the glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungi M.grisea
Discussion
results
Targeted gene replacement of
M. grisea ICL1
Introduction
Glyoxylate Cycle -> TCA Cycle -> gluconeogenesis = glucose
lipid metabolism as predominant source for ATP generation
Plant pathogenic fungi evolved
b-oxidation of fatty acids and the production of acetyl CoA
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To investigate the reasons why plant infection might be delayed and result in less acute disease symptoms
acetyl CoA -> glyoxylate cycle via isocitrate lyase-mediated production of glyoxylate and the production of malate by malate synthase
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the ability of icl1 mutants to perform a number of virulence-associated functions, including completion of the prepenetration stage of development
To breach tough plant cuticles and invade underlying tissues
required for fully virulent and produce acute rice blast symptoms
the emergence of germ tubes after conidial
germination was delayed in icl1 mutants
icl1 mutants impaired in virulence-associated functions
GUY 11
I-10
To withstand extensive battery of chemical & physical plant defense mechanism
affect germ tube emergence, appressorium development and cuticle
penetration
96.19 ± 1.06% conidia had formed a germ tube 2 h
after inoculation onto plastic surfaces
29.06 ± 16.25% in the icl1 mutant
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By 4 h after inoculation,the rates of germ tube emergence were, however, similar
The formation of appressoria was
also substantially delayed
Magnaporthe grisea
retain the capacity to cause disease
formed appressoria from 74.17 ± 18.64%
of germ tubes within 8 h of germination
Causal agent of rice blast disease
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appear to be unaffected in their ability to invade and proliferate normally
The rate of successful appressorium-mediated penetration of plants was determined by allowing conidia to germinate on onion epidermis and form appressoria
Infects host by means of specialized structure called appresorium
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Dome-shaped
virulence defect results
Melanin-pigmented cells
I-11
Guy 11, 70.5 ± 3.28% of appressoria had
successfully penetrated epidermal layers after 24 h
Develop enormous turgor pressure
(To generate invasive force to rupture rice leaf cuticle)
44.7 ± 6.36% of appressoria had completed
infection
delay in completing the prepenetration stages of development causing lower frequency of appressorium-mediated
infection events
observed a delay in turgor generation by appressoria when assayed using an incipient cytorrhysis test
M. grisea uses lipid metabolism extensively during appressorium formation
measures the number of
cells collapsing after exposure to solute of varying concentrations
ATP generation and fuel secondary metabolic pathways
Hyperosmotic solutions cause the cells to
collapse allowing an assessment of their internal turgor
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melanin biosynthesis for appressorium function
there was a significant difference in the number of appressoria that were collapsed after incubation in 1 M glycerol
Experimental procedures
Plant infections
Quantification of ICL1p:sGFP expression by ELISA
Identification and targeted gene replacement of
M. grisea ICL1
Phenotypic analysis of Dicl1 mutants
Fungal isolates and culture conditions
wild-type M. grisea strain Guy 11 was
used
Restriction digests, gel electrophoresis and
DNA gel blot hybridizations were carried out
Media composition, fungal
growth, nucleic acid extraction and DNA-mediated transformation
Genomic DNA from M. grisea Guy 11 was used for PCR
Gene replacement
5.3 kb EcoRI fragment spanning ICL1 cloned into
modified pBluescript vector
XhoI and SalI sites had been removed from the multiple cloning site.
pZWICL1 digested with XhoI and SalI releasing 1.65 kb fragment containing ICL1 protein-coding sequence.
Guy 11 (19.9 ± 8.6
icl1 mutant (30.3 ± 9.4).
Entry to plant tissue
lipolysis is important for turgor generation in appressoria -> synthesis of glycerol -> accumulates to very high
concentrations in appressoria
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indicates that appressoria of icl1 mutants do not develop turgor as rapidly as wild-type M.grisea
Grows rapidly and bring about observable disease symptoms within 3 days
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Turgor levels after 48 h of appressorium maturation were almost identical between the icl 1 mutant and Guy11
In heavy infections, disease can be so severe that whole seedlings die
linearized plasmid ligated to 1.4 kb XhoI–SalI-linked hygromycin phosphotransferase gene cassette tcreate gene replacement vector pDICL1
In older plants, fungus can prevent grain-filling or destroy the grain-bearing structures of the plant
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targeted deletion of ICL1 results in retardation and impairment of a number of virulence-associated processes in M. grisea
EcoRI insert liberated and transformed into M grisea strain Guy 11.
appressorium turgor generation in M.grisea
Glycerol
High expression of ICL1
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during conidial germination
and appressorium formation
expression vector, 1.5 kb promoter fragment from 5¢ end of ICL1 amplified from pZWICL1 with primers T3 and ICL1-N
accompanied by movement of lipid droplets into developing appressoria
penetration peg formation and
during the generation of invasive hyphae
coalescence of droplets and take-up by vacuoles, followed by rapid lipolysis
Accumulation of it in large quantities cause turgor pressure in the cell
guy 11 & icl1 mutant(I-10) able to form appressoria
Highly soluble osmolyte, causing rapid rapid influx of water into appresorium to generate hydrostatic turgor
nile red staining
introduced NcoI site at 3¢ end allow fusion of promoter fragment to the start codon of sGFP
abundant lipid droplets in both strains, which degraded
during spore germination
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required by C. albicans to be fully virulent in a mouse
model of candidiasis
Movement of lipid droplets to developing appressoria occurred in the icl1 mutant, but was delayed compared with Guy 11
Biosynthesis of glycerol occurs rapidly during appresorium morphogenesis
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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.
Involves mobilization of storage reserves from the conidium
required by pulmonary bacterium Mycobacterium tuberculosis shows attenuation in virulence
co-opted the glyoxylate cycle into fulfilling a specific role in pathogenesis
highly expressed during infection by the human pathogenic fungus Cryptococcus neoformans
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support growth of the intracellular pathogenic bacterium Rhodococcus equi on lipids
to determine whether ICL1
encodes isocitrate lyase and to characterize its role in
M. grisea
Glycerol is synthesized predominantly via triacylglycerol lipase activity, which is abundant in mature appresoria, using lipid reserves mobilized from conidium
DNA gel blot analysis carried out to ensure single-copy integration of plasmid DNA
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5.3 kb EcoR1 fragment spanning the ICL1 gene locus was selected
This rapid lipolysis is likely to be the generation of fatty acids and subequently after b-oxidation, of acetyl CoA
1.8 kn Xhol-Sal fragment that contain the majority of protein-coding sequence was removed and replaced with hygromycin phosphotransferase gene cassette
M.grisea may require a mechanism for using acetyl-CoA to allow subsequent development of the pathogen after plant infection
I-7, I-10, I-11, gene probe derived from protein-coding region of ICL1 failed to hybridize
They fail to grow but wild-type Guy 11and ectopic transformants; I-6 and I-8 grew normally on media containing either sodium acetate or olive oil as sole carbon source
ICL1 (encoding isocitrate lyase)
Highly expressed during appresorium development, escpecially in mature appresoria & invasive hyphae
Targeted mutation showed a significant delay in spore germination & disease symptom expression
M. grisea ICL1 encodes isocitrate lyase, that involve in proliferation of the fungus on two carbon compounds or after lipid metabolism
Magnaporthe grisea mycelium grown in complete medium
Mycelium then removed by filtration and transferred to minimal medium
proteins were extracted by maceration in 1 ml of bicarbonate
buffer
Appropriate absorbance values for conversion to GFP equivalents determined from each dilution series of extract and concentrations corrected according to dilution factor
This eliminated
bias resulting from steric hindrance in ELISA
Recent studies showed significance of isocitrate lyase in two distinct microbial pathogens of human hosts
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Targeted deletion of ICL1 did not cause observable effects on hyphal elongation or mycelial growth.
Indicate that glyoxylate cycle may be of wodespread importance to fungal pathogens in both animal and plants
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reduction in conidiogenesis by ICL1 mutants 50% compared with the isogenic wild type
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M.grisea ICL1 is highly expressed during appresorium-mediated plant infection
reduction in observable disease symptoms on all seedlings inoculated with ICL1 mutants I-7 or )-10 compared with plants inoculated with Guy 11 or transformants I-8 or I-30
gene replacement vector had integrated at an ectopic
site in the genome
Isocitrate lyase catalyse the production of glyoxylate from isocitrate
First step in glyoxylate pathway that allows acetyl-CoA to be converted to pyruvate & glucose (gluconeogenesis)
In rice seedlings inoculated with
Guy 11, rice blast disease symptoms began to appear as early as 48 h after inoculation with spore suspensions
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seedlings inoculated with Dicl1 mutants did not exhibit rice blast symptoms until 96 h after inoculation
M.grisea ICL1 was initially recognized from a collection of expressed sequence tags (ESTs) derived from cDNA clone (appresorium-stage cDNA library)
Putative ICL1 gene product showed
85.5% amino acid identity to the isocitrate lyase gene acu3 of Neurospora crassa
76.3% identify to acuD of Aspergillus nidulans
Vegetative growth assessed by measurement of colony
diameter on plate cultures
Conidial development assessed by harvesting conidia from surface of 12-day
investigate the utilization of carbon sources by
Dicl1 mutants
growth was measured after 12 days on minimal medium agar with 6 mM glucose
sodium acetate or 2 g 1 olive oil as sole carbon source
Appressorium development
allowing conidia to germinate on hydrophobic plastic coverslips and incubating in humid environment for 24 h at 24∞C
frequency of conidial germination and appressorium formation
determined at different time intervals by counting number of germ tubes and/or appressoria formed from 300 conidia
Appressorium turgor
estimated using an adaptation of the incipient cytorrhysis (cell collapse) assay
Lipid mobilization
assessed by Nile red staining
Cuticle penetration
assessed by recording the frequency of penetration
peg formation from appressoria on onion epidermis
frequency of cuticle penetration
determined microscopically by counting the penetration events from 100 appressoria.
performed using 14-day-old seedlings of rice cultivar CO-39 or 10-day-old seedlings of barley cultivar Golden Promise
M. grisea conidial suspension containing 104 conidia ml-1 was prepared in 0.2% (v/v) gelatin solution.
suspension was sprayed evenly onto rice plants
seedlings incubated in controlled environment chamber until disease symptoms appeared.
Disease lesion densities recorded from 20 to 30 infected leaves using 5 cm section of each leaf
To investigate the temporal & spatial pattern of ICL1 expression during infection-related development
A 1.5 kb promoter fragment upstream of the ICL1 protein-coding sequence was fused to the green fluorescent protein-encodimg gene sGFP
Conidia, harvested from plate cultures of a ICL1(p):sGFP transformant, exhibited high levels of GFP fluorescence
Appresoria was highly fluorescent during turgor generation, and emerging penetration pegs also fluoresced brightly
After cuticle penetration, M.grisea formed highly vacuolated, bulbous, branched, infected hyphae, which also showed a high level of ICL1(p):sGFP expression