. oryzae
        
        carried out targeted gene replacements using the ku70 mutant
        as recipient strain
        
        generate a set of 22
        isogenic mutants differing with respect to a single ATG gene
      - Atg muntants impaired in nonselective autophagy were nonphatogenic or highly reduced in virulence
        
        Moatg13 and Moatg18 mutants able to cause disease
      - Cytological analysis of prepenetration structures
        
        Conidial cell collapse was prevented in
        all cases by inhibition of nonselective autophagy
      - To confirm the role of these genes in autophagy
        
        GFP-MoATG8 gene fusion was introduced into subset of ATG gene deletion set and distribution of autophagosomes was assessed
        
        Moatg4 mutant expressing GFP-MoATG8
        
        autophagosomes did not accumulate in fungal spores or
        appressoria and were very significantly reduced in number
        
        Autophogy is arrested by the absence of MoATG4-encoded cysteine protease - disrupting appressorium maturation
    - - 6 genes specifically associated w pexophagy and mitophagy
        
        Moatg11, Moatg24, Moatg26, Moatg27, Moatg28 and
        Moatg29 mutants
        
        All mutants are able to cause rice blast disease and did
        not affect conidial or appressorial autophagy
        
        Selective autophagy is dispensable for appresorium mediated plant infection
- - - - Breach the plant cuticle
        
        Gain entry into internal tissue
      - accumulation of high conc. glycerol
        
        Hydrostatic turgor (rapid influx of water into appressorium).
        
        Melanin prevent efflux of glycerol
        
        Allow increase turgor pressure
      - conidium germinates develop germ tube
        
        Development requires activation of PMK1 mitogen-activated protein kinase pathwasy and reegulate by cell cycle
        
        Completion mitosis leads to collapse and death of fungal conidium, content migrate to appressorium
        
        Suggest ATG8 gene (type 2 autophagic cell death is needed for appressorium formation and plant infection
      - Autophagy
        
        S. cerevesiae
        
        30 ATG genes
        
        Encodes protein neccessary for autophagy
        
        TOR kinase regulates initiation autophagy leading to formation of a single membrane structure, the phagophore, which surrounds and engulfs cytoplasm, organelles, and other cellular components, developing into a spherical, double-membrane autophagosome
        
        The autophagosome expands and then fuses with a vacuole, the lytic compartment (lysosome equivalent) of fungal cells, sequestering its contents and inner membrane for degradation by hydrolases
        
        degrade S. cerevesiae
        
        Peroxisomes
        
        Mitochondria
        
        endoplasmic reticulum
        
        during the biosynthetic cytoplasm-to-vacuole-targeting
        (Cvt) pathway
        
        transport
        the inactive precursor of the vacuolar hydrolase aminopeptidase Ito the vacuole
        
        Selective forms of autophagy require a distinct set of proteins, such as Atg11, which encodes a peripheral membrane protein that is the adaptor required for cargo loading in pexophagy and for delivery of aminopeptidase I to the vacuole in the Cvt pathway
- - - - MoATG8 has alsobeen shown to be involved in regulation of glycogen metabolismduring conidiogenesis
      - Moatg8 mutants produce less conidia than an isogenic wild-type strain of M. oryzae
    - - phosphoprotein
        Atg13 is part of a multiprotein regulatory protein complex with Atg17, Vac8, and other proteins, which activate the Atg1 protein kinase in S. cerevisiae
      - necessary for induction of autophagy
      - Atg13 mutants show a reduction but not elimination of autophagy
  - - - the Ku80-encoding gene in M. oryzae has a similar effect and we were able to validate use of the ku70 mutant in this systematic analysis of autophagy in M. oryzae.