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THE GLYOXYLATE CYCLE IS REQUIRED FOR TEMPORAL REGULATION OF VIRULENCE BY…

- - - - icl1 mutants 1-7, 1-10 and 1-11 failed to grow whereas the wild-type Guy 11 and two ectopic transformants, 1-6 and 1-8 grew normally
    - - the mean lesion density on leaves of rice seedlings inoculated with Guy11 was higher than in seedlings inoculated with icl1 mutant
        
        in rice seedlings inoculated with Guy11, rice blast disease symptoms began to appear as early as 48h after inoculation with spore suspensions.
    - - the emergence of germ tube after conidial germination was delayed in icl1 mutants.
        
        In Guy 11, the number of conidia form germ tube 2h after is larger compared to in icl1 mutantss 1-10.
        
        after 4h, the rates of germ tube emergence were similar.
    - - in 24h old apppresoria, there were dofference in the number of appressoria that were collapsed after incubation in 1M glycerol and icl1.
        
        this indicates appressoria of icl1 mutants do not develop turgor as rapidly as wild-type M.grisae
        
        turgor level between icl1 and Guy 11 almost identical after 48h
        
        this show that deletion of ICL1 result in retardation and impairment of number of virulence-associated proceses in M.grisea.
    - - there is observation that movement of lipid to form appressoria in icl1 mutant delayed compared to Guy 11
      - by 8h, appressoria in icl1 mutant 1-10 have abundant lipid bodies while degraded in Guy 11
      - by 24h, lipid depleted in both Guy11 and 1-10
      - the conclusion is absence of ICL1 genes delays appressoria maturation and turgor generation and delay in disease symptoms expression by icl1 mutants.
  - - - :checkered_flag: conclude : M. grisea ICL1 express in response to the presence of 2 C compound
        
        highly express during appresorium morphogenesis & invasive growth
      - M. grisea ICL 1 promoter:
        
        respond to the presence of acetate very rapid
        
        led to abundant GFP production within 24 hours
      - GFP:
        
        quantified by ELISA using a monoclonal antiGFP antibody
        
        antibody do not show any cross reaction with mycelial protein extracts from Guy11
      - Cultures of Guy11 and ICL1(p):sGFP transformant ZWI1-4:
        
        prepared in rich medium
        
        mycelium recovered and transferred to minimal growth medium with glucose/ sodium acetate
        
        :red_flag: mycelium removed at 4 intervals after transfer to these cond & made protein extract
      - A calibration curve generated using purified recombinant GFP
      - to measure the induction of ICL1 expression in response to acetate:
        
        used ELISA to quantify GFP abundance in M. grisea transformants
    - - M. grisea transformant:
        
        expressing sGFP under the control of the A. nidulans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter
        :recycle: showed very low levels of sGFP fluorescence
      - after cuticle pentration:
        
        M. grisea highly vacuolated, bulbous, branched infection hyphae
        (high level of ICL1(p):sGFP expression)
      - Appressoria were highly fluorescent:
        
        during turgor penetration
        
        during emerging penetration pegs
    - - conidia were incubated on a surface that can be breached by M. grisea appreso
        eg: sterile onion epidermis
      - conidia harvested from ICL1(p):sGFP transformant:
        
        exhibited high levels of GFP fluorescence
    - - 240 bp fragment of the
        gene:
        
        was amplified and used to identify a corresponding
        cDNA and genomic clone spanning the gene locus
      - EST sequence:
        
        used to design
        ICL1-specific DNA primers and
- - - - the production of acetyl CoA
        
        channelled through the glyoxylate cycle via
        
        isocitrate lyase-mediated production of glyoxylate
        
        production of malate by malate synthase
      - B-oxidation of fatty acid
    - - co-opted the cycle into fulfilling a specific role in pathogenesis in response to a glucose-deficient env.
    - - ICL1 highly expressed
    - - uses in phagolysosome
  - - - turgor generation
        
        induction of the glyoxylate cycle to provide a mechanism of generating glucose
  - - - germ tube emergence
      - cuticle penetration
      - appressorium development
    - - unaffected in ability to invade & proliferate in normal tissue
    - - delay in completing prepenetration stages of development
      - lower frequency of appresorium-mediated infection
    - - need isocitrate lyase to be fully virulent
- - - - 1.5 kb promoter fragment from the 5¢
        end of ICL1 was amplified from pZWICL1
        
        primers T3 and ICL1-N : introduced an 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 Nco1
        
        cloned into pMJK80, which contains the sGFP allele and A. nidulans trpC terminator
        
        resulting ICL1(p):sGFP fusion was excised as a EcoRI–XhoI fragment and cloned into an M. grisea transformation vector pCB1532
      - complementation of Dicl1 mutants: 5.3 kb EcoRI
        fragment spanning the ICL1 locus was cloned from pZWICL1 into pCB1532, which carries a selectable marker bestowing resistance to sulphonylurea
      - The resulting plasmid, pICL1532, was introduced into M. grisea Dicl1 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.