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Peroxisomal carnitine acetyl transferase is required for elaboration of…

- - - - fatty acid b-oxidation
        occurs only in peroxisomes
    - - Cat2
        
        provides a means for peroxisomally generated acetyl CoA to be transferred to the cytoplasm
      - Cat1
        
        channels acetyl CoA into mitochondria
        for oxidation via the tricarboxylic acid cycle
    - - encodes the major CAT activity in M. grisea, based on enzymatic assays and the inability of pth2 mutants to utilize acetate or lipids as a sole carbon source
      - inability of Dpth2
        
        there may be a
        separate cytosolic pool of Pth2
    - - generation of targeted PTH2 mutant shows significance of CAt on fungal pathogenicity
      - cytological analysis
        
        pth2 mutants are
        impaired in rice cuticle penetration
      - three possible virulence associated
        functions for the enzyme
        
        Pth2 is required for turgor generation necessary
        for appressorium-mediated penetration
        
        Acetyl CoA from fatty acid b-oxidation could, for example, be processed via the glyoxylate cycle and gluconeogenesis to yield precursors to glycerol biosynthesis,which is necessary for turgor generation
        
        cytorrhysis assays
        
        no observable difference in appressorium
        turgor in Dpth2 mutants
        
        PTH2 is involved in the supply of acetate units for melanin biosynthesis via the dihydroxynaphthalene pathway
        
        appressorium melanization is vital for
        plant infection by M. grisea
        
        not the primary reason for loss of pathogenicity in
        Dpth2.
        
        appressorial melanin biosynthesis does
        occur in Dpth2 mutants
        
        appressorium turgor
        generation was normal in Dpth2 mutants,
        
        1 more item...
        
        Dpth2 mutants clearly showed a reduction
        in melanin production in appressoria
        
        Pth2 activity contributes to melanin biosynthesis , CAT not essential for turgor generation in appressoria
        
        peroxisomal fatty acid b–oxidation is vital for facilitating appressorium mediated infection in M. grisea
        
        Pth2 acts to make acetyl CoA available to the glyoxylate cycle which feeds through reactions of the gluconeogenesis pathway to support glucan and chitin biosynthesis, which are required for cell wall generation and growth of penetration hyphae
        
        addition of exogenous glucose or sucrose to appressoria of Dpth2 partially restores their ability to cause disease
        
        calcofluor white
        staining
        
        reduced chitin in infection hyphae in PTH2 mutants
        
        hyphae don't form on host leaves
        
        Pth2 is necessary for efficient cell wall biosynthesis during penetration peg development
        
        mutant phenotype PTH2 mutants completely non-pathogenic
  - - - importance of acetyl CoA as a substrate for several
        metabolic and biosynthetic pathways
  - - - highlights the dependence of foliar plant pathogens on lipid metabolism for supporting initial growth and development on the leaf surface
    - - Deletion of the PEX6 peroxin encoding prevents peroxisome biogenesis and renders M.grisea completely non-pathogenic, demonstrating the significance of peroxisomal activities in appressorium function
    - - woronin essential for appressorium function in M. grisea
- - - - presence of glucose in medium did not affect PTH2 transcript levels when the fungus exposed to acetate or glycerol
    - - Dmac1sum1–99 mutant which lacks the regulatory
        subunit of PKA
      - lacks catalytic subunit of PKA
  - - - mitochondria
      - peroxisomes
    - - provides the major exit route from fatty acid metabolism in oilseed plants
      - contrast to dependency on peroxisomal
        carnitine shuttle
    - - complement all Dpth2 mutant phenotypes
      - fully functional
    - - observed with a punctate distribution in
        vegetative hyphae grown in axenic culture
    - - To investigate the cellular distribution
      - an A. nidulans gene that encodes multifunctional fatty acid b-oxidation enzyme containing type-1 peroxisomal-targeting signal
  - - - hygromycin-resistant transformants selected and analysed by DNA gel blot
  - - - spores germinated normally
        and developed appressoria
    - - After 48 h, 61 ± 4% of the Dpth2 mutant had successfully penetrated.
  - - - liberate fatty acids in the appressorium
    - - to test whether Pth2 is required for turgor generation in the appressorium
      - No significant difference in appressorial turgor between pth2 mutant
      - PTH2 is not needed for generation or maintenance of appressorium turgor
  - - - buf1 mutant= thick, diffuse layer of
        microfibrillar melanin precursors
      - pth2 mutant= melanin
        layer was observed, but was reduced in thickness
      - Guy 11 and pth2 mutants
        
        Appressorium melanization can readily observed in both copy
    - - no restoration of lesion formation in
        pth2 mutants by addition of scytalone
      - reduction in melanin production observed in pth2 mutants is not the primary reason for their lack of pathogenicity
  - - - glycogen and lipid reserves move into the developing infection structures
      - Lipid bodies then coalesce and are taken-up by vacuoles by autophagocytosis resulting in rapid lipolysis
      - Are these processes are altered in pth2 mutant?
        
        conidia were germinated on plastic coverslips and appressorium development allowed to occur over 48 h
        
        Nile Red solution to
        visualize lipid bodies
        
        Guy 11
        
        Large numbers of lipid bodies were present within conidia, germ tubes and developing appressoria
        
        After 8 h,lipid bodies were predominantly mobilized to appressoria
        
        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
        
        Large number of lipid bodies were still present in germ tubes after 24 h incubation
        
        lipid bodies are not metabolized within appressoria at the same rate as in a wild-type M. grisea strain
  - - - rapid cell wall biosynthesis occurs at the apex of penetration hyphae enabling fungal proliferation in plant tissue
    - - generation of precursors for cell wall biosynthesis must result from degradation of conidial storage products
      - rice cuticle penetration assays
        
        performed using a pth2 mutant in the presence of 2.5% glucose or sucrose solution
        
        ability to cause rice blast symptoms was partly restored to pth2 mutant in presence of either glucose or sucrose
        
        glucose take-up by the fungus can partly restore the functional competence of appressoria, and in particular their ability to form penetration hyphae
  - - - cell wall biosynthesis might be affected in the mutant
    - - Incubation of pth2 mutant on onion epidermis
      - The fungus was allowed to form appressoria and then stained with calcofluor white, which binds to chitin in the cell wall
      - Calcofluor white fluorescence was observed in the cell walls of appressoria of both Guy 11 and Dpth2, but was not observed in penetration hyphae of pth2 mutant even after 48 h of development
      - Chitin synthesis during invasive hypha formation is impaired in the pth2 mutant.