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Regulation of Appressorium Development in Pathogenic Fungi (Appresorium…
Regulation of Appressorium Development in Pathogenic
Fungi
Appresorium Maturation and Cuticle Rupture
appresoria change their axis of polarity and re-establish polarised growth in the interface between the fungus and plants.
focus turgor in appresorium:
associated with isotrophic expansion of the cell into physical force
leading to generation and protrusion of the penetration peg into cuticle
appresorium pore at the base of infection cell from which penetration hypha emerges
M. oryzae
and
Colletotrichum
sp.:
appreosrium pore clearly distinct from rest of infection cell
thinner cell wall
absence of melanin (visible by ultra-structural analysis)
appresorium pore:
site of remodelling of the actin cytoskeleton
remodelling actin require morphogenetic septin GTPases
during penetration peg formation:
rapid F-actin polymerisation occurs leading to rapid polarised growth of penetration hypha
septin ring:
approximately 5.9 um at the appressorium
pore of
M. oryzae
composed 4 core septins (sep3, sep4, sep5 & sep6)
necessary for scaffolding actin, leading to formation of a toroidal Factin network at the base of the appressorium
acts as a lateral diffusion barrier, tethering in place proteins implicated in F-actin polymerisation like Las17 component of the arp2/3 complex
ezrin, radixin, moesin (ERM) domain proteins:
require for actin membrane interactions at the cortex of cells
found within septin ring at the appresorium pore
BAR
domain proteins:
imply in the control of membrane curvature generation
eukaryotic cells:
undergo membrane curvature generation to generate invaginations associated
endocytosis and also cellular
protrusions. eg: lamellipodia found in epithelial cells
reactive oxygen species burst catalysed by the Nox2 NADPH oxidase is necessary for septin-mediated appressorium repolarisation:
Nox2 and NoxR (reg subunit) require for septin ring formation
second NADPH oxidase encoded by NOX1 gene: - necessary for maintanence of the polarised growth and organisation of the toroidal F-actin network
mutation of genes encoding any septin components:
NOX2 and NOXR genes is sufficient to prevent plant infection
indeed, appressorium pore fails to differentiate from
the rest of the infection cell
by contrast, mutation of NOX1 leads to arrest of the penetration process just after differentiation of a stunted penetration peg (fails to elongate and breach the cuticle)
reactive oxygen species (ROS):
generated within the appresorium act in at least two different ways to stimulate cytoskeletal remodelling
act directly on proteins such as gelsolin (involved in actin severing and formation of free barbed ends that stimulate rapid F-actin polymerisation
action of latrunculin (actin depolymerising agent) can competitively inhibited by the presence of ROS in
M. oryzae
appressoria lead to penetration peg formation
acts on signalling components that operate downstream of a turgor sensor to define the point at which re-polarisation needs to be triggered
likely to be upstream of the formation of the hetero-oligomeric septin ring
Early Appressorium Development
Maturation & melanisation controlled by entry into G2 and mitosis.
Cytokinesis nad contractile actomysin ring forms at neck of appresorium.
-Autophagy- intracelluar content of conidium degraded and trafficked to incipient appresorium.
cell cycle arrest
is required for plant infection
cooperation of at least two distinct underlying mechanisms
activation of the DNA damage response cascade
transcriptional regulation of a gene called HSL1
protein kinase that modulates the G2 to M transition
Appressorium formation also relies on perception of
physical
and
biochemical cues
at the leaf surface
fundamental
for
penetration
of the cuticle by diverse plant pathogens
the control of nuclear division
coordination with morphogenesis at the leaf surface
in
M. oryzae
appressorium morphogenesis involves
the
Pmk1 MAP kinase pathway
necessary for
appressorium development
a number of
potential receptors
are involved in perception of surface signals
PTH11
CFEM domain G-protein coupled receptor
necessary for perception of
the hydrophobic leaf surface
if
absence
, appressoria do not form
widely conserved in other plant pathogenic fungi
required for infection related morphogenesis
the
cAMP response pathway
RAS signalling
generation of a dominant-active allele
of Ras2 (RAS2G17V)
abnormal appressorium
formation in the absence of a surface
Mac1 adenylate cyclase interacts with Cap1
( cyclase-associated protein)
Cap 1
re-modelling the actin cytoskeleton
activates adenylate cyclase
Pmk1 kinase cascade
(Mst11, Mst7 and Pmk1)
scaffolded by a protein called
Mst50
interacts with
Mst11
upon activation and phosphorylation of its components
a phosphor-relay is triggered leading to
the detachment of Pmk1
its traversal to the nucleus
during appressorium maturation
several
transcription factors
operate
downstream of
Pmk1, including Mst12 and Mcm1
activate a large set of gene products
involved in
cell wall differentiation
and
the physiological changes
Pmk1
regulate microconidia formation by
M. oryzae
Pmk1 and Mst12 mutants show
reduced microconidia production
Mcm1
is essential for their development
Controlled by perception on hydrophobicity
surface water contact angles greater than 90 degree( Teflon)
hardness
Fungal react to wax monomer (1,16 hexadecanediol- powerfull inducer)
Fully functional if undergo mitosis
Spore land on surface of host
M. oryzae: Conidium germinates within hours (adhesive to hydrophobic waxy leaf
Spore germinates, send out a germ tube (emerging from tapering end of three-celled conidum.
Germ tube extend 10-15 um
Germ tube flattening tip, hooking, differentiate into unicellular appresorium.
Single round nuclear division
Conidium nucleus enter DNA-replication (S phase).
Inhibition DNA replication by hydroxyurea/ temp sensitive mutant nim1 (Abberant mitosis)
Cell cycle is conserved ( cell cycle arrest necessary for infective filament to penetrate) Ex: U. maydis- self/nonself recognition process: two monokaryotic sporidia fuse, form infectious dikaryotic filament.
Turgor generation up to 8 Mpa (Blocked by targeted mutation of any 16 genetic component of non-selective- caused to be non-pathogenic.
APPRESSORIUM TURGOR GENERATION
accompanied by synthesis of glycerol and other polyols.
tugor generation and melanin formed
function melanin: 1) maintain turgor pressure.
melanin may not provide the barrier for
osmolytes in C. graminicola
function turgor pressure: 1) to retard efflux of glycerol from expanded appressorium. 2) provide rigidity. 3) provide resilience to infection cell
anthracnose pathogen of corn, Colletotrichum graminicola
turgor accumulates when melanin biosynthesis is inhibited and the penetration of intact
leaves and artificial substrates still occurs
Mach-Zehnder interferometry method
cell collapse assays (cytorrhysis) analysis of the appressorial osmolyte content
melanin is not required
for solute accumulation and turgor generation
Melanin
albino mutants, lacking the CgPKS1 polyketide synthase gene involved in 1,3,6,8-tetrahydroxy-naphthalene biosynthesis,
prone to
rupture and impaired in their ability to cause disease
Phakopsora pachyrhizi
high turgor, of up to
5.13 MPa, could be observed in its non-melanised appressoria
requires accumulation
of osmotically active polyols,
can be
retained even in the absence of melanin.
THE PENETRATION PEG AS SITE OF EFFECTOR DELIVERY
plant infection by pathogen
involves deployment of effector protein
function effector protein: 1) surpress plant immunity responses. 2) facilitate proliferation of pathogen within plant tissue
C.
higginsianum
Ultra-structural analysis
effectors within the appressorium pore at the point of plant infection
this shows how the penetration peg allows rapid deployment of effector
this is evidence that specialised focal secretionmechanisms for effectors are likely to be present in both Colletotrichum orbiculare and M. oryzae
U. maydis
retrograde
early endosome-mediated, long-distance signalling pathway is necessary for:
1) transcriptional regulation of effector genes. 2) effector secretion from the hyphal tip during plant tissue
colonisation