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The biology of blast: Understanding how Magnaporthe oryzae invades rice…
The biology of blast:
Understanding how
Magnaporthe oryzae
invades rice plants
INTRODUCTION
Understand rice blast disease to develop new and durable disease strategies
Rice blast disease symptoms
In rice seedlings that have blast disease appear
small necrotic lesions
then, become
large and coalesce
Under humid condition, the
aerial hyphae
which generated from each lesions, release
conidia
which disperse to new rice plant.
In older plant, the disease spread from neck to
panicle
cause devasting symptoms and yield losses.
The symptoms can be observed at the nodes and neck holding the panicles.
Highlights
How
appressorium development in
M. oryzae
constitutes a highly orchestrated process that occurs in response to
physical cues
and
starvation stress
Which links
cell cycle progression
and the
molecular control of cytokinesis with cellular differentiation
,
programmed cell death
, and
turgor-driven entry into rice tissue
CARBON AND NITROGEN STARVATION IN M. ORYZAE INFECTION
Appressorium
Genes being expressed upon development of appressorium are MPG1 hydrophobin, Hex1 Woronin body associated gene and MSP1 gene
Appressorium development occurs in the absence of exogenous nutrients
Nitrogen source utilization M. oryzae
Regulated by the wide domain GATA-factor encoding gene NUT1
Domain GATA-factor encoding gene NUT1 is a functional homologue of AreA/nit-2 genes of A. nidulans and N. crassa
AreA/nit-2 genes are transcriptional activators of the nitrogen source utilisation pathway
Transcriptional activators are protein (transcription factor) that increases gene transcription of a gene or set of genes. Most activators are DNA-binding proteins that bind to enhancers or promoter-proximal elements.
Absence or Presence of Nitrogen source
When ammonium is present, binding of the GATA factor to promoter regions is inhibited by a transcriptional repressor NMR, which blocks their ability to undergo transcriptional activation
When ammonium is absent, GATA proteins are activated and bind to the promoter regions of genes (necessary for nitrate assimilation)
Nitrogen catabolite repression (NCR) is regulated in part by NUT1
MPG1 promoter shows the presence of GATA motifs that appear to be important in its regulation during nutrient starvation
might represent putative binding sites for the Nut1 transcription factor
Δnut1 mutants
Cannot grow on a variety of nitrogen sources including nitrate, nitrite, formamide, histidine and uric acid
Can grow on other nitrogen sources such as proline, glutamate and alanine, implying the potential for another wide domain nitrogen regulatory gene in M. oryzae
Δnut1 mutant is non pathogenic to roots, but infects leaves just like wild type strain
NUT1
positive regulator of nitrate reductase gene in M. oryzae
activates expression of nitrogen-regulated genes such as MPG1
involved in the regulation of the biotrophic to necrotrophic switch
occurs during proliferation of the fungus in rice tissue, prior to the onset of disease symptoms
AreA-like GATA factors
Effect of AreA like GATA factors
On pathogenicity
Depend upon the duration of the biotrophic phase of infection
Domain regulators of nitrogen source utilisation (NPR1 and NPR2 genes) for nitrogen pathogenicity regulation
involved in regulating nitrogen metabolism during nitrogen or carbon starvation
required for pathogenicity by M. oryzae
unlinked to NUT1 (because the genes are yet to be cloned and characterised)
Genes expressed during during rice infection under nitrogen limiting conditions in M. oryzae
global nitrogen regulator
NUT1
known pathogenicity genes;
MPG1
, the integral membrane protein
PTH11
, tetrahydroxynaphalene reductase
T4HR
, alternative oxidase
AOX
and neutral trehalase
NTH1
novel gene
SPM1
(involved in pathogenicity)
encodes a putative subtilisin serine protease involved in conidiation, normal appressorium development and invasive growth
Transcriptional regulation of SPM1 is also subject to NCR in M. oryzae
REACTIVE OXYGEN SPECIES GENERATION DURING APPRESSORIUM DIFFERENTIATION IN M. ORYZAE
M.oryzae have to overcome plant defenses such as the production of reactive oxygen species (early defense)
Reactive oxygen species are toxic molecules which trigger programmed cell death and strengthen plant cell walls
M.oryzae evolve by means of ROS detoxification
For instance, the colonization of M. oryzae is reduced by the deletion of SSD1 gene which is the regulator cell wall biogenesis.
To avoid the basal plant defenses, SSD1 assembly in the cell wall is needed to establish the early infection
CELL CYCLE CONTROL OF
APPRESSORIUM
DIFFERENTIATION IN
M. ORYZAE
appressorium differentiation occur by single round mitosis in germ tube following conidial germination
Conidium - tear-drop shaped cell with three cells, each containing a single nucleus
Initiation of appressorium
controlled by progression of germ tube > S-phase
DNA replication - by application of hydroxyurea (chemical inhibitor for DNA replication)
Hyroxyurea - cause M.oryzae to arrest growth with undifferentiated germ tube
Time course exposure of hydroxyurea > Progression into S-phase occured within 2-4h of spore germination on hydrophobic surfaces
Direct genetic evidence for role of DNA replication come from generation of temp-sensitive Monim1 mutant.
Monim1 > related to Aspergilus nidulans NimO gene (never mitosis mutant, nimO18), > also Saccharomyces cerevisiae DBF4 gene, (encodes regulatory subunit of Cdc7p-Dbf4p kinase complex, required for Cdc7p kinase activity and initiation of DNA replication)
although aberrant segregation of chromatin still occurs DNA unable to replicate > to cells advancing into mitosis with un-replicated DNA at restrictive temperature.
germlings arrested growth prior to hooking or swelling of the germ tube tip, consistent with initiation of appressorium formation requiring DNA replication
NimA temperature-sensitive mutant generated and exposed to the restrictive temperature, germ tubes became hooked & swollen, but > unable to differentiate mature appressoria
encodes a protein kinase necessary for entry into mitosis, suggesting that appressorium differentiation is controlled at the G2-M border
blocking mitosis by conditional mutation of MoBIM1 gene > functional homologue of A. nidulans BimE and S. cerevisiae APC1 (encodes large subunit of the anaphase-promoting complex, did not affect appressorium formation)
blocking mitotic exit by expressing mutant alleles of CYC1 and CYC2 >encoded stabilised versions of cyclinB, resistant to ubiquitin-mediated breakdown, did not affect appressorium differentiation.
Conclusion - Initiation of appressorium formation requires an S-phase control point, - appressorium differentiation requires control at the G2-M transition
differentiation appressorium
cytokinesis occurs and the site of cell division is spatially uncoupled from the site of mitosis (NOT EFFECT MORPHOGENESIS & PREVENT PLANT INFECTION)
site of septum formation is initially defined by a septin collar, laid down prior to mitosis in the germ tube.
Formation of the actomyosin contractile ring that initiates septum formation however occurs post-mitotically and linked to transit of the daughter nucleus to the incipient appressorium
by collapse and death of the conidium
three nuclei in conidium degraded due to plant infection result in single daughter nucleus in the mature apprpessorium from which all subsequent nuclei in invasive hyphae are formed
How conidial cell death?? > strong experimental evidence suggests that conidial cell death will occur by autophagy
Appressorium formation by
M. oryzae
Disease cycle of
M. oryzae
Appressorium
Unicellular
A mechanical force to cause rupture of the rice cuticle and entry into plant tissue
Dome-shaped structure which generates cellular turgor
Formation depends on
Environmental cues, such as
hydrophobicity
and
hardness of the contact surface
Plant cutin monomers
Nutrient starvation
nutrient depleted especially nitrogen sources
M. oryzae
can
formed appressoria on artificial plastic surfaces
away from host plant
Within 6-10h, appressorium is diifferentiate, it became darkly pigemented due to
melanin
.
Melanin
is essential for appressorium turgor generation and mutants that do not synthesise melanin correctly – which can be readily selected based on their different colours (albino, rosy and buff coloured) – are non-pathogenic
Appressorium turgor is generated by
accumulation of compatible solutes
Glycerol: a very high concentration within the infection cell, it focused at the leaves make it swollen which lead the
enormous pressure build-up
Allow penetration of hyphae at the base of appressorium
rupture the host plant cuticles
Gylcerol synthesized by mobilisation of lipid bodies from the
three-celled conidium
to the developing
appressorium
and
rapid breakdown of lipid and glycogen
LIpid ajnd glycogen contribute to
penetration hypha developmen
t and further fungal growth
Mutants lack of
Critical
multi-functional fatty acid β-oxidation protein Mfp1
, for instance, show a
substantial decrease in virulence
Examples: Mutants lacking c
arnitine acetyl transferase
, which is responsible for transport of acetyl CoA across the mitochondrial and/or peroxisomal membrane, are non-pathogenic
INFECTION-ASSOCIATED AUTOPHAGY
Autophagy
= cell survival mechanism that often triggered by nutrient starvation and results in breakdown of cytoplasm or organelles and recycling of their constituents
large increase in autophagosomes in conidia due to absence of nutrients during conidial germination on a surface inductive to appressorium formation
Autophagy does not occur when
PMK1 MAP kinase mutant. PMK1 is known to regulate appressorium formation and infectious hyphal growth in the rice blast fungus
ATG1 or any of the 16 genes necessary for non-selective macroautophagy
prevented rice blast disease
Autophagy-related protein 8
(Atg8) is a ubiquitin-like protein required for the formation of autophagosomal membranes
No effect on plant infection when sub-type specific genes essential for mitophagy or pexophagy deleted
infection-associated autophagy in
M. oryzae
requires bulk recycling for entire contents of the three-celled conidium > appressorium for further differentiation, turgor generation and subsequent growth during the initial stages of tissue invasion
Plays role during conidiogenesis for regulation of glycogen metabolism
CONCLUSION
Rice blast fungus undergoes morphological changes which leads to development and action of appressoria at the leaf surface
TREHALOSE-6-PHOSPHATE SYNTHASE, AND NADH-DEPENDENT GENETIC SWITCH IN M.ORYZAE
Trehalose synthesis
mediated by trehalose-6-phosphate synthase (T6PS), encoded by TPS1 gene
TPSI gene - required for trehalose synthesis, operation of functional appressoria and for invasive growth
Δtps1 mutants can use nitrite and ammonium, but not nitrate as sole nitrogen source irrespective of the carbon source available
TPS1 controls expression of the negative regulators of NCR, which have been designated NMR1, NMR2, and NMR3
Δtps1 mutant shows high levels of NMR1 transcript in nitrate-containing media, while expression of nitrate and nitrite reductase genes is also reduced
Trehalose-6-phosphate synthase
integrates control of glucose-6-phosphate metabolism and nitrogen source utilisation
by regulation of the oxidative pentose phosphate pathway
where NADPH is generated by glucose-6-phosphate dehydrogenase and is necessary for nitrate reductase activity
Tps1 directly binds to NADPH and regulates a set of related transcriptional co-repressors, comprising the three proteins, Nmr1, Nmr2, and Nmr3, which can each bind NADP
targeted deletion of any of the Nmr-encoding genes suppresses the non-pathogenic phenotype of a Δtps1 mutant
Tps1-dependent Nmr co-repressors control the expression of a set of virulence-associated genes, regulated by two additional GATA factors, as well as NUT1, that are de-repressed during appressorium-mediated plant infection
One of the GATA factors, Asd4, is essential for rice blast disease
Rice blast disease by M. oryzae requires a regulatory mechanism involving an
NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional co-repressors, and the balance of NADPH and NADP acting as the signal transducer
