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The Molecular Biology of Appressorium Turgor Generation by the Rice Blast…
The Molecular Biology of Appressorium Turgor Generation by the Rice Blast Fungus
Magnaporthe grisea
Glycogen metabolism in
M. grisea
Glycogen
Abundant within the spores of
M. grisea
Mobilized very quickly on germination
Rosettes accumulate within appressoria
during development
During onset of turgor
generation
Disappears from appressoria
during melanization and turgor generation
Degrade by two enzyme activities
Glycogen phosphorylase
Amyloglucosidase
Glycogen mobilization
Regulated by the cAMP
response pathway
∆cpkA mutants show retarded degradation of glycogen during conidial germination and during initiation of appressorium development
PKA mutant
∆mac1 sum1-99
Rapid degradation of glycogen before onset of melanin
production
pls1 mutant
Non-pathogenic and produces completely non-functional
appressoria
Accumulates glycogen deposits within infected
cells
Encodes a tretraspanin
Novel membrane protein required to control translation of turgor into physical force for penetration hypha production.
MST12
-encoded transcription factor
Causes penetration hyphae emergence to occur independent of turgor generation
Enzymes which glycerol could be synthesized from storage carbohydrates
NADH-dependent glycerol-3-phosphate dehydrogenase
NADPH-dependent glycerol dehydrogenase
Present in appressoria, not induced during turgor generation
Triacylglycerol lipase activated during onset of turgor generation
Trehalose metabolism in
M. grisea
common storage product within microbial cells.
act as a stress metabolite
cellular protectant from desiccation
synthesis of trehalose
M. grisea
UDP-glucose and glucose-6-phosphate are the
substrates in this process
synthesis of trehalose in
Saccharomyces cerevisiae
multienzyme complex is responsible for the synthesis of T6P
T6P is the catalytic subunit of which is encoded by a gene
called TPS1
T6P synthase activity is a means by which the influx of glucose into glycolysis is regulated
M. grisea
∆tps1 mutants
non-pathogenic
potential for trehalose metabolism to lead to glycerol synthesis in the appressorium was explored to determine why this mutant is non pathogenic
appressorium turgor generation was severely
affected in these strains
it also displayed a number of pleiotropic effects.
∆tps1 mutants were unable to grow on glucose or a variety of other rapidly fermentable carbon sources,
unable to utilize acetate or lipids as sole carbon sources.
unable to grow on acetate or
lipids,
Yeast tps1∆ mutants
unable to grow on glucose due to the
absence of T6P synthase activity
this leading to an uncontrolled
influx of glucose into glycolysis
leads to rapid utilization
of ATP
first two steps of glycolysis and a subsequent
drop in ATP and phosphate levels within the cell
glucose is effectively toxic to these cells
The
M. grisea
genome sequence
revealed the presence of two
trehalase-encoding genes,
NTH1
predicted to encode a neutral trehalase,
highly expressed during conidiogenesis and spore germination
TRE1
encoding the main trehalase activity
during M. grisea spore germination
completely dispensable for pathogenesis
TRE1 encodes an unusual
trehalase
it does not show significant similarity to other fungal neutral or acidic trehalases.
Mutants lacking NTH1
reduced in virulence,
decreased ability to perform invasive growth within rice tissues.
Mutants lacking TRE1
produce rice blast symptoms
are identical to those of an isogenic wild-type strain of the fungus
trehalose synthesis is required for
appressorium function
Process of appressorium turgor generation
Appressorium form on surface of rice blast & free water is essential pre-requisite for cell generation
Solutes (glycerol that accumulates in excess 3M) are accumulated within appresssorium
Which cause water flow against concentration gradient into appressorium
Accumulation of glycerol shown by vapour-pressure psychrometry
which require for generation of substantial turgor pressure
Appressorium form on leaf surface in the absence of external nutrients
So, appressorium must be accumulated
de novo
from storage production present in conidia.
Analysis of conidia= reveal that they accumulate lipids, glycogen & dissaccharide trehalose as predominant storage production
Mannitol has reported to accumulate within spores
Recent investigation : focus on trehalose, lipid & glycogen as potential sources of glycerol
compatible solutes during appressorium turgor generation in
M.grisea
Appressorium development by M. grisea
three-celled, teardrop-shaped conidia land on the surface of a rice leaf.
spores germinate
adhere tightly to the hydrophobic surface
extension of a narrow germ tube that emerges from the conidium (
1 hour
)
germ tube starts to swell at its apex, and flattens against the surface of the rice leaf.
develops into a swollen dome-shaped cell, called the appressorium
appressoria become separated from
the germ tube and conidium by a thick septum
become
melanin-pigmented.
deposited in a layer within the
cell wall of the appressorium.
Mutants lacking melanin
2 more items...
buf1 mutant
due to mutation of a gene encoding dihydroxynapthalene
reductase
buff colour
RSY1 gene
encodes scytalone dehydratase
pink colour
ALB1
gene
mutations in a polyketide synthase
albino mutant phenotype
tightly coupled to cell
division
mitosis is always observed within germ tubes
form in response
hydrophobic
surfaces
absence of external nutrients
hard surface
e presence
of constituents of cuticular wax
cAMP
response pathway
after the attachment of
the fungus on the leaf surface.
Mutants lacking adenylate
cyclase (
unable to accumulate cAMP
)
, do not form
appressoria
frequent reversion by a second-site mutation in the regulatory subunit of PKA
cAMP-independent triggering of PKA
restoration of
appressorium development
mutants
lacking the catalytic subunit of PKA
A form small, mis-shaped
and non-functional appressoria
MAP kinase (mitogen-activated protein kinase) cascade
Pmk1
MAP kinase
required for the production of appressoria
mutants lacking the PMK1 gene
arrest the growth before
development of infectious structures
Introduction
Rice blast disease= most serious disease of cultivated rice
that cause serious impact on rice growing plants
Fungus cause blast disease is
Magnoporthe grisea
(ascomycete)
Individual host-limited form of
M.grisea
responsible for blast disease
wheat blast is a serious problem in Brazil
M.grisea
undergoes series of morphogenetic developmental steps
leading to production of specialized infection structure=appressorium
It produce on the surface of rice leaves & brings out plant infection
primarily by physical breakage of lea cuticle
Appressoria generates substantial turgor
Incipient cytorrhysis experiments were carried out by increasing concentrations of polyethylene gel of
M.grisea
To determine the rate of cell collapse
In a way, equivalent turgor within appressoria estimated
Experiment results shows: appressoria of
M.grisea
generate up to 8MPa pressure during plant infection
Appressorium produce narrow penetration hypha at cell base
which forced through underlying cuticle
later develops into hypha that covers leaf
Symptom of rice blast is apparent to 4-5 days after initial infection
Lipid metabolism during
appressorium turgor generation
During
M. grisea
spore germination
Lipid bodies are mobilized quickly from the conidium,
Can be observed accumulating in the germ tube apex
. Lipid bodies are surrounded by a single unit membrane in M.grisea
Appear highly refractile by phase contrast microscopy
Can readily visualized by staining with Nile Red
During appressorium maturation
lipid bodies
coalesce
and taken up by vacuoles
by a process that resembles autophagocytosis
Lipolysis appears
to occur in vacuoles that also coalesce to form a large central vacuole
within the appressorium during turgor generation
Triacylglycerol lipase activity in
M. grisea appressoria
is cAMP-regulated
a regulatory subunit PKA
mutant
carries out rapid lipid degradation in the appressorium
before melanization of the infected cell
4 genes encode intracellular triacylglycerol lipases
These enzymess producing glycerol
from lipid droplets
2 homologues of gene (YMR313c)
in the M. grisea genome,
currently being investigated
Appressorium lipolysis and glycerol
generation
is generation of fatty
acids
glyoxylate cycle
required for pathogenicity in
M. grisea
Mutants lacking the ICL1 gene
encoding isocitrate lyase
reduced in virulence
due to a temporal regulation effect on the development of
appressorium
ICL1 gene expression
shown to be very high
during appressorium morphogenesis
during appressorium morphogenesis
invasive growth of
M. grisea
∆icl1 mutants were capable of causing rice blast symptoms, albeit
in a delayed manner
compared with a wild-type strain of M. grisea,
fatty
acid β-oxidation
for the physiology of appressoria.
because produced
melanin-pigmented appressoria
may be generally important
Conclusion
Importance of turgor generation to appressorium
capable of penetrating into inert plastic membranes
Extra-cellular depolymerizing enzymes act to accelerate the process in living tissue
Rapid generation of turgor
Appressoria accumulates high concentration of compatible solutes such as glycerol
synthesized in large quantities in the appressorium
M.grisea
has evolved a remarkable mechanism involving production of appressoria
for attachment of to the rice leaf surface
for generation of mechanical force to penetrate the rice leaf cuticle
The availability of the full
M. grisea
genome sequence,coupled with the ability to perform gene functional analysis
Relatively high throughput, means that there is a clear opportunity to perform a systems biology approach
To the dissection of fungal pathogenesis in
M. grisea
and, in particular, to resolve the mechanism by which appressorium turgor is generated.
