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

- - - - a GFP-MoATG8 gene fusion was construct and introduced to wild-type strain of M. oryzae Guy11 and Moatg8 mutant
        
        expression of the GFP-MoATG8 fusion was sufficient to complement the Moatg8 mutant phenotypes
        
        GFP-MoATG8-labeled autophagosomes accumulated in conidia during germination and then steadily 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 and vacuole expansion was associated with mature appressoria
    - - expressed GFP-MoATG8 in a pmk1 MAP kinase mutant that does not elaborate appressoria and is consequently non pathogenic
        
        In the 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, indicating that conidial programmed cell death does not occur in the absence of appressorium formation
  - - - ku70-encoding gene of M. oryzae was deleted 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 cause the identifying mutants was straightforward
      - homologous gene replacement occurred at a frequency of 80% (n=100) in the ku70 mutant background
      - highly variable frequency of gene replacement in M.oryzae, which is locus-dependent and ranges from 1% to 25%
  - - - By reference to a series of molecular analyses in yeast these genes could be functionally separated into those that putatively play a role
        
        in the initiation of autophagy
        
        MoATG1
        
        MoATG13
        
        MoATG17
        
        nucleation
        
        MoATG6
        
        phagophore, and autophagosome expansion
        
        MoATG3
        
        MoATG4
        
        MoATG5
        
        MoATG7
        
        MoATG8
        
        MoATG10
        
        MoATG12
        
        MoATG16
        
        recycling
        
        MoATG2
        
        MoATG9
        
        MoATG15
        
        MoATG18
      - The most significant differences compared to the S.
        cerevisiae autophagy gene family was the absence of clear orthologue gene required for the selective Cvt
        and pexophagy pathways in yeast
        
        ATG20
        
        ATG21
        
        ATG23
        
        ATG14
        
        encodes the autophagy-specific subunit of phosphatidylinositol
        3-kinase complex
        
        ATG31
        
        encodes a protein that interacts with Atg17p and Atg29p forming a complex involved in localizing other Atg proteins to the phagophore assembly site
    - - were able to generate a set of 22
        isogenic mutants differing with respect to a single ATG gene
        
        Two putative orthologues of ATG22 were encoded in the M. oryzae genome, precluding analysis of this gene function by a single gene deletion
        
        To check the efficacy of using the ku70 mutant, we also deleted MoATG8, MoATG4, MoATG9, and MoATG12 in Guy11 so that comparative phenotype analysis could be carried out
        
        Phenotypic analysis of mutants generated in both genetic backgrounds gave identical results.
      - To confirm the role of these genes in autophagy
        
        the GFPMoATG8 gene fusion was introduced into a subset of the ATG gene
        deletion set and the distribution of autophagosomes
        
        autophagosomes did not accumulate in fungal spores or
        appressoria and were very significantly reduced in number
        
        Large aggregates of GFP-MoAtg8 were observed
        and were excluded from vacuoles
        
        MoATG4 encodes a cysteine protease necessary for processing of Atg8 and therefore required for phagophore initiation, autophagosome formation, and vacuole fusion
        
        Consistent with this predicted role, GFP-MoAtg8 puncta were significantly reduced in number (P 0.0001) in both appressoria and conidia of Moatg4 when compared to Guy11 and excluded from vacuoles
        
        conidia did not collapse and die during appressorium formation, and there was no intense burst of autophagic activity in appressoria
        
        indicate that autophagy is arrested by the absence of MoATG4-encoded cysteine protease,
        disrupting appressorium maturation
    - - spores were collected from Moatg1, Moatg2, Moatg3, Moatg4, Moatg5, Moatg6, Moatg7, Moatg8, Moatg9, Moatg10, Moatg12, Moatg13, Moatg15, Moatg16, Moatg17, and Moatg18 mutants and used to inoculate 21-day-old seedlings of the blast-susceptible rice cultivar CO-39
        
        The isogenic ku70 mutant and the wild-type Guy11 strain were both able to cause severe rice blast symptoms
        
        In contrast, Atg mutants impaired in nonselective autophagy were nonpathogenic or highly reduced in virulence. Only the Moatg13 and Moatg18 mutants were able to cause any disease symptoms, and lesion numbers were significantly reduced when compared to the wild type (t-tests, P 0.001 and P 0.01 respectively)
        
        cytological analysis of prepenetration structures revealed that conidial cell collapse was prevented in
        all cases by inhibition of nonselective autophagy
  - - - Moatg11
      - Moatg24
      - Moatg26
      - Moatg27
      - Moatg28
      - Moatg29
- - - - A mechanism to recycle cytoplasm, organelles and intracellular proteins
      - Cell survival response that is triggered by startvation stress
      - Function
        
        Hemeostasis
        
        Cellular differentiation
        
        Defence from infection
        
        ETC(involved in many aspects of development in multicellular organisms)
      - In fungi
        
        few reports
        
        The osmotrophic, mycelial growth of fungi and their ability to grow in heterogeneous nutrient scarce environment strongly suggests involvement of autophagy
        
        important in
        
        Podospora anserina
        
        heterokaryon incompatibility
        
        Aspergillus fumigatus
        
        metal ion hemeostasis
      - M.oryzae
        
        MoATG8 involved in autophagic cell death of the conidia during appressorium development
        
        MoATG8
        
        involved in the regulation of glycogen metabolilsm during conidiogenesis
        
        :small_red_triangle:Moatg8
        
        unable for conidia to collapse
        
        appressoria still developed
        
        nonfunctional, and cannot cause disease
        
        produce less conidia than WT
        
        MoATG1
        
        independently identified as a differentially expressed gene during appressorium formation
        
        shown necessary for pathogenicity
      - Pro-survival response essential for cells to deal with nutrient shortage in the extra-cell
  - - - could not be determined by analysis of MoATG8
      - genome level analysis
      - targeted gene replacement
        
        identifying new virulence factors
        
        gene replacement
        
        time-consuming
        
        poor levels of efficiency
        
        ku70 mutant
        
        enhanced the frequency of homologous recombination
      - nonselective
        autophagy
        
        appressoria formation
      - Deletion of any of the 16 gene products
        
        macroautophagy
        
        unable to cause blast disease
        
        impairment in appressorium
      - cause any disease symptoms
        
        Moatg13
        
        Moatg18
      - phosphoprotein Atg13
        
        multiprotein regulatory protein
        complex
        
        Atg17, Vac8
        
        activate the
        Atg1 protein kinase
        
        induction
        of autophagy
        
        Atg13 mutants
        
        reduction, but
        not elimination of autophagy
        
        Atg18
        
        recycling of Atg9
      - Conidial collapse
        
        impaired in all
        macroautophagy mutants
        
        Moatg4
        
        disruption of
        autophagosome generation
        
        autophagy
        
        conidia
        
        appressoria
      - deletion of any
        of the 6 specific genes
        
        pexophagy or mitophagy
        
        did not affect fungal pathogenicity
        
        selective forms of autophagy
        
        not necessary for
        plant infection
      - Pexophagy play role in plant tissue colonization
        
        breach of the cuticle
        
        peroxisome biogenesis
        
        appressorium function & pysiology
        
        fatty acid -oxidation
      - Controlling the initiation of fungal autophagy provide an effective target
        
        development of new and novel antifungal strategies