Regulation of Appressorium Development in Pathogenic
Fungi

Appresorium Maturation and Cuticle Rupture

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.

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cell cycle arrest is required for plant infection

APPRESSORIUM TURGOR GENERATION

cooperation of at least two distinct underlying mechanisms

appresoria change their axis of polarity and re-establish polarised growth in the interface between the fungus and plants.

activation of the DNA damage response cascade

transcriptional regulation of a gene called HSL1

appresorium pore at the base of infection cell from which penetration hypha emerges

accompanied by synthesis of glycerol and other polyols.

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

tugor generation and melanin formed

protein kinase that modulates the G2 to M transition

function melanin: 1) maintain turgor pressure.

function turgor pressure: 1) to retard efflux of glycerol from expanded appressorium. 2) provide rigidity. 3) provide resilience to infection cell

Appressorium formation also relies on perception of physical and biochemical cues at the leaf surface

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)

fundamental for penetration of the cuticle by diverse plant pathogens

anthracnose pathogen of corn, Colletotrichum graminicola

the control of nuclear division

turgor accumulates when melanin biosynthesis is inhibited and the penetration of intact
leaves and artificial substrates still occurs

coordination with morphogenesis at the leaf surface

Mach-Zehnder interferometry method

in M. oryzae

cell collapse assays (cytorrhysis) analysis of the appressorial osmolyte content

appresorium pore:

  • site of remodelling of the actin cytoskeleton
  • remodelling actin require morphogenetic septin GTPases

appressorium morphogenesis involves

melanin is not required
for solute accumulation and turgor generation

  • Controlled by perception on hydrophobicity
  • surface water contact angles greater than 90 degree( Teflon)
  • hardness

during penetration peg formation:

  • rapid F-actin polymerisation occurs leading to rapid polarised growth of penetration hypha

melanin may not provide the barrier for
osmolytes in C. graminicola

Mac1 adenylate cyclase interacts with Cap1
( cyclase-associated protein)

Melanin

albino mutants, lacking the CgPKS1 polyketide synthase gene involved in 1,3,6,8-tetrahydroxy-naphthalene biosynthesis,

Fungal react to wax monomer (1,16 hexadecanediol- powerfull inducer)

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

Cap 1

re-modelling the actin cytoskeleton

prone to
rupture and impaired in their ability to cause disease

activates adenylate cyclase

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 Pmk1 MAP kinase pathway

the cAMP response pathway

THE PENETRATION PEG AS SITE OF EFFECTOR DELIVERY

ezrin, radixin, moesin (ERM) domain proteins:

  • require for actin membrane interactions at the cortex of cells
  • found within septin ring at the appresorium pore

necessary for appressorium development

Fully functional if undergo mitosis

a number of potential receptors are involved in perception of surface signals

plant infection by pathogen

involves deployment of effector protein

PTH11

function effector protein: 1) surpress plant immunity responses. 2) facilitate proliferation of pathogen within plant tissue

BAR domain proteins:

  • imply in the control of membrane curvature generation

CFEM domain G-protein coupled receptor

necessary for perception of
the hydrophobic leaf surface

C.
higginsianum

if absence, appressoria do not form

eukaryotic cells:

  • undergo membrane curvature generation to generate invaginations associated
  • endocytosis and also cellular
    protrusions. eg: lamellipodia found in epithelial cells

Ultra-structural analysis

effectors within the appressorium pore at the point of plant infection

RAS signalling

generation of a dominant-active allele
of Ras2 (RAS2G17V)

abnormal appressorium
formation in the absence of a surface

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

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

  • 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.

Pmk1 kinase cascade (Mst11, Mst7 and Pmk1)

U. maydis

retrograde

scaffolded by a protein called Mst50

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

interacts with Mst11

  • Single round nuclear division


  • Conidium nucleus enter DNA-replication (S phase).

  • Inhibition DNA replication by hydroxyurea/ temp sensitive mutant nim1 (Abberant mitosis)

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)
  • 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.

upon activation and phosphorylation of its components

  • Turgor generation up to 8 Mpa (Blocked by targeted mutation of any 16 genetic component of non-selective- caused to be non-pathogenic.

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

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

Pmk1 regulate microconidia formation by M. oryzae

Pmk1 and Mst12 mutants show
reduced microconidia production

Mcm1 is essential for their development

widely conserved in other plant pathogenic fungi

required for infection related morphogenesis