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The glyoxylate cycle is required for temporal regulation of virulence by…

- - - - to generate glucose
    - - lipid metabolism for ATP generation
        
        involve B-oxidation & production of acetyl CoA.
        
        acetyl CoA is channelled through glyoxylate cycle via isocitraye lyase & malate synthase
    - - impaired in virulance-associated functions
      - retain the capacity to cause disease
        
        uneffected in their ability to invade and proliferate normally in plant tissue
        
        Defect in virulence results in delay in penetration of development. Leading to a lower frequency of appressorium-mediated infection events.
    - - occured in a nutrient-free environment lipid bodies are known to be mobilized from the conidium to the germ tube apex
    - - M. grisea uses lipid metabolism extensively during appressorium formation
        
        Lipid metabolism may contribute to ATP generation and fuel secondary metabolic pathways, (melanin biosynthesis) which critical for appressorium function.
    - - glyoxylate
        cycle is stimulated
      - during penetration peg formation and the generation of invasive hyphae
    - - Mycobacterium tuberculosis, in which isocitrate
        yase mutants show attenuation in virulence
        
        both M. grisea and C. albicans have co-opted the glyoxylate cycle into fulfilling a specific role in pathogenesis in response to a glucose-deficient environment, the leaf surface in the case of M. grisea, and the phagocytic macrophage invaded by C. albicans
    - - during infection by the human pathogenic fungus Cryptococcus neoformans (although dispensable for pathogenesis),
        
        to support growth of the intracellularpathogenic bacterium Rhodococcus equi on lipids, which it uses in the phagolysosome
      - Foliar pathogens of plants need a means of germinating
        
        developing on the leaf surface in the absence of external nutrients
        
        many species carry out morphogenetic programmes resulting in the development of
        specific infection structures in this environment.
    - - fundamental to allowing these development to proceed before host invasion.
      - a requirement for ICL in virulence has been observed very recently based on the results of an insertional mutant screen in the brassica pathogen Leptosphaeria maculans, in which a virulence mutant was found to carry an insertion in an ICL-encoding gene
- - - - to withstand the extensive battery of chemical and physical defence mechanisms deployed by plants.
  - - - Appressoria are dome-shaped, melanin-pigmented cells, which develop enormous turgor in order to generate an invasive force to rupture the rice leaf cuticle
      - Having gained entry to the plant tissue, the fungus then grows rapidly, invading plant cells and bringing about observable disease symptoms within 3 days
      - the fungus can prevent grain-filling or destroy the grain-bearing structures of the plant
    - - enormous turgor in appressoria
        
        Glycerol acts as a highly soluble osmolyte, causing rapid influx of water into the
        appressorium to generate hydrostatic turgor.
        
        biosynthesis of glycerol occurs rapidly during appressorium morphogenesis and involves mobilization of storage reserves from the conidium
        
        glycerol is synthesized predominantly via triacylglycerol lipase activity in mature appressoria, using lipid reserves mobilized from the conidium
        
        rapid lipolysis in M. grisea
        
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  - - - Targeted mutation of ICL1 led to mutants that showed a significant delay in spore germination and disease symptom expression.
- - - - Isocitrate lyase catalyses the production of glyoxylate from isocitrate and is the first step in the glyoxylate pathway
        
        allows acetyl CoA to be converted to pyruvate and, subsequently, via gluconeogenesis, to glucose.
    - - used to identify a corresponding cDNA and genomic clone spanning the gene locus
        
        The putative ICL1 Gene product showed 85.5% amino acid identity to the isocitrate lyase gene acu3 Of Neurospora crassa and 76.3% identify to acuD of Aspergillus nidulans
    - - The ICL1(p):sGFP promoter fusion was introduced into a wild-type strain of M. grisea , Guy 11, and transformants with a single integration of the plasmid were selected by DNA gel blot analysis
    - - Expression declined initially during germ tube emergence and elongation, before appressorium development
        
        Once formed, appressoria of ICL1(p):sGFP transformants were highly fluorescent, particularly at the onset of turgor generation, 24–48 h after spore germination
        
        When conidia were incubated on a surface that can be breached by M. grisea appressoria, such as sterile onion epidermis, the expression of ICL1(p):sGFP was followed throughout the infection process.
        
        Appressoria were highly fluorescent during turgor generation and emerging penetration pegs also fluoresced brightly.
        
        After cuticle penetration, M. grisea formed highly vacuolated, bulbous, branched infection hyphae, which also showed a high level of ICL1(p):sGFP expression.
        
        An M. grisea transformant expressing sGFP under the control of the A. nidulans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, which gives constitutive expression in most ascomycete fungi showed very low levels of sGFP fluorescence throughout appressorium formation and invasive growth
- - - - Mycelium was removed by filtration and transferred to minimal medium (glucose or acetate (6mM) as sole carbon source)
        
        Replicate samples of mycelium were removed by filtration through Miracloth, washed thoroughly with dH2O, snap frozen in liquid nitrogen and lyophilized
        
        Samples (1 mg) of
  - - - PCR
        
        gene replacement
        
        The resulting plasmid, pZWICL1, was digested with Xhol and SalI releasing a 1.65 kb fragment containing the ICL1 protein-coding sequence
        
        The linearized plasmid was ligated to a 1.4 kb XhoI–SalI-linked hygromycin phosphotransferase gene cassette to create the gene replacement vector pDICL1
        
        The EcoRI insert was liberated and transformed into M. grisea strain Guy 11
        
        construction of the ICL1p:sGFP expression vector, a 1.5 kb promoter fragment from the 5' end end of ICL1 was amplified from pZWICL1 and ICL-N which introduced an NcoI site at the 3' end to allow fusion of the
        promoter fragment to the start codon of sGFP.
        
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        5.3 kb EcoRI fragment spanning ICL1 was cloned into a modified pBluescript vector (Stratagene) from which XhoI
        and SalI sites had been removed from the multiple cloning site.
  - - - Media composition, fungal
        growth, nucleic acid extraction and DNA-mediated transformation
        
        Restriction digests, gel electrophoresis and
        DNA gel blot hybridizations