Genomic DNA from M.grisea Guy 11 used for PCR amplification with the primers ZW-1 (5'-ACTACCTGCGACCCATTATTGC-3') and ZW-2 (5'-GAGAAGGTCGACACCTGAGTC-3'),designed based on available M.grisea EST sequence

Isocitrate lyase catalyses production of glyoxylate from isocitrate (first step in glyoxylate pathway).

amplicon was cloned and sequenced and used to identify corresponding genomic and cDNA clones

gene replacement, 5.3kb EcoR1 fragment spanning ICL1 was cloned into modified pBluescript vector from which vhol and Sall sites has been removed from multiple cloning site

resulting plasmid, pZWICL1 digested with Xhol and Sall release 1.65kb fragment contain ICL1 protein-coding sequence

ICL1 gene translated product = 85.5% and 76.3% identity to isocitrate lyase acu3 gene of N. crassa and acuD gene of A. nidulans.

linearized plasmid was ligated to 1.4 kb Xhol-Sall-linked hygromycin phosphotransferase gene cassette to create the gene replacement vector p mutant ICL1

1.5 kb promoter upstream of ICL1 fused with GFP gene and introduced into wild-type M. grisea (Guy11) and collect transformants identified by DNA gel blot analysis.

Ecor1 insert was liberated and transformed into M. grisea strain Guy 11

ICL1p:sGFP expression vector, 1.5kb promoter fragment from 5' end of ICL1 was amplified from pZWICL1 with primers T3 and ICL-N that introduced an Ncol site at the end 3' end -to allow fusion of the promoter fragment to start codon of sGFP

Expression of GFP decreased initially during germ tube development but appressorium formed later highly fluorescence especially during turgor generation.

Upon exposure to plant cuticle, ICL1-GFP highly expressed (appressorium and penetration peg together with vacuolated, bulbous, branched infection hyphae were highly fluorescence)

amplified DNA fragment was digested with EcoR1 and Ncol and cloned into pMJK80 that contain sGFP allele and A.nidulans trpC terminator

resulting ICL1(p):sGFP fusion was excised as EcoR1-Xhol fragment and cloned into an M.grisea transformation vector pCB1532

Not ICL1 but GAPDH promoter low expression during infection.

complementation of Dicl1 mutants, a 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 mutant cl1 mutant I-10, and transformants were selected on
150 mg ml-1 chlorimuron ethyl

all M.grisea transformatants, DNA gel blot analysis was carried -to ensure single-copy integration of plasmid DNA

Guy11 and ZWI1-4 transformants growth in rich medium and recovered mycelium to be transferred into minimal medium (only glucose or sodium acetate as sole carbon source).

Mycelium were removed and transferred at 4 intervals and protein extracts were made. GFP quantified by ELISA with monoclonal anti-GFP Ab (specific; no cross-reaction with Guy11 protein).

ICL1 promoter responded to acetate rapidly -> abundant GFP production.

vegetative growth was assessed by measurement of colony diameter on plate cultures of M.grisea grown on complete medium

Results consistent with the RNA gel blot analysis therefore, 2-carbon compound as sole carbon source required for high expression of ICL1 which is also needed for infection.

conidial development was assessed by harvesting conidia from surface of 12-day-old plate cultures and determining the conc. of resulting conidial suspension using haemocytometer

investigate utilization of carbon sources by mutants icl1 mutants-growth was measured after 12 days on minimal medium agar with glucose, sodium acetate or 2 gl-1 olive oil as sole carbon source.

Aim: To determine whether ICL1 encodes for isocitrate lyase and its role in M. grisea.

appressorium development was assessed by allow conidia to germinate on hydrophobic plastic ceverslips

EcoRI fragment was selected but XhoI-SalI fragment (with majority of protein-encoding sequence) were removed and replaced with hygromycin phosphotransferase gene cassette (function for resistance to hygromycin B).

appressorium turgor was estimated using an adaptation of incipient cytorrhysis (cell collapse) assay

glyoxylate cycle assimilate 2 carbon compound into tricarboxylic acid cycle (TCA)and gluconeogenesis to generate glucose.

cuticle penetration was assessed by recording the frequency on penetration peg formation from appressoria on onion epidermis

The resulting construct were introduced into wild-type M. grisea pathogenic strain. DNA extracted from identified transformants (I-7. I-10 and I-11 with confirmed gene replacement as gene probe failed to hybridised) were analysed using DNA gel blots.

plant infections were performed using 14-day-old seedlings of rice cultivar CO-39 or 10-day-old seedlings of barley cultivar Golden Promise that both are very susceptible to blast disease

glyoxylate cycle using lipid metabolism as main source for ATP germination, invole beta-oxidation of fatty acid n production of acetyl CoA.
acetyl CoA ->glyoxylate cycle via isocitrate lyase
the production of malate by malase synthase.

M.grisea conidial suspension containg 104 conidia ml-1 was prepared in 0.2% (v/v) gelatin solution

suspension was sprayed evenly onto rice plants and seedlings were incubated in a controlled environment chamber until disease symptoms appeared

The I-7. I-10 and I-11 transformants were cultured on NaAc or olive oil sole carbon source containing media to determine whether the glyoxylate cycle were affected by ICl1 mutant. FAILED to grow.

disease lesion densities were recorded from 20 to 30 infected leaves using 5cm section of each leaf.

Guy11 and other ectopic transformants grow normally concluding ICL1 which codes for isocitrate lyase is important for growth on 2-carbon compounds.

during conidial germination n appressorium formation: occurs in nutrient-free environment
lipid body mobilized from conidium to germ tube apex

during the presence of turgor genertion, lipid bodies taken up by vacuoles before lipolysis.
M.grisea use lipid metabolism during appressorium formation which conribute to ATP generation and melanin biosynthesis.
lipolysis important for turgor generation via synthesis of glycerol which accumulate in appresoria

media composition, fungal growth, nucleic acid extraction and DNA-mediated transformation were carried out

restriction digests, gel electrophoresis and DNA gel blot hybridizarions- suing standard procedure

during the prepenetration stage induction of the glyoxylate cycle to provide generation of glucose.

high-level express of ICL 1 during conidia germination n apressorium formation indicates glyoxylate cycle is stimulated.
ICL 1 express during penetration peg formation n invasive hyphae.

initial stage of infection:
-lipid metabolism ploriferate
-glucose acquisite from plant tissue

Candida albicans: require isocitrate lyase to be virulent
M.grisea n C.albicans