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

- - - - A reduction in disease symptoms on all seedlings inoculated with icl1 mutants I-7 or I-10 compared with plants inoculated with Guy 11 or transformants I-8 or I-30, which the gene replacement vector had integrated
      - In rice seedlings inoculated with Guy 11, rice blast disease symptoms began to appear as early as 48 h after inoculation with spore suspensions, and they were very obvious after 72 h
        
        In contrast, seedlings inoculated with icl1 mutants did not exhibit rice blast symptoms until 96 h after inoculation
      - Why plant infection might be delayed
        and result in less acute disease symptoms?
        
        The emergence of germ tubes after conidial
        germination was delayed in icl1 mutants
        
        The formation of appressoria was also substantially delayed in icl1 mutants compared with Guy 11
        
        The emergence of a penetration hypha at the base of the appressorium and rupture of the epidermis was then assayed by light microscopy
        
        a delay in turgor
        generation by appressoria when assayed using an incipient cytorrhysis test was observed
        
        This indicates that appressoria of icl1 mutants do not develop turgor as rapidly as wild-type M. grisea
      - Therefore, targeted deletion of ICL1 results in retardation and
        impairment of a number of virulence-associated processes in M. grisea
        
        The 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
  - - - identification and characterization of putative isocitrate lyase gene.
        
        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.
    - - 1.5 kb promoter fragment upstream of the ICL1 protein-coding sequence was fused to the green ﬂuorescent protein-encoding gene sGFP ( ICL1(p):sGFP)
        
        (A-B) = Conidia, harvested from plate cultures of a ICL1(p):sGFP transformant, exhibited high levels of GFP ﬂuorescence.
        
        (C-D)= Expression decline initially during the emergence and elongation of germ tube, before appressorium development
        
        (E-F)= appressoria of ICL1(p):sGFP transformants were highly ﬂuorescent, at the onset of turgor generation, (24–48 h after spore germination)
        
        (G-H)= M. grisea transformant expressing sGFP under the control of the A. nidulans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, showed very low levels of sGFP ﬂuorescence throughout appressorium formation and invasive growth.
        
        (I-J)= Appressoria were highly ﬂuorescent during turgor generation when conidia were incubated on a surface that can be breached by M. grisea appressoria follow with the expression of ICL1(p):sGFP (infection process)
        
        (M-N)= emerging penetration pegs also ﬂuoresced brightly
        
        (O-P)= After cuticle penetration, M. grisea formed highly vacuolated, bulbous, branched infection hyphae, which also showed a high level of ICL1(p):sGFP expression
      - High levels of ICL1(p):sGFP expression were also observed when mycelial cultures were incubated with sodium acetate or olive oil as sole carbon source
        
        To measure the induction of ICL1 expression in response to acetate, an enzyme-linked immunosorbent assay (ELISA) was used to quantify GFP abundance in M. grisea transformants
        
        A calibration curve was ﬁrst generated using puriﬁed recombinant GFP to allow subsequent quantiﬁcation of GFP within mycelial extracts of ICL1(p):sGFP transformants.
        
        GFP was then quantiﬁed by ELISA using a monoclonal antiGFP antibody
        
        3F= The antibody did not show any cross-reaction with mycelial protein extracts from Guy11.
        
        3G= M. grisea ICL1 promoter responded to the presence of acetate very rapidly and led to abundant GFP production within 24 h.
        
        M. grisea ICL1 is expressed in response to the presence of two carbon compounds as a sole carbon source, and is also highly expressed during appressorium morphogenesis and invasive growth by M. grisea.
  - - - concluded that ICL1 is necessary for
        full virulence in M. grisea
  - - - A 5.3 kb EcoR1 fragment spanning the ICL1 gene locus was selected, and a 1.8 kb XhoI–SalI fragment containing the majority of the protein-coding sequence was removed and replaced with a hygromycin phosphotransferase gene cassette ) bestowing resistance to hygromycin B
        
        4C= Transformants were selected and analysed using DNA gel blots. In these transformants, I-7, I-10 and I-11, a gene probe derived from the protein-coding region of ICL1 failed to hybridize.
        
        To determine whether the glyoxylate cycle was affected in these mutants, the three D icl1 transformants were cultured on media containing either sodium acetate or olive oil as sole carbon source,
        
        D icl1 mutants I-7, I-10 and I-11, failed to grow, whereas the wild-type Guy 11 and two ectopic transformants, I-6 and I-8, grew normally.
        
        All strains grew normally on glucose
      - conclusion
        
        M. grisea ICL1 encodes isocitrate lyase, which is involved in the proliferation of the fungus on two carbon compounds or after lipid metabolism.
- - - - indicates that a cell is using lipid metabolism as
        its predominant source for ATP generation
      - involving boxidation
        of fatty acids and the production of acetyl CoA
      - acetyl CoA is then channelled through the glyoxylate
        cycle via isocitrate lyase-mediated production of glyoxylate
      - production of malate by malate synthase
      - conclusion- M. grisea
        requires the glyoxylate cycle enzyme isocitrate lyase
        
        to be fully virulent
        
        acute rice blast
        symptoms.
  - - - germ
        tube emergence
      - appressorium development
      - cuticle
        penetration
    - - retain the capacity to
        cause disease
        
        unaffected in their ability to invade and proliferate normally in plant tissue
        
        delay in completing the prepenetration stages of dev., leading to a lower freq. of appressorium-mediated infection events
  - - - Genomic DNA from M. grisea Guy 11 was used for PCR amplification with the primers designed based on an available M. grisea EST sequence.
        
        The following PCR conditions were 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.
        -An initial denaturation step of 3 min at 94°C and a final elongation step at 72°C for 10 min were performed.
        
        For gene replacement, a 5.3 kb EcoRI fragment spanning ICL1 was cloned into a modified pBluescript vector from which XhoI and SalI sites had been removed from the multiple cloning site.
        
        The resulting plasmid, pZWICL1, was digested with XhoI 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.
        
        For construction of the ICL1p:sGFP expression vector, a 1.5 kb promoter fragment from the 5’ end of ICL1 was amplified from pZWICL1 with the primers T3 and ICL1-N, which introduced an NcoI site at the 3’ end to allow fusion of the promoter fragment to the start codon of sGFP.
        
        The amplified DNA fragment was digested with EcoRI and NcoI and cloned into pMJK80, which contains the sGFP allele and A. nidulans trpC terminator.
        
        The resulting ICL1(p):sGFP fusion was excised as a EcoRI–XhoI fragment and cloned into an M. grisea transformation vector pCB1532. -For complementation of Dicl1 mutants, a 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 µg ml-1 chlorimuron ethyl.
        
        For all M. grisea transformants, DNA gel blot analysis was carried out to ensure single-copy integration of plasmid DNA.
    - - Plant used : 14-day-old seedlings of rice cultivar CO-39 or 10-day-old seedlings of barley cultivar Golden Promise.
        
        Procedure:
        
        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.
        
        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.
    - - Culture used : Wild-type rice pathogenic M. grisea strain Guy 11.
        
        Media composition, fungal growth, nucleic acid extraction and DNA-mediated transformation were all carried out as described.
        
        Restriction digests, gel electrophoresis and DNA gel blot hybridizations were all carried out using standard procedures.
  - - - High-level expression of ICL1
        
        during conidial germination and appressorium formation indicates that the glyoxylate cycle is stimulated at this time
        
        during penetration peg formation and
        during the generation of invasive hyphae
    - - lipid bodies coalesce and
        are taken up by vacuoles, before rapid lipolysis.
      - M. grisea uses lipid metabolism extensively during appressorium formation.
    - - melanin biosynthesis- critical for appressorium
        function.
    - - may be induction of the glyoxylate
        cycle to provide a mechanism of generating glucose