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Peroxisomal carnitine acetyl transferase is required for elaboration of…

- - - - unreported SKL type I peroxisomal targeting sequence at its C-terminus
    - - most closely related to FacC from Neurospora crassa and Aspergillus nidulans
  - - - PTH2 expression during growth of the fungus on lipids and fatty acids
        
        Elevated expression of a 2.1 kb PTH2 transcript 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
        
        contrast to the regulation of FacC expression in A.
        nidulans,
        
        glucose repression
        
        Substrate induction of PTH2 expression was also rapid, with elevated transcript levels observed as soon as 1 h after transfer to acetate containing growth medium, with peak transcript 8 h after transfer to acetate
        
        mutant strains of M. grisea
        
        affected the cyclic AMP response pathway
        
        Acetate-induction of PTH2 expression was lacks the catalytic
        subunit of PKA
        
        Dmac1sum1–99 mutant which lacks the regulatory
        
        subunit of PKA and therefore shows constitutive cAMPindependent
        PKA activity
        
        2 more items...
        
        PTH2 expression in mycelium is normally repressed in the presence of glucose, which requires the cAMP-dependent PKA signalling pathway
  - - - appressorium function may be
        compromised
        
        PTH2 is not
        needed for generation or maintenance of appressorium turgor.
  - - - a melanin layer was observed, but was reduced in thickness when compared with Guy 11
        
        pth2 mutant
        appressoria still generate turgor,
  - - - The gene disruption construct
        was introduced into Guy 11, and hygromycin-resistant transformants
        
        analysed by DNA gel blot
        
        The wild type PTH2 allele was contained in a 13 kb HindIII fragment
        
        Other transformants showed hybridization to both HindIII fragments indicating ectopic insertion of the gene replacement vector
        
        pth2 mutants were unable to utilize acetate, trigtriglycerides
        (triolein) or long chain fatty acids (olive oil) as carbon sources
  - - - pth2 mutants, lipid bodies are not metabolized within appressoria at the same rate as in a wild-type M. grisea strain.
  - - - that glucose take-up by the fungus can partly restore the functional competence of appressoria, and in particular their ability to form penetration hyphae.
  - - - To determine the subcellular location of Pth2 we constructed expressed a Pth2–GFP fusion protein in M.
        grisea under the control of its native promoter
        
        To investigate the cellular distribution in more detail
        a FoxARFP was introduced fusion protein to the same Pth2–GFP transformant of M. grisea.
        
        foxA-RFP and Pth2–GFP fusion proteins accumulated at the cortex of cells
- - - - Coordinately regulated cell wall-degrading enzymes
      - Generating enormous invasive forces - appressoria
    - - Cause disease using appressorium break through the tough cuticle
        
        Appressorium
        
        Dome-shaped cell, differentiated at the end of fungal germ tube
        
        Bounded by melanin-rich cell wall and accumulates glycerol to very high concentrations
        
        Generation of hydrostatic turgor and prodcution of substantial invasive force
        
        Narrow penetration hypha is formed at the base of appressorium
        
        Enter rice epidermal cell by invagination of the plasmalemma
        
        Epidermal cells become filled with invasive hyphae
        
        Proliferates throughout the rice leaf - kill plant cells produce necrotic lesions
      - Spores mobilize lipid bodies to developing appressorium under control of Pmk1 MAP kinase pathway
        
        Rapid lipolysis occur in mature infection cell at the onset of turgor generation
        
        Isocitratte lyase activity required - glyoxylate cycle is important
        
        Triacylglycerol lipase acivity has been measured in appressoria - regulated by the cAMP response pathway
      - Consequence of lipolysis and fatty acid B-oxidation in appressorium
        
        Generation of acetyl CoA across intracellular membrane - catalysed y carnitine acetyl transferases (CATs)
        
        Conver acetyl CoA units to acetyl-carnitine for transport across the membrane
        
        CATs - Member of the carnitine acyltransferase family
        
        Carnitine palmitoyltransferases and Carnitine octanoyltransferases
        
        Highly conserved throughout eukaryotes
        
        In M. grisae
        
        PTH2 encode CAT
  - - - Appressorium development
      - Succesful plant infection
    - - Necessary for appressoria to function correctly
      - Pth2 regulated by cyclic AMP response pathway - located in peroxisomes
      - Pth2 contributes to effective lipid reserve mobilization
        
        During appressorium maturation
        
        Necessary for penetration hypha formation and host invasion
    - - Intracellular transport of peroxisomally generated acetyl CoA within appressorium
        
        Critical to plant infection by the rice blast fungus