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

- - - - Mycelium were removed by ﬁltration and transferred to minimal medium containing glucose or sodium acetate (6 mM) 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
        
        1 mg sample of freeze-dried mycelium (FDM) placed in Eppendorf tubes, and proteins extracted by maceration in 1 ml of bicarbonate buffer (pH 9.6) using a hand-held homogenizer.
        
        Debris pelleted by centrifugation, 100 ml of supernatant containing solubilized proteins transferred to wells of Immulon II HB microtitre plates
        
        Extracts were double diluted into 50 ml volume of fresh buffer, and proteins were immobilized by incubating the plates for 16 h at 4 degree C in sealed plastic bags
        
        The wells washed four times with PBST [PBS + 0.05% (v/v) Tween 20 (polyoxyethylene-sorbitan monolaurate); P1379; Sigma] and once each with PBS and dH20 and air dried at 23∞C in a laminar ﬂow hood. - Standard calibration curve of recombinant GFP protein was prepared in bicarbonate buffer in microtitre wells as described.
        
        For enzyme-linked immunosorbent assays, wells containing immobilized GFP incubated with mouse anti-GFP monoclonal antibody (mAb) ascites ﬂuid, diluted 1:2500 in PBST for 1 h followed by goat anti-mouse polyvalent (immunoglobulin classes IgG, IgA and IgM) peroxidase conjugate diluted 1:1000 for a further hour.
        
        Bound antibody visualized by incubating wells with tetramethyl benzidine (TMB) substrate solution for 30 min. - Reactions were stopped by addition 3 M H2SO4, and absorbance values were determined at 450 nm with an MRX automated microplate reader.
        
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  - - - 50 ml drop of conidial suspension at a concentration of 1 ¥ 104 conidia ml-1 was placed on the surface of onion epidermis and incubated in a humid environment at 24∞C for 24 h.
        
        The frequency of cuticle penetration was determined microscopically by counting the penetration events from 100 appressoria. The experiments were repeated three times.
    - - For D icl1 mutants, growth was measured after 12 days on minimal medium agar with 6 mM glucose, sodium acetate or 2 g l-1 olive oil as sole carbon source to determine the utilization of carbon sources
    - - The frequency of conidial germination and appressorium formation was determined at different time intervals by counting the number of germ tubes and/or appressoria formed from 300 conidia. Each test was repeated three times.
    - - Appressoria were allowed to form on plastic coverslips incubated in a humid chamber for 24 or 48 h, and the surface water was removed carefully with a pipette and replaced with an equal volume of glycerol solution varying in concentration from 0.5 to 5.0 M
  - - - M. grisea conidial suspension containing 104 conidia ml-1 was prepared in 0.2% (v/v) gelatin solution
        
        The suspension was sprayed evenly onto rice plants as described previously & seedlings were incubated in a controlled environment chamber until disease symptoms appeared.
        
        Disease lesion densities were recorded from 20 to 30 infected leaves using a 5 cm section of each leaf. Infection assays were carried out three times using 45 plants per assay.
  - - - PCR conditions used: 30 cycles of denaturation at 94∞C for 10 s, annealing at 58∞C for 30 s and extension at 72∞C for 3 min.
        
        Initial denaturation step of 3 min at 94∞C and a ﬁnal elongation step at 72∞C for 10 min were performed.
        
        The amplicon was cloned and sequenced and used to identify corresponding genomic and cDNA clones
    - - A 5.3 kb EcoRI fragment spanning ICL1 cloned into modiﬁed pBluescript vector (Stratagene) from which XhoI and SalI sites had been removed from the multiple cloning site.
        
        Plasmid, pZWICL1, digested with XhoI and SalI release 1.65 kb fragment containing 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.
        
        EcoRI insert was liberated and transformed into M.grisea strain Guy 11
    - - 1.5 kb promoter fragment from the 5¢ end of ICL1 was ampliﬁed from pZWICL1 with the primers T3 & ICL1-N
        
        Introduced NcoI site at the 3 end to allow fusion of the promoter fragment to the start codon of sGFP.
        
        Ampliﬁed DNA fragment was digested with EcoRI and NcoI and cloned into pMJK80, that contains sGFP allele & A.nidulans trpC terminator
        
        ICL1(p):sGFP fusion was excised as a EcoRI–XhoI fragment and cloned into M. grisea transformation vector pCB1532
    - - 5.3 kb EcoRI fragment spanning the ICL1 locus was cloned from pZWICL1 into pCB1532, that carries a selectable marker bestowing resistance to sulphonylurea
        
        Resulting plasmid, pICL1532, was introduced into M.grisea** D icl1 mutant I-10, and transformants were selected on 150 mg ml-1 chlorimuron ethyl.
        
        For all M. grisea transformants, DNA gel blot analysis was carried out to ensure single-copy integration of plasmid DNA.
  - - - Use Media composition, fungal growth, nucleic acid extraction and DNA-mediated transformation
        
        Carried out restriction digests, gel electrophoresis and DNA gel blot hybridizations using standard procedures
- - - - Transformants, I-7, I-10 and I-11, a gene probe derived from the protein-coding of ICL1 failed to hybridize
    - - ICL1 mutants, I-7, I-10 and I-11, failed to grow
      - The wild-type, Guy 11 and two ectopic transformants, I-6 and I-8, grew normally
      - M. grisea ICL1 encodes isocitrate lyase,
        
        Involved in proliferation of the fungus on 2 carbon compounds or after lipid metabolism
  - - - susceptible rice cultivar, CO-39
      - susceptible barley cultivar, Golden Promise
    - - with ICL1 mutants I-7 or I-10 compared with plants inoculated with Guy 11 or transformants I-8 or I-30
        
        In rice seedlings inoculated with
        Guy 11, rice blast disease symptoms began to appear as early as 48h
        
        In seedling inoculated with ICL1 mutants not exhibit rice blast symptoms
    - - ICL1 mutants
        
        Emergence of germ tubes after conidial
        germination was delayed
        
        Formation of appressoria delayed
      - Guy 11
        
        Conidia had formed a germ tube 2 h
        after inoculation onto plastic surfaces
        
        Formed appressoria within 8 h germination
        
        Appressoria had successfully penetrated epidermal layers after 24 h
      - Cytorrhysis test
        
        To observe a delay in turgor
        generation by appressoria
        
        By 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
        
        Number of appressoria collapsed after incubation of 1M glycerol between Guy 11 and ICL1 mutants
        
        Appressoria of ICL1 mutants not develop turgor as rapidly as wild-type M. grisea
        
        Turgor levels after 48 h of appressorium maturation were almost identical between the ICL1 mutants and Guy 11
        
        Targeted deletion of ICL1 results in retardation and
        impairment of a number of virulence-associated processes in M. grisea.
      - Rapid lipolysis
        
        To determine whether these processes were affected in
        ICL1 mutants, conidia of Guy 11 and the ICL1 mutant I10 were allowed to form appressoria
        
        Nile Red staining of triacylglycerol
        
        Movement of lipid droplets to develop appressoria in the ICL1 mutant, but delayed compared with Guy 11
        
        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
  - - - recognized from a collection of expressed sequence tags (ESTs) derived from cDNA clones of an appressorium-stage cDNA library
        
        85.5% amino acid identity to the isocitrate lyase gene acu3 of Neurospora crassa
        
        EST sequence ( to design ICL1-specific DNA primers)
        
        240 bp fragment of the gene was amplified and used to identify a corresponding cDNA and genomic clone spanning the gene locus
        
        DNA sequencing revealed a 1970 bp coding region interrupted by four introns (verified by sequencing of genomic and cDNA clones), which was capable of encoding a 547-amino-acid protein of 60 kDa
        
        76.3% identify to acuD of Aspergillus nidulans
      - Isocitrate lyase catalyses the production of glyoxylate from isocitrate ( at the same time, acetyl CoA converted to pyruvate and, subsequently, via gluconeogenesis, to glucose
      - Different carbon sources
        
        Mycelial cultures incubated with sodium acetate or olive oil as sole carbon source - High levels of ICL1(p):sGFP expression
        
        Mycelial cultures grown in glucose-rich medium - no expression
    - - ICL1(p):sGFP promoter fusion
        ( 1.5 kb promoter fragment upstream of the ICL1 protein-coding sequence was fused to the green fluorescent protein-encoding gene sGFP )
        
        introduced into a wild-type strain of M. grisea, Guy 11
        
        transformants with a single integration of the
        plasmid were selected by DNA gel blot analysis
        ( 3 independent tranformants were used )
        
        Once appressoria formed - ICL1(p):sGFP transformants highly fluorescent (particularly at the onset of turgor generation)
        
        When conidia were incubated on a surface that can be breached by M. grisea appressoria, such as sterile onion epidermis - expression of ICL1(p):sGFP was followed throughout the infection process
        
        During germ tube emergence and elongation, before appressorium development - Expression declined
        
        During turgor generation - Appressoria and emerging penetration pegs highly fluorescent
        
        Conidia (from plate cultures of a ICL1(p):sGFP transformant - High levels of GFP fluorescence
        
        After cuticle penetration - M. grisea formed highly vacuolated, bulbous, branched infection hyphae - high level of ICL1(p):sGFP expression
        
        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 - very low levels of sGFP fluorescence throughout appressorium formation and invasive growth
    - - Enzyme-linked immunosorbent assay (ELISA)
        
        to quantify GFP abundance in M. grisea transformants
        
        All the observed expression patterns
        were consistent with results from RNA gel blot analysis ICL1 expression
        
        M. grisea ICL1 promoter responded to the presence of acetate very rapidly and led to abundant GFP production within 24 h
        
        The antibody did not show
        any cross-reaction with mycelial protein extracts from Guy11
- - - - 1) Invade tissues directly or move within the host via its circulatory system
      - 2) In the host, pathogen proliferate while contending with both innate immunity and
        the adaptive immune response
    - - Evolved distinct developmental processes
        1) to breach tough plant cuticles and invade
        underlying tissues
        2) to withstand the extensive battery of chemical and physical defence mechanisms deployed by plants
  - - - melanin pigmented cells
        ( develop turgor to generate an invasive force to rupture the rice leaf cuticle )
      - dome-shaped
    - - 3) invading plant cells
        
        4) brings about observable disease symptoms within 3 days
  - - - Glycerol
        1) highly soluble osmolyte
        2) causing rapid influx of water into the appressorium to generate hydrostatic turgor