Regulation of Appresorium Development in Pathogenic Fungi

Introduction

Penetration peg as site of effector delivery

effector proteins : suppress plant immunity responses and facilitate proliferation of the pathogen within plant
tissues

Pathogenic Fungi

Cause of many world's most devastating crop diseases

within the appressorium pore at the point of plant infection

Increase expenditure significantly each year

allows rapid deployment of effectors early during the infection process

Developing world --> High cost of fungicides --> Disease outbreaks cause serious concerns for farmers

communication between the extending hyphal tip and the fungal nucleus

30% of global harvest is lost each year to plant disease

retrograde - early endosome-mediated, long-distance signalling pathway

Appresorium tugor generation

Identifying durable solutions is important for plant productivity in a sustainable way

Accompanied
by rapid synthesis of glycerol and other polyols

transcriptional regulation of effector genes

effector secretion from the hyphal tip

Appressorium maturation and cuticle rupture

Changes axis of polarity & re-established polarised growth at the interface between host & fungi

melanin serves a role to maintain turgor pressure due to lowering the porosity of the appressorium cell wall

Appresoria

Lead to formation of a thick differentiated melanin layer on the inner side of the appressorium cell wall

For turgor to for physical force to generate protrusion of the penetration peg into the cuticle.

Fungi has evolved capacity to breach intact cuticles of plant host by elaborating specialized structures of appresoria

required to retard efflux
of glycerol from the rapidly expanding appressorium

provide structural rigidity and resilience to the
infection cell

appressorium pore

thin cell wall , lacks melanin

Can take various forms

site to remodel actin skeleton

requires morphogenetic septin GTPases

Single-celled structures

Compound appresoria composed of numerous cells

composed of septin rings

Can form structures known as infections cushions

in M.oryzae = sep3, sep4, sep5, sep6

cell collapse assays (cytorrhysis)

appressorial osmolyte content using a method called Mach-Zehnder interferometry

for scaffolding actin to form toroidal F-actin network at the base of the appressorium

showed that melanin is not required
for solute accumulation and turgor generation

Usually are simple terminal swellings at the tips of germ tubes that emerge from spores on leaf surface

Lateral diffusion barrier- binding proteins implicated in F-actin polymerisation

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

In Magnaporthe oryzae (Rice blast fungus) and Colletotrichum (Anthracnose)

high turgor, of up to 5.13 MPa, could be observed in its non-melanised appressoria

Appresoria are melanin-pigmented, septate structures that form at tips of germ tubes, then differentiate into dome-shaped

Las17 component of arp2/3 complex

Turgor generation requires accumulation
of osmotically active polyols but can apparently be
retained even in the absence of melanin

Early appressorium development

contains ERM Domain protein ( ezrin, radixin,moesin) used in actin membrane interactions

occurs soon after a spore lands on the surface of its host

cell walls of appressoria must have evolved in different ways to maintain turgor, some of which do not require melanin

What this review is about

BAR domain protein: utilized in the membrane curvature generation

the spore rapidly germinates and sends out a germ tube upon hydration and surface contact.

clear role for melanin in structural rigidity and turgor generation in fungi such as C.graminicola and M. oryzae

Comparison & evaluation of recent studies about the biology of appresorium development in plant pathogenic fungi

emerging from the tapering end of
the three-celled conidium.

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non-melanised fungi may still
undertake mechanical appressorium-mediated infection

undergo membrane curvature generation in order to generate invaginations associated with endocytosis & cellular protusions (happens in eukaryotes)

Control
of initiation of appressorium development is based on:

Many of the studies focused on two diverse species M.oryzae (ascomycete) & U.maydis (basidiomycete)

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

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cellular protrusions require membrane curva-ture to be stimulated, followed by rapid membrane biogenesis & F-actin polymerisation

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penetration peg formation rapid F-actin polymerisation occurs at this point leading to rapid polarised growth of the penetration hyphae

the cell from which the germ tube
emerges undergoes a single round of nuclear division, before appressorium development

An emerging picture of a highly orchestrated developmental process requiring perception of physical and chemical cues from the plant leaf surface

Reactive oxygen species

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

Nox2 & regulatory subunit NoxR

catalysed by the Nox2 NADPH
oxidase

necessary for septin-mediated appressorium repolarisation

Targeting fundamental morphogenetic processes ,may be important in developing anti-penetrant fungicides or targeting plant-based methods to control most cereal diseases

required for septin ring formation at the base of the
appressorium

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

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appressorium maturation and melanisation is controlled by entry of the nucleus into G2 and mitosis

mutation Nox1 & Nox2

if mitosis occurs does
the appressorium become fully functional.

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cytokinesis occurs and a contractile actomyosin ring forms at the neck of the appressorium

prevent plant infection and,indeed, the appressorium pore fails to differentiate from the rest of the infection cell

Future prospects

Autophagy is then stimulated within the conidium

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NOX1 leads to arrest of the penetration process just after differentiation of a stunted penetration peg, which fails to elongate and breach the cuticle

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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

may act
in at least two different ways to stimulate cytoskeletal remodelling.

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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

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

e.g

action of latrunculin(actin depolymerising agent) competitively inhibited by the presence of ROS in M. oryzaeappressoria, leading to penetration peg formation

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

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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

Common themes emergence

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M. oryzae appressorium morphogenesis involves the Pmk1 MAP kinase pathway and the cAMP response pathway

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Mac1 adenylate cyclase interacts with Cap1, a cyclase-associated protein that activates adenylate cyclase and is potentially involved
in re-modelling the actin cytoskeleton

Importance of cell cycle control and the operation of a widely conserved MAP kinase pathway for appressorium differentiation

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M. oryzae, the Pmk1 MAPK pathway is necessary for appressorium development

To study how isotropic expansion of appresoria is translated into the generation of invasive forces necessary to breach the leaf surface

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CFEM domain Gprotein Gprotein
coupled receptor, is necessary for perception of the hydrophobic leaf surface by M. oryzae

Emerging picture of the appresorium pore as a key signalling hub during plant infection

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:

Future experiments

Mst11, Mst7 and Pmk1, which appear to be scaffolded by a protein called Mst50, which interacts with Mst11

To define precisely how focal effector secretion is regulated and the likely conservation of early endosome-mediated signalling

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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

The precise mechanism by which the appressorium monitors turgor, to determine the optimal point for re-polarisation and host cell penetration

Pmk1 pathway appears to regulate microconidia formation by M. oryzae

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