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

- - - - may be necessary for cellular differentiation
      - defense from infection
      - many aspects of development in multicellular organisms
    - - fundamental to the fungal lifestyle.
      - important in heterokaryon
        incompatibility in Podospora anserina
      - for metal ion
        homeostasis in Aspergillus fumigatus
    - - a role for MoATG8
        in autophagic cell death of conidia during appressorium development
        
        involved in regulation of glycogen metabolism
        during conidiogenesis
      - Moatg8 mutant
        
        unable to undergo conidial cell
        collapse
        
        could form appressoria,
        nonfunctional and unable to cause plant disease
        
        produce less
        conidia than an isogenic wild-type strain of M. oryzae
      - MoATG1
        
        identified as a differentially
        expressed gene during appressorium formation
        
        necessary for pathogenicity
    - - large burst of autophagy activity continues in the conidium until it collapses and undergoes cell death.
    - - it is necessary both for conidial cell death and also
        for maturation and differentiation of functional appressoria
  - - - powerful means of identifying new virulence factors
      - less successful in defining cellular processes critical for plant diseases to occur
    - - ku70 mutant generated and showed it significantly enhanced the frequency of homologous recombination to 80%
    - - Moatg13
      - Moatg18
- - - - Use yeast as reference
        
        Genes functionally separated into those role in initiation of autophagy
        
        (MoATG1, MoATG13, MoATG17), nucleation (MoATG6), phagophore, and autophagosome expansion (MoATG3, MoATG4, MoATG5, MoATG7, MoATG8, MoATG10, MoATG12, and MoATG16), and recycling (MoATG2, MoATG9, MoATG15, and MoATG18)
      - Significant difference: absence of clear orthologues of ATG20,ATG21, andATG23
        
        for selective Cvt and pexophagy pathways in yeast
        
        Absence in genome sequence of filamentous fungi or other multicellular eukaryotes.
        
        Absence ATG19: Cvt pathway-specific receptor protein, confined to S. cerevisiae.
      - ATG14 absence from M.oryzae
        
        encode for autophagy-specific subunit pof phosphatidylinositol 3-kinase complex
        
        ATG31, encodes protein interact with Atg17p and Atg29p, forming complex involved in localizing other Atg proteins to the phagophore assembly site
    - - Nonselective autophagy
      - Pexophagy, mitophagy or Cvt pathway
    - - targeted gene replacement ku70 mutant as recipient strain
        
        able to generate a set of 22
        isogenic mutants differing with respect to a single ATG gene
        
        Two putative orthologues of ATG22 (25) were encoded in the M. oryzae genome
    - - M. oryzae Moatg4 mutant expressing GFP-MoATG8, we found that 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
      - 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 vacuole
      - conidia did not collapse and die during appressorium formation, and there was no intense burst of autophagic activity in appressoria
      - results indicate that autophagy is arrested by the absence of MoATG4-encoded cysteine protease,
        disrupting appressorium maturation
    - - Isogenic ku70 mutant and the wild-type Guy11 strain were both able to cause severe rice blast symptoms
      - 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
      - Lesion numbers were significantly
        reduced when compared to the wild type
      - Cytological analysis of prepenetration structures revealed that conidial cell collapse was prevented in
        all cases by inhibition of nonselective autophagy
      - The loss of rice blast symptoms was found to be due to impaired appressorium function
      - Inoculation of wounded seedlings with either conidia or hyphae also failed to produce rice blast symptoms, indicating a role for autophagy in proliferation of the fungus in plant tissue
      - Moatg6 mutant showed reduced viability upon storage and a
        severe impairment in sporulation
      - Complementation analysis was performed on a subset of the atg mutants and in all cases tested led to restoration of the wild-type phenotype
      - Conclude that each individual gene product that is necessary for nonselective fungal autophagy in M. oryzae is also required for rice blast disease
- - - - a layer of melanin in the appressorium cell wall prevents the efflux of glycerol, allowing turgor to increase to a level sufficient to rupture the plant suface
      - A narrow penetration hypha enters the rice epidermis
        and differentiates into bulbous A narrow penetration hypha enters the rice epidermis
        and differentiates into bulbous
      - regulated genetically by control of the cell cycle
    - - conidium germinates and
        develops a short cylindrical germ tube
  - - - undergoes mitosis
        
        one
        of the resulting daughter nuclei migrates into the incipient appressorium
        
        the other migrates back into the conidium
        
        necessary for appressoria to develop and for plant infection to occur
        
        . Completion of
        mitosis
        
        leads to collapse and death of the fungal
        conidium
    - - type II autophagic cell death is necessary
        for appressorium maturation and plant infection (7
  - - - leading to formation of a single membrane
        structure, the phagophore
        
        developing into a spherical, double-membrane autophagosom
        
        autophagosome expands and then fuses with a vacuole
        
        sequestering its contents and
        inner membrane for degradation by hydrolases (
  - - - encodes a peripheral membrane protein
- - - - using the split marker strategy
    - - the primers used were M13 F with HY and M13R with YG
  - - - primers ATG8.5 and ATG8.3
      - amplicon was digested and
        cloned into pCB1532
      - ClaI and XhoI sites
        at the ends of the fragment
    - - primers ATG8p5 and Atg8p3
      - SpeI and NcoI sites
      - fragment was sub cloned into pMJK142.2
    - - primers ATG8p5 and GFPrev
  - - - Ku70Ff & Ku70LFr*
        
        from the start codon and introduce a NdeI site at the 3' end
      - Ku7-RFf & Ku70RFr
        
        downstream of the gene and introducing a NdeI site at the 5' end
    - - SurF and SurR
    - - to liberate the gene disruption cassette from pMJG2
    - - deletion mutants were obtained