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Peroxisomal carnitine acetyl transferase is required for elaboration of…

- - - - Targeted gene replacement was performed in which an 80 bp EagI-NcoI fragment of the PTH2 gene was removed and replaced with a 1.4 kb hygromycin phosphotransferase gene cassette
    - - The gene disruption construct was introduced into a rice pathogenic wild-type strain of M. grisea, Guy 11, and hygromycin-resistant transformants selected and analysed by DNA gel blot
      - wildtype PTH2 allele was contained in a 13 kb HindIII fragment
      - 2,3,4,6.= gene replacement event was verified by the presence of a 14.5 kb HindIII fragment
      - 5,7= Other transformants showed hybridization to both HindIII fragments indicating ectopic insertion of the gene replacement vector
    - - Dpth2 mutants were unable to utilize acetate, triglycerides (triolein) or long chain fatty acids (olive oil) as sole carbon sources
      - consistent with loss of carnitine metabolism.
    - - The wild-type M. grisea strain Guy 11 showed CAT activity of 25 nmol min-1 mg-1 protein compared with a level of 2.4 nmol min-1 mg-1 protein observed in a Dpth2 mutant
      - Reintroduction of the PTH2 gene into a Dpth2 mutant
        restored CAT activity to normal levels
  - - - Elevated expression of a 2.1 kb PTH2 transcript was observed when the fungus was grown on ethanol, acetate, olive oil, oleic acid, triolein, or glycerol as sole carbon sources compared with growth on glucose
    - - Elevated expression of PTH2 was associated with substrate induction as opposed to glucose repression because the presence of glucose in the medium did not affect PTH2 transcript levels when the fungus was exposed to acetate or glycerol
    - - Substrate induction of PTH2 expression was also rapid
    - - PTH2 expression was de-repressed in the presence of
        glucose in the DcpkA mutant
  - - - To investigate the cellular distribution in more detail we introduced a FoxARFP fusion protein to the same Pth2–GFP transformant of M. grisea
      - Colocalization of the foxA-RFP and Pth2–GFP fusion proteins was observed in intracellular vesicles, which accumulated at the cortex of cells
      - GFP expression was observed with a punctate distribution in vegetative hyphae grown in axenic culture and then transferred to medium containing 50 mM sodium acetate, and within conidia
- - - - Glucose take-up by the fungus can partly restore the functional competence of appressoria, and in particular their ability to form penetration hyphae
  - - - After 8 h, lipid bodies were predominantly mobilized to appressoria and between 12 and 24 h, lipid bodies became depleted at the onset of turgor generation
      - pth2 mutant, very few lipid bodies were present in appressoria after 8 h indicating a delay in lipid body mobilization to incipient appressoria
- - - - using either a battery of
        coordinately regulated cell wall-degrading enzymes
      - generating enormous invasive forces by means of specialized structures known as appressoria