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The glyoxylate cycle is required for temporal regulation of virulence by…
The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic Magnaporthe grisea
Introduction
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- recent studies: significant of isocitrate lyase in two distinct microbial pathogens of human host
- this experiment : indicate that the glyoxylate cycle may be of widespread importance to fungal pathogens in animals & plants
- glycogen is synthesized predominantly via triacylglycerol lipase activity
- lipolysis in M.grisea is from generation of fatty acid and after B-oxidation of acetyl CoA :-
-M.grisea required mechanism for using acetyl CoA to allow development of pathogen after plant infection
-investigate the role of glyoxylate cycle
Discussion
TCA cycle with glyoxylate cycle
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ICL1
- Less virulent than isogenic wild-type strain
- Impaired in virulence associated function that associated with preparation stage of development resulting in lower frequency of appressorium-mediated infection events
~ Germ tube emergence
~ Appressorium development
~ Cuticle penetration
- Retain capacity to cause disease because they can still invade & proliferate normallyin plant tissue
- Highly expressed during this means that glyocylate cycle is stimulated at this time
~ Conidial germination
~ Appressorium formation
- Also highly expressed during:
~ Penetration peg formation
~ generation of invasive hyphae
Lipid metabolism
- During conidial germination and appressorium development, lipid bodies are transported from conidium to germ tube apex
- At onset of turgor generation, lipid bodies merge and taken up by vacuoles before rapid lipolysis - lipid metabolism
- Lipid metabolism contribution
~ ATP generation
~ Fuel secondary metabolic pathway (eg, melanin biosynthesis - critical for appressorium function)
- Lipolysis
~ Important for turgor generation in appressoria via synthesis of glycerol that highly accumulated in glycerol
- Lipid metabolism induce gyoxylate cycle
~Consequence of reliance upon lipid metabolism for turgor generation during penetration stage of development
~ To provide a mechanism of generating glucose
Candida albicans
- Requires isocitrate lyase to be fully virulent in mouse model of candidiasis
Mycobacterium tuberculosis
- Isocitrate mutants show attenuation in virulence
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Procedure
Quantification of ICL1p:sGFP expression by ELISA
- Magnaporthe grisea mycelium from Guy 11 and
ICL1(p):sGFP transformants ZWI1-2, 1-4 and 1-6 were grown
- ELISA was carried out
Phenotypic analysis of Dicl1 mutants
- colony diameter grown on complete media are measured
- for various carbon source utilization; grown on glucose, sodium acetate or olive oil
- appressorium development; conidia was allowed to germinate on hydrophobic plastic cover slip, number of germ tubes were counted at various time intervals
- appressorium turgor; estimated using an adaptation of the incipient cytorrhysis assay
- lipid mobilization during appressorium development; assessed using Nile red staining
- cuticle penetration; frequency of penetration peg formation on onion epidermis
Identification and targeted gene replacement of
M. grisea ICL1
- Genomic DNA from M. grisea Guy 11 was used for polymerase chain reaction (PCR) amplification
- The amplicon was cloned and sequenced and used to identify corresponding genomic and cDNA clones
- For gene replacement, a 5.3 kb EcoRI fragment spanning ICL1 was cloned into a modified pBluescript vector
- ICL1p:sGFP expression vector was constructed
Plant infections
- M. grisea conidial suspension was sprayed evenly on rice plant and incubated until symptoms appear