The molecular biology of appresorium turgor generation by rice blast…
The molecular biology of appresorium turgor generation by rice blast fungus
Appressorium development by M. grisea
Initiated when three-celled conidia land on the surface of rice leaf
Spores germinate immediately on contact with the rice leaf
Spore tip adhere tightly to the hydrophobic surface
extension of narrow germ tube occur
exntesion emerged from conidium within an hour if its landing on the leaf surface.
Germ tube apex develops into a appressorium.
process of appressorium morphogenesis is tightly coupled to cell division.
M. grisea appressoria form in response to:
absence of external nutrients
of constituents of cuticular wax
cAMP response pathway is triggered after the attachment of fungus on the leaf surface.
Mutants lacking adenylate cyclase unable to accumulate cAMP and do not form appressoria
mutation is because of frequent reversion by a second-site mutation in regulatory subunit of PKA
CAMP-independent trigger PKA
Appressorium development is restored.
At the same time, mutants lacking the catalytic subunit of PKA form non-functional appressorium.
MAP kinase cascade involving Pmk1 MAP kinase is also required for the production of appressorium
mutants lacking PMK1 gene arrest the growth before the development of appressoria.
appresorium development can be divided into 2 different stages:
The initiation of appressorium development
requires cAMP signalling
Requires the presence of the Pmk1 MAP kinase
once formed, appressoia become separated from germ tube and conidium by a thick septum.
melanin is present within the cell wall of appressorium.
mutant-lacking melanin is identified in M.grisea because of alteration of colour of fungus
buf1 mutant occur because of gene encoding dihydroxynapthalene reductase.
RSY1 gene encodes scytalone dehydratase and identified because of their pink colour.
ALB1 gene lead to albino mutant phenotype.
all these three mutants are non-pathogenic because they produce appressoria that unable to accumulate tugor.
The process of appressorium
occurs due to rapid influx of water into the infected cell.
occurs normally in dewdrops of the surface of a rice leaf and free water (essential pre-quisite for the generation of these cells).
water flows into appresorium against a concentration gradient.
biochemical analysis demonstrated that the number of solutes accumulate within these cells including glycerol that accumulate to concentration in excess of 3 M
accumulation of high concentration of glycerol shown by vapour-pressure psychrometry
appresorium form in free water on the leaf surface in the absence of :
other solutes within appresorium
all these must be accumulate
from storage products present in the conidia fungus
as predominant storage products
mannitol has accumulate within the spores
have all found to be degraded or transported to the germ tube apex during conidial germination
in recent investigation, trehalose, lipid and glycogen as potential sources of glycerol and other solutes during turgor generation
Trehalose metabolism in
act as a stress metabolite
cellular protectant from desiccation
accumulation of trehalose in many eukaryotes = sugar metabolism can be regulated
synthesized in M. grisea using UDP-glucose and glucose-6-phosphate
directly converted into trehalose
multienzyme complex is responsible for the synthesis of T6P
T6P synthase activity = the influx of glucose into glycolysis is regulated
mutants are unable to grow on glucose
absence of T6P synthase activity
influx of glucose into glycolysis
of ATP in the first two steps of glycolysis
drop in ATP and phosphate levels within the cell
targeted gene replacement to be required for rice blast disease
M. grisea ∆tps1
appressorium turgor generation was severely
mutants were unable to grow on glucose / other
rapidly fermentable carbon sources
mutants unable to utilize acetate / lipids as sole carbon sources
mutants are unable to grow in
the presence of glucose under any conditions (toxic)
unable to grow on acetate or lipids
gluconeogenesis may also be affected
by loss of T6P synthase activity
glucose utilization can be restored by the presence of alternate carbon sources
the presence of complex nitrogen sources
genome sequence revealed presence of
encoding the main trehalase activity
completely dispensable for pathogenesis
produce rice blast symptoms, identical to those of an isogenic wild-type strain of the fungus
an unusual trehalase that does not show significant similarity to other fungal neutral or acidic trehalases
similar to a novel trehalase gene found in the
encode a neutral trehalase
highly expressed during conidiogenesis and spore germination
reduced in virulence
decreased ability to perform invasive growth within rice tissues
trehalose synthesis is
possible, trehalose accumulates as an accessory-compatible solute in appressoria
sugar signalling role of T6P synthase is essential for the regulation of appressorium turgor generation in
Lipid metabolism during appressorium
Lipid bodies mobilized quickly from conidium
(accumulating in germ tubes apex & incipient appesorium)
surrounded bu single unit membrane
highly refractile (phase contrast microscopy)
lipid bodies coalease
taken up by vacuole (by process resembles autophagocytosis)
lipolysis occur in vacuole (coalease) form large central vacuole
Presence of triacylglycerol lipase act
Triacylglycerol lipase act.
:recycle:cpkA mutant (lacks PKA) : lipase act. decrease , lipid bodies fail coalease/ degradation during morphogenesis
PKA mutant :
rapid lipid degradation
completed before melanization of infected cells
under Pmk1 MAP kinase
mutant's lipid bodies failed to move to germ tube apex
Genome encodes at least 4 genes (encode intracellular triacylglycerol lipases
enzyme: rapidly producing glycerol from lipid droplets (transported to developing appresorium
novel triacylglycerol lipases
directly asst. with lipid bodies.
responsible for rapid lipolysis during maturation
gene YMR313c (2 homolog)
Consequences lipolysis and glycerol generation:
generation of fatty acids.
requirement for fatty acid B-oxidation and subsq activation of glyoxylate shunt & gluconeogenesis
If Lacks ICL1 genes lacks virulence:
due to temporal regulation effect of appressorium: spore germination retarded
spore germination retarded in :warning:icl1 mutants
Hence, cell-wall biosynthesis and compatible solution required glyoxylate cycle to active
ICL1 gene expression high during appresorium morphogenesis, penetration peg formation and invasive growth of M.grisea
:warning:icl1 mutants causing rice blast symptoms( albeit in delayed manner)
wild type strain have alternative pathway for
appressorium maturation and penetration hyphae development must be present in the fungus
required for pathogenicity of several other phytopathogenic and human
reflects shared needs to develop intially within glucose-deficient env.
for appresorium-mediated plant infection by antracnose fungus ( Colletotrichum lagenarium)
produced melanin-pigmented appresoria (fatty acid b-oxidation generally important for appresoria physiology)
Glycogen metabolism in M. grisea
regulatory subunit PKA mutant mac1 sum1-99 showed:
rapid degradation of glycogen before onset of melanin production within appresoria
glycogen degraded by two major enzyme activities:
:check:both of enzyme required for full virulence of the fungus
mutant lacking genes encoding these enzymes have been generated in M. grisea and being characterised
is non pathogenic
produces completely non-functional appresoria
accumulates glycogen deposits within infected cells
which a novel membrane protein that may required for controlling translation of turgor into physical force for penetration hypha production
glycogen, tehalose and lipid metabolism each contribute to glycerol formation in the appressorium is, as yet, unresolved
relative contribution of lipid and glycogen in glycerol synthesis:
require further biochemical analysis
enzymes by which glycerol could be synthesized from storage carbohydrates –NADH-dependent glycerol-3-phosphate dehydrogenase and NADPH-dependent glycerol dehydrogenase
:check: are present in appresoria nut not included during turgor generation
triglycerol lipase activity is activated during on set of turgor generation
glycogen rosetts found to accumulate within appresoria during development
but on set of turgor generation, glycogen quick disappears from appresoria during melanization and turgor generation
mobilized very quick on germination
glycogen immobilization appears to be regulated by cAMP response pathway
abundant within the spores of M. grisea