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

- - - - required for fully virulent and produce acute rice blast symptoms
  - - - appear to be unaffected in their ability to invade and proliferate normally
  - - - melanin biosynthesis for appressorium function
- - - - To investigate the reasons why plant infection might be delayed and result in less acute disease symptoms
        
        the ability of icl1 mutants to perform a number of virulence-associated functions, including completion of the prepenetration stage of development
        
        the emergence of germ tubes after conidial
        germination was delayed in icl1 mutants
        
        GUY 11
        
        96.19 ± 1.06% conidia had formed a germ tube 2 h
        after inoculation onto plastic surfaces
        
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        formed appressoria from 74.17 ± 18.64%
        of germ tubes within 8 h of germination
        
        Guy 11, 70.5 ± 3.28% of appressoria had
        successfully penetrated epidermal layers after 24 h
        
        I-10
        
        29.06 ± 16.25% in the icl1 mutant
        
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        The formation of appressoria was
        also substantially delayed
        
        I-11
        
        44.7 ± 6.36% of appressoria had completed
        infection
        
        The rate of successful appressorium-mediated penetration of plants was determined by allowing conidia to germinate on onion epidermis and form appressoria
        
        observed a delay in turgor generation by appressoria when assayed using an incipient cytorrhysis test
        
        measures the number of
        cells collapsing after exposure to solute of varying concentrations
        
        Hyperosmotic solutions cause the cells to
        collapse allowing an assessment of their internal turgor
        
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      - targeted deletion of ICL1 results in retardation and impairment of a number of virulence-associated processes in M. grisea
      - appressorium turgor generation in M.grisea
        
        accompanied by movement of lipid droplets into developing appressoria
        
        coalescence of droplets and take-up by vacuoles, followed by rapid lipolysis
        
        guy 11 & icl1 mutant(I-10) able to form appressoria
        
        nile red staining
        
        abundant lipid droplets in both strains, which degraded
        during spore germination
        
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        absence of the ICL1 gene delays appressorium maturation and turgor generation and may contribute to the reduction and delay in disease symptom expression by icl1 mutants.
    - - to determine whether ICL1
        encodes isocitrate lyase and to characterize its role in
        M. grisea
      - 5.3 kb EcoR1 fragment spanning the ICL1 gene locus was selected
      - 1.8 kn Xhol-Sal fragment that contain the majority of protein-coding sequence was removed and replaced with hygromycin phosphotransferase gene cassette
      - I-7, I-10, I-11, gene probe derived from protein-coding region of ICL1 failed to hybridize
        
        They fail to grow but wild-type Guy 11and ectopic transformants; I-6 and I-8 grew normally on media containing either sodium acetate or olive oil as sole carbon source
        
        M. grisea ICL1 encodes isocitrate lyase, that involve in proliferation of the fungus on two carbon compounds or after lipid metabolism
      - Targeted deletion of ICL1 did not cause observable effects on hyphal elongation or mycelial growth.
        
        reduction in conidiogenesis by ICL1 mutants 50% compared with the isogenic wild type
        
        reduction in observable disease symptoms on all seedlings inoculated with ICL1 mutants I-7 or )-10 compared with plants inoculated with Guy 11 or transformants I-8 or I-30
        
        gene replacement vector had integrated at an ectopic
        site in the genome
        
        In rice seedlings inoculated with
        Guy 11, rice blast disease symptoms began to appear as early as 48 h after inoculation with spore suspensions
        
        seedlings inoculated with Dicl1 mutants did not exhibit rice blast symptoms until 96 h after inoculation
  - - - First step in glyoxylate pathway that allows acetyl-CoA to be converted to pyruvate & glucose (gluconeogenesis)
    - - Putative ICL1 gene product showed
        
        85.5% amino acid identity to the isocitrate lyase gene acu3 of Neurospora crassa
        
        76.3% identify to acuD of Aspergillus nidulans
    - - A 1.5 kb promoter fragment upstream of the ICL1 protein-coding sequence was fused to the green fluorescent protein-encodimg gene sGFP
        
        Conidia, harvested from plate cultures of a ICL1(p):sGFP transformant, exhibited high levels of GFP fluorescence
        
        Appresoria was highly fluorescent during turgor generation, and emerging penetration pegs also fluoresced brightly
        
        After cuticle penetration, M.grisea formed highly vacuolated, bulbous, branched, infected hyphae, which also showed a high level of ICL1(p):sGFP expression
- - - - Dome-shaped
      - Melanin-pigmented cells
      - Develop enormous turgor pressure
        (To generate invasive force to rupture rice leaf cuticle)
    - - Grows rapidly and bring about observable disease symptoms within 3 days
      - In heavy infections, disease can be so severe that whole seedlings die
      - In older plants, fungus can prevent grain-filling or destroy the grain-bearing structures of the plant
  - - - Involves mobilization of storage reserves from the conidium
      - This rapid lipolysis is likely to be the generation of fatty acids and subequently after b-oxidation, of acetyl CoA
        
        M.grisea may require a mechanism for using acetyl-CoA to allow subsequent development of the pathogen after plant infection
  - - - Indicate that glyoxylate cycle may be of wodespread importance to fungal pathogens in both animal and plants
- - - - This eliminated
        bias resulting from steric hindrance in ELISA
  - - - 5.3 kb EcoRI fragment spanning ICL1 cloned into
        modified pBluescript vector
      - XhoI and SalI sites had been removed from the multiple cloning site.
    - - introduced NcoI site at 3¢ end allow fusion of promoter fragment to the start codon of sGFP
  - - - growth was measured after 12 days on minimal medium agar with 6 mM glucose
      - sodium acetate or 2 g 1 olive oil as sole carbon source
    - - allowing conidia to germinate on hydrophobic plastic coverslips and incubating in humid environment for 24 h at 24∞C
    - - determined at different time intervals by counting number of germ tubes and/or appressoria formed from 300 conidia
    - - estimated using an adaptation of the incipient cytorrhysis (cell collapse) assay
    - - assessed by Nile red staining
    - - assessed by recording the frequency of penetration
        peg formation from appressoria on onion epidermis
    - - determined microscopically by counting the penetration events from 100 appressoria.