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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
Introduction
Magnaporthe grisea
Causes rice blast disease
Prevalent in temperate-flooded and tropical-upland rice cropping system
Cause serious crop losses of approx. 10-30% of the rice harvest
Cause by individual host-limited forms of
M. grisea
Undergoes series of defined morphogentic developmental stage
Form infection structure: appressorium
Large number of grass hosts: such as barley and wheat
Appressorium
Generate substantial tugor
Result in production of narrow penetration hypha
At the base of the cell, forced through underlying cuticle
Develop into invasive hyphae filling the epiderman cells of the leaf
Appressorium development by M. grisea
Spore germination proceeds with the germ tube development towrads the conidium till the leave surface
germ tube swell at its apex, and flattens against the surface of
the rice leaf.
Develops into a swollen
dome-shaped cell, called the appressorium
initiated when three-celled, tear
drop-shaped conidia land on the surface of a rice leaf
spores germinate when in contact with the leave and adhere tightly
spore tip mucilage that is released from the apex of the spore
Appressorium Morphogenesis
tightly coupled to cell
division, as mitosis
response to(SIGNALS)
hydrophobic surface
the absence of external nutrients
constituents of cuticular wax
signalling pathways regulate appressorium formation
Triggers cAMP pathways right after the attachment (1ST MUTATION)
Mutants lacking adenylate
cyclase unable to accumulate cAMP do not form
appressoria
frequent reversion occurs to trigger second site mutation in regulatory subunit of PKA(cAMP-dependent protein kinase A)
Leads to
cAMP-independent triggering of PKA
restoration of
appressorium development [
mutants
lacking the catalytic subunit of PKA
form small, mis-shaped
and non-functional appressoria
MAP kinase (mitogen-activated protein kinase) involving Pmk1 MAP kinase is required for appressorium development
mutants lacking the PMK1 gene arrest the growth before
development of infectious structures
Divided into 2 different stages
appressorium morphogenesis
requires the presence of the Pmk1MAPkinase
pathway
initiation of appressorium development
requires cAMP signalling
Conclusion
M. grisea
Evolved remarkable mechanism
Production of cell
Attachment to the rice leaf surface
Generation of mechanical force to penetrate the rice leaf cuticle
Analysis of genome sequences and cytological studies
produce extra-cellular depolymerizing enzymes
Accelerate the infection process on living plant tissue
Capable of penetrating inert plastic membranes
Shows the importance of tugor generation by appressorium
Appressorium accumulates high concentration of compatible solutes such as glycerol in generating tugor
Glycogen metabolism
Glycogen
abundant within the fungi spores
mobilized on spores germination
found accumulate within appressoria during development
mobilization regulated by cAMP response pathway
cpkA
mutants show retarded degradation of glycogen
during conidial germination
during initiation of appressorium development
regulatory subunit PKA muntant
mac1 sum1-99
showed rapid degradation of glycogen together with melanin production within appressoria
Degraded by two major enzymes: glycogen phosphorylase and amyloglucosidase
Both enzymes needed for full virulence of fungus
pls1
mutant is non-pathogenic nad produces non-functional appressoria
accumulates glycogen deposits within infected cells
PSL1
encode tretraspanin
required for controlling the translation of tugor into physical force
Appressoria becomes seperated from the germ tube and conidia by a thick septum
Mutants lacking melanin
mutations in gene-encoding enzymes in the dihydroxy napthalene melanin biosynthetic pathway(dihydroxynapthalene reductase)
Can be identified by the colour change of the fungus
M. grisea RSY1 gene
encodes scytalone dehydratase(mutants are pink colour)
buf1
mutant-buff colour
of mutants lacking this enzymatic activity.
Becomes melanin-pigmented within the cell wall of the appressorium
mutations in a polyketide synthase encoded by the ALB1
gene, lead to an albino mutant phenotype.
ALL THE 3 TYPE MUTANTS ARE NON-PATOGENIC BECAUSE CANNOT ACCUMULATE TURGOR
Trehalose metabolism in
M. grisea
Major role: stress metabolite & cellular protectant form dessication.
storage product within microbial cells
accumulation of trehalose in many eukaryotes: sugar metabolism can be regulated
synthesis of T6P (Trehalose-6-phosphate)
Substrates: UDP-glucose and glucose-6-phosphate
Converted into trehalose
multienzyme complex is responsible for the synthesis of T6P
the catalytic subunit of which is encoded by a gene
called TPS1
M. grise
a Δtps1 mutants
non-pathogenic, appressorium turgor generation was severely
affected in these strains
displayed a number of pleiotropic effects
Δtps1 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.
In S. cerevisiae, T6P synthase activity is a means
by which the influx of glucose into glycolysis is regulated
Yeast tps1Δ mutants are unable to grow on glucose due to the absence of T6P synthase activity
-leading to an uncontrolled
influx of glucose into glycolysis.
The differences between
yeast tps1Δ mutants and M. grisea Δtps1 mutants
S. cerevisiae tps1Δ mutants are unable to grow in
the presence of glucose under any conditions -toxic
M. grisea, glucose utilization can be restored by the presence of alternate carbon sources, and in particular, the presence of complex nitrogen sources such as yeast extract or peptone
M. grisea Δtps1 mutants are unable to grow on acetate or lipids, indicating that gluconeogenesis may also be affected by loss of T6P synthase activity
M. grisea genome sequence revealed the presence of two
trehalase-encoding genes
TRE1
NTH1
The process of appressorium
turgor generation
occurs due to
the rapid influx of water into the infected cell.
free water is an essential pre-requisite for the
generation of these cells
Water flows into the appressorium
against a concentration gradient
generated by the accumulation
of a compatible solute within the appressorium
Biochemical analysis of M. grisea appressoria
number of solutes accumulate within these cells
glycerol through vapour-pressure psychrometry
required for
generation of the substantial turgor pressure
storage
products present in conidia of the fungus
in absence of nutrients
predominant storage products
lipids, glycogen and the
disaccharide trehalose
degraded rapidly or transported to the germ tube
apex during conidial germination
Mannitol accumulation in spores