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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
Rice blast disease is the most serious disease of cultivated rice and prevalent in temperate-flooded and tropical upland
Fungus that cause blast disease is
Magnaporthe grisea
(Hebert) Barr
M. grisea
undergoes a series of defined morphogenetic developmental steps
Production of specialized infection structure called appressorium
The cells produced on surface of rice leaves
Plant infection by physical breakage of leaf cuticle
Appressoria of rice blast fungus generate substantial turgor
Experiment to estimate equivalent turgor within appressoria
Incipient cytorrhysis experiments
Applying increasing concentrations of polyethylene glycol to appressoria of
M. grisea
The rate of cell collapse is determined
Through this experiments, appressoria of
M. grisea
generate up to 8 MPa during plant infection
Resulting in a narrow penetration hypha at the base of cell
Force through the underlying cuticle and develops into invasive hyphae
Glycogen metabolism
Glycogen
abundant within the spore
mobilized very quickly on germination
Glycogen rosettes
accumulate within appressoria during their development
glycogen quickly disappears from the appressoria
during melanization
during turgor generation
Glycogen mobilization
regulated by the cAMP response pathway
∆cpkA
show retarded degradation of glycogen
during conidial germination
during initiation of appressorium development
subunit PKA mutant=
∆mac1
and
sum1-99
show rapid degradation of glycogen
before the onset of melanin production within appressoria
Glycogen degradation
degraded by two major enzyme activities
glycogen phosphorylase
amyloglucosidase
required for full virulence of the fungus
PLS1
encodes a tretraspanin
novel membrane protein
required for controlling the
translation of turgor
into physical force
for penetration hypha production
pls1 mutant
non- pathogenic
produces completely non-functional appressoria
accumulates glycogen deposits within infected cells
penetration hyphae emergence
requires the MST12-encoded transcription factor
occurs independent of turgor generation
enzymes in synthesizing glycerol from storage carbohydrates
NADH-dependentglycerol-3-phosphate dehydrogenase
NADPH-dependent glycerol dehydrogenase–
present in appressoria but are not induced during turgor generation
triacylglycerol lipase activity
rapidly activated
during the onset of
turgor generation
Appressorium development by
M. grisea
Rice infection is initiated when three-celled, tear-drop shaped conidia landed on surface of rice leaf
The spores germinate immediately on contact with rice leaf
adhere tightly to the hydrophobic surface by means of a spore tip mucilage that is released from the apex of the spore
Germination proceeds by extension of a narrow germ tube
emerges from the conidium within an hour of its
landing on the leaf surface
The germ tube
starts to swell at its apex, and flattens against the surface of the rice leaf
mitosis is always observed within germ tubes
of the fungus before the appressorium development
The germ tube apex then develops into a swollen dome-shaped cell =
appressorium
What triggers development of appressorium?
Hard and hydrophobic
surfaces
Absence of external nutrients
Presence of constituents of cuticular wax
The signalling pathways
responsible for regulating appressorium formation
In
M. grisea
, it is cAMP response pathway
Mutants lack adenylate cyclase, unable to accumulate cAMP, do not form appressoria
subject to frequent reversion by a second-site mutation in the regulatory subunit of PKA (cAMP-dependent protein kinase A)
leads to cAMP-independent triggering of PKA and restoration of appressorium development
A MAP kinase (mitogen-activated protein kinase) cascade involving the Pmk1 MAP kinase is also required for the production of appressoria
mutants lacking the PMK1 gene arrest the growth before development of infectious structures
2 different stages of a appressorium development
The initiation of appressorium development
requires cAMP signalling
Appressorium morphogenesis
requires the presence of the Pmk1 MAP kinase
pathway
Mutants
buf1 mutant
mutation of a gene encoding dihydroxynapthalene
reductase
M. grisea RSY1
gene encodes scytalone dehydratase, and mutants are identified because of their pink colour.
Mutations in a polyketide synthase encoded by the
ALB1
gene, lead to an albino mutant phenotype
All these three mutants are non-pathogenic
the appressoria that they produced are unable to accumulate turgor
The Process of Appressorium, Turgor Generation
Appressorium turgor generation occurs due to rapid influx of water into infected cells.
Occurs normally, in dewdrop on the surface of rice leaf
free-water is essential pre-requisite for this cell generation
water flow into the appressorium against concentration gradient
generated by accumulation of compatible solute within appressoria
number of solutes accumulate including the glycerols.
High concentration of glycerol was required for generation of substantial turgor pressure.
Appressoria form in free water on the leaf surface in absence of external nutrient
glycerol and other solute within the appressoria must be accumulated
de novo
from the storage product in conidia.
conidia was accumulated with lipid, glycogen and disaccharide trehalose as predominant storage product.
onset of conidial germination, trehalose, glycogen and lipid found either bening degraded rapidly or transported into germ tube apex.
Lipid Metabolism During Appressorium Turgor Generation
Lipid bodies
Mobilized from conidium and accumulate in the germ tube apex.
Surrounded by single unit membrane in
M. grisea
Appear highly refractile by phase contrast microscopy.
Can be readily visualize using Nile Red stain.
Appressorium development - Lipid bodies accumulate at the germ tube apax and in the incipient appressorium.
Appressorium maturation - Autophagocytosis (Lipid bodies taken up by vacoule) anf triacylglycerol lipase activiti present.
Turgor ggeneration - Lipolysis occur to produce large central vacuole.
Triacylglycerol lipase activity in
M. grisea
: cAMP -regulated because
cpkA
mutant lack catalytic subunit of PKA.
Low lipase activity. Lipid bodies fail to coalese undergo the degradation during appressorium morphogenesis.
Initial trafficking of lipid bodies to appressorium (PMK1 MAP kinase).
Lack of PMK1, lipid bodies fail to move
Genome of
M. grisea
Encodes atleast 4 genes that code for intracellular triacylglycerol lipases.
Provide meas of rapidly producing glycerol from lipid droplets (transported to the developing appressorium).
Generation of fatty acid
Consequence of appressorium lipolysis and glycerol generation.
Glyoxylate cycle (requires for pathogenicity in
M. grisea
.
Mutant lack of
ICLI
gene that encodes for isocitrate lyase wee shown to be less virulence.
Due to temporal effect on the development of appressorium.
Cell wall biosynthesis and compatible solute generation required the glyoxylate cycle to be active in fungus during appressorium development.
ICLI
: high during appressorium mophogenesis, penetration peg formation and invasive growth of
M. grisea
.
the glyoxylate shunt has been shown to be requires for pathogenicity of several other phytopathogenic and human pathogenic fungi.
Peroxisomal function ; required for appressorium-mediated plan infectionn by the entrance of
Colleotrichum lagenarium
: produce melanin-pigmented appressoria. - suggest fatty acid beta-oxidation in generally imprtant in appressoria physiology.
Trehalose metabolism in M.grisea
non-reducing disaccharide trehalose is the common storage product
act as stress metabolite
cellular protectant against dessication
accumulation of trehalose in many eukaryotes means the sugar metabolism can be regulated
Trehalose synthesis
T6P (trehalose-6-phosphate) is synthesized using UDP-glucose and glucose-6-phosphate as substrate and convert directly into trehalose
TPS1 gene encode for trehalose synthesis
require for rice blast disease
Δtps1 is non pathogenic and ppressorium turgor generation was severely impaired.
dislay number of pleiotrophic effect
mutant unable to grow on or a variety of other
rapidly fermentable carbon sources
unable to utilise acetate or lipid as sole of carbon sources
glucose utilisation can be restored by presence of alternate carbon sources such as yeast extract or peptone.
mutant unable to grow on acetate or lipid indicate the gluconeogenesis was affected by the loss of T6P synthase activity
NTH1 gene encode neutral trehalase
highly expressed during conidiogenesis and spore germination
mutant lack of virulence due to inability to perform invasive growth within rice tissue.
TRE1 gene encode for main trehalase activity
during spore germination and involve in pathogenesis
mutant produce rice blast symptom that identical to isogenic wild type strain.
mutant tps1, nth1 and tre1 suggest that trehalose synthesis is require for appressorium function not the trehalosm metabolism
