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Regulation of Appresorium Development in Pathogenic Fungi (Early…
Regulation of Appresorium Development in Pathogenic Fungi
Introduction
Pathogenic Fungi
Cause of many world's most devastating crop diseases
Increase expenditure significantly each year
Developing world --> High cost of fungicides --> Disease outbreaks cause serious concerns for farmers
30% of global harvest is lost each year to plant disease
Identifying durable solutions is important for plant productivity in a sustainable way
Appresoria
Fungi has evolved capacity to breach intact cuticles of plant host by elaborating specialized structures of appresoria
Can take various forms
Single-celled structures
Compound appresoria composed of numerous cells
Can form structures known as infections cushions
Usually are simple terminal swellings at the tips of germ tubes that emerge from spores on leaf surface
In
Magnaporthe oryzae
(Rice blast fungus) and
Colletotrichum
(Anthracnose)
Appresoria are melanin-pigmented, septate structures that form at tips of germ tubes, then differentiate into dome-shaped
What this review is about
Comparison & evaluation of recent studies about the biology of appresorium development in plant pathogenic fungi
Many of the studies focused on two diverse species
M.oryzae
(ascomycete) &
U.maydis
(basidiomycete)
An emerging picture of a highly orchestrated developmental process requiring perception of physical and chemical cues from the plant leaf surface
Targeting fundamental morphogenetic processes ,may be important in developing anti-penetrant fungicides or targeting plant-based methods to control most cereal diseases
Penetration peg as site of effector delivery
effector proteins : suppress plant immunity responses and facilitate proliferation of the pathogen within plant
tissues
within the appressorium pore at the point of plant infection
allows rapid deployment of effectors early during the infection process
communication between the extending hyphal tip and the fungal nucleus
retrograde - early endosome-mediated, long-distance signalling pathway
transcriptional regulation of effector genes
effector secretion from the hyphal tip
Appresorium tugor generation
Accompanied
by rapid synthesis of glycerol and other polyols
Lead to formation of a thick differentiated melanin layer on the inner side of the appressorium cell wall
required to retard efflux
of glycerol from the rapidly expanding appressorium
provide structural rigidity and resilience to the
infection cell
melanin serves a role to maintain turgor pressure due to lowering the porosity of the appressorium cell wall
cell collapse assays (cytorrhysis)
appressorial osmolyte content using a method called Mach-Zehnder interferometry
showed that melanin is not required
for solute accumulation and turgor generation
melanin may not provide the barrier for
osmolytes in C. graminicola
melanin plays a
structural role because albino mutants, rupture and impaired in their ability to cause disease
high turgor, of up to 5.13 MPa, could be observed in its non-melanised appressoria
Turgor generation requires accumulation
of osmotically active polyols but can apparently be
retained even in the absence of melanin
cell walls of appressoria must have evolved in different ways to maintain turgor, some of which do not require melanin
clear role for melanin in structural rigidity and turgor generation in fungi such as C.graminicola and M. oryzae
non-melanised fungi may still
undertake mechanical appressorium-mediated infection
Appressorium maturation and cuticle rupture
Changes axis of polarity & re-established polarised growth at the interface between host & fungi
For turgor to for physical force to generate protrusion of the penetration peg into the cuticle.
appressorium pore
thin cell wall , lacks melanin
site to remodel actin skeleton
requires morphogenetic septin GTPases
composed of septin rings
in
M.oryzae
= sep3, sep4, sep5, sep6
for scaffolding actin to form toroidal F-actin network at the base of the appressorium
Lateral diffusion barrier- binding proteins implicated in F-actin polymerisation
Las17
component of arp2/3 complex
contains ERM Domain protein ( ezrin, radixin,moesin) used in actin membrane interactions
BAR domain protein: utilized in the membrane curvature generation
undergo membrane curvature generation in order to generate invaginations associated with endocytosis & cellular protusions (happens in eukaryotes)
cellular protrusions require membrane curva-ture to be stimulated, followed by rapid membrane biogenesis & F-actin polymerisation
penetration peg formation rapid F-actin polymerisation occurs at this point leading to rapid polarised growth of the penetration hyphae
Reactive oxygen species
Nox2 & regulatory subunit NoxR
required for septin ring formation at the base of the
appressorium
catalysed by the Nox2 NADPH
oxidase
necessary for septin-mediated appressorium repolarisation
Nox1 gene
encodes 2nd NADPH oxidase
to maintain polarised growth and organisation of toroidal F-actin network at the base of the appressorium during penetration
peg formation
mutation Nox1 & Nox2
prevent plant infection and,indeed, the appressorium pore fails to differentiate from the rest of the infection cell
NOX1 leads to arrest of the penetration process just after differentiation of a stunted penetration peg, which fails to elongate and breach the cuticle
may act
in at least two different ways to stimulate cytoskeletal remodelling.
act directly on proteins such as gelsolin, which are involved in actin severing and formation of free barbed ends that stimulate rapid F-actin polymerisation
e.g
action of latrunculin(actin depolymerising agent) competitively inhibited by the presence of ROS in
M. oryzae
appressoria, leading to penetration peg formation
acts on signalling components that operate down-
stream of a turgor sensor (or sensors) that must operate in
the appressorium to define the point at which re-polarisation
needs to be triggered
Chm1
, a protein kinase implicated in septin phosphory-
lation
Early appressorium development
occurs soon after a spore lands on the surface of its host
the spore rapidly germinates and sends out a germ tube upon hydration and surface contact.
emerging from the tapering end of
the three-celled conidium.
Control
of initiation of appressorium development is based on:
perception of hydrophobicity
surface hardness
fungus is able to respond to wax monomers
such as 1,16-hexadecanediol; powerful inducers of appressorium development at the leaf surface
the cell from which the germ tube
emerges undergoes a single round of nuclear division, before appressorium development
Entry of this conidial nucleus into DNA replication (S-phase) is necessary for:
initiation of appressorium development
inhibiting the DNA replication either by hydroxyurea or nim1 mutant
appressorium maturation and melanisation is controlled by entry of the nucleus into G2 and mitosis
if mitosis occurs does
the appressorium become fully functional.
cytokinesis occurs and a contractile actomyosin ring forms at the neck of the appressorium
Autophagy is then stimulated within the conidium
Blocking autophagy by targeted mutation of any of the 16 genetic components of the non-selective macroautophagy pathway is sufficient to render the fungus non-pathogenic
U.maydis undergoes a self-/non-self-recognition process onthe corn leaf surface in which two monokaryotic sporidia fuse to form an infectious dikaryotic filament
The
cell cycle arrest results by cooperation of two mechanism:
activation of the DNA damage response cascade,
transcriptional regulation of a gene called
HSL1 that encodes a protein kinase that modulates G2 to M transition
M. oryzae appressorium morphogenesis involves the Pmk1 MAP kinase pathway and the cAMP response pathway
Mac1 adenylate cyclase interacts with Cap1, a cyclase-associated protein that activates adenylate cyclase and is potentially involved
in re-modelling the actin cytoskeleton
M. oryzae, the Pmk1 MAPK pathway is necessary for appressorium development
CFEM domain Gprotein Gprotein
coupled receptor, is necessary for perception of the hydrophobic leaf surface by M. oryzae
RAS signalling is likely to act upstream of the Pmk1 and cAMP response pathways because generation of a dominant-active allele of Ras2 (RAS2G17V) leads to abnormal appressorium formation
Pmk1 kinase cascade is composed of three protein kinases:
Mst11, Mst7 and Pmk1, which appear to be scaffolded by a protein called Mst50, which interacts with Mst11
Pmk1 pathway appears to regulate microconidia formation by M. oryzae
Expression of the dominant M. oryzae RAS2G17V allele in Colletotrichum graminicola and C. gloeosporioides also led to aerial appressoria suggesting conservation of the surface perception signalling mechanism
Future prospects
Common themes emergence
Importance of cell cycle control and the operation of a widely conserved MAP kinase pathway for appressorium differentiation
To study how isotropic expansion of appresoria is translated into the generation of invasive forces necessary to breach the leaf surface
Emerging picture of the appresorium pore as a key signalling hub during plant infection
Future experiments
To define precisely how focal effector secretion is regulated and the likely conservation of early endosome-mediated signalling
The precise mechanism by which the appressorium monitors turgor, to determine the optimal point for re-polarisation and host cell penetration
To identify the turgor sensing mechanisms of appresoria, as well as an understanding how this triggers regulated synthesis of ROS and lead to septin-mediated cytoskeletal re-organisation
To identify new components of the regulatory networks by rapid progress in generating mutants by high-throughput genome editing and silencing
Utilising the new genome editing techniques, such as CRISPR-Cas9 methodologies which numerous fungal genes could be tested to establish their role in appresorium biology and plant infection