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The biology of blast: Understanding how Magnoporthe oryzae invades rice…
The biology of blast: Understanding how
Magnoporthe oryzae
invades rice plants
Introduction
Rice blast causes 10-30% harvest losses to the global rice production
Investigating the response of this fungus to the existing environmental condition where it come into contact on rice leaf surface
This research focus on early stages of rice infection by blast fungus;
Magnoporthe oryzae
or originally known as
Magnoporthe grisea
Conclusion
Cell cycle checkpoint govern the progression of appressorium development and the infection-associated autophagy is very important for biological role of these cells
Magnoporthe oryazae
undergo specific sequence of morphogenetic events that lead to the development and action of appressoria on the rice leaf surface
Appressorium formation by
Magnoporthe oryazae
Appressorium
dome shape
generates cell turgor( ruptures the rice cuticle and makes a entryway.)
unicellular
the appressorium only forms during environmental cues. however also able to grow in different pplaces far from host
the higher the environmental cues, the higher the virulence. these cues allows critical development of the appressorium and subsequnt pathogens
Disease cycle
spore germination
germ tube development
appressorium formation
penetration peg emergence
conidial attachment
invasive growth in host
becomes darkly pigmented due to formation of melanin in the cell wall
generates appressorium turgor and the formation of other mutants
compatible solutes(glycerol) is accumulated. in high concentration it provides a higher pressure on the leaf surface that allows penetration of the hypha at the base of appressorium to rupture the hosts cuticle.
Cell cycle control of appressorium differentiation in
M. oryzae
NimA
Encode protein kinase
Appressorium differentiation attached at G2-M border
It's locking within MoBim1 gene
Appressorium found at restrictive temperature
Germination
From tapering end of spore and nucleus passes into germ tube (mitosis occur).
One of daughter nuclei moves to swollen germ tube tip while others moves back into spore cell.
Appressorium differentiation
single
round of mitosis in the germ tube following conidial germination.
tear-drop shaped cell with three cells, each containing a single
nucleus.
MoBim1 gene
Alleles of CYC1 and CYC2
Encoded stabilized version of cyclin B
resistant to ubiquitin-mediated breakdown and not affect appressorium differentiation
MoNIM1 mutant
Related to NimO gene and also DBF4 gene
Encodes regulatory subunit of Cdc7p-Dbf4p kinase complex
Required for Cdc7p kinase activity and initiation of DNA replication
Appressorium differentiation
Cytokinesis occurs and sites of cell division spatially uncoupled from site of mitosis
Altering spatial control of cytokinesis prevent plant infection
Site of septum formation defined by septin collar
Formation of actomyosin contractile ring initiates septum formation
It's accompanied by collapse and death of conidium
Conidial cell death requires autophagy
Trehalose-6-phosphate synthase, an NADPH-dependent genetic switch in
M.oryzae
Trehalose synthesis
Operation of functional appresoria & for invasive growth
Mediated by
Trehalose-6-phosphate synthase (T6PS)
Encoded by
TPS1 gene
TPS1 controls expression of
negative regulators of NCR
NMR1
NMR2
NMR3.Δtps1 (without TPS1 gene)
In a nitrate-containing media: NMR3.Δtps1 mutants shows high levels of NMR1 transcript
Shows a reduced expression of nitrate & nitrite reductase genes
Irrespective of C source available, Δtps1 mutants use N source of nitrite and ammonium, but not nitrate
Integrates control of glucose-6-phosphate metabolism & nitrogen source utilisation
By regulation of
oxidative pentose phosphate pathway
Where NADPH is generated by glucose-6-phosphate dehydrogenase, which is necessary for nitrate reductase activity
Directly binds to NADPH & regulates
a set of related transcriptional co-repressors protein
(which can bind NADP)
Nmr1
Nmr2
Nmr3
Targeted deletion of any of the Nmr-encoding genes, supresses the non-pathogenic phenotype of Δtps1 mutant
Tps1-dependent Nmr co-repressors appear to control the expression of a set of
virulence associated genes
Regulated by two additional
GATA factors
, NUT1 that are de-repressed during appresonium-mediated plant
Asd4 (one of the GATA factors) is essential for rice blast disease
In conclusion,
M.oryzae
requires a regulatory mechanism which involves
NADPH sensor protein
Tps1
A set of NADP-dependent transcriptional co-repressors
Balance of NADPH & NADP acting as the signal transducer
Reactive oxygen species generation during appresorium differentiation in
M.oryzae
In an infection by
M.oryzae
, plant responses attack is by production of
Reactive Oxygen Species (ROS)
surrounding the infection sites
Its a toxic molecules that can trigger programmed cell death in infected plant cells
It can also toughen plant cell walls
But, the fungus has evolved to effective means of rapid ROS detoxification
Deletion of
SSD1 gene
(regulator of cell wall biogenesis)
Causes the colonisation of rice leaves by the fungus to be severely reduced
Host cells that are infected by hyphae of Δssd1 mutants shows an accumulation of ROS
Suggest that assembly of cell wall by SSD1 is essential for the initial establishment of infection, by avoiding the induction of basal plant defence responses
Interestingly, ROS production by fungal NOX1 and NOX2 NADPH oxidases is also essential for appresorium function & pathogenicity of
M.oryzae
Δnox1 & Δnox2 mutants are unable to carry out appresonium cuticle penetration
It appear to be necessary for formation of polarised penetration hypha at base of appresonium
Metallothionein
- a cellular redox sensors
MMT1 gene
It showed reduced expression in Δpmk mutant
Encodes for 22 amino acids metallothionein in
M.oryzae
MMT1 metallothionein-like protein acts as powerful antioxidant
Probably plays a role during oxidative cross-linking within fungal cell wall during development & polarity re-establishment during plant infection
Δmmt1 mutants are non-pathogenic, and unable to cause penetration-mediated penetration on host surface
CATB gene
ΔcatB mutant is severely impaired in pathogenicity and on exposure to ROS, the virulence is further reduced
Its also possible that CATB is involved in
Fungal cell wall biogenesis
Differentiation during appresorium formation
Penetration hypha production
During plant invasion, encodes secretion of catalase B which helps in maintenance of fungal cell wall integrity of
M.oryzae
infection associated autophagy
autophagy
the breakdown of the cytoplasm and organelles due to nutrient starvation
regulate glycogen metabolism during conidiogenesis
conidial germination
in conidial germination, the concentration of autophagosomes in the conidia increases due to lack of nutrients
induces the surface that allows appressorium formation
when PMK1 kinase mutant or any nutrient is presented: autophagy does not occur
Autophagy Inhibitory
prevents conidia cell death
impairs the ability of the fungus to cause rice blast
appressorium becomes non functional
some subtype specific gene (needed for mitophagy/pexophagy) when deleted it does not have an affect on plant infection
infection associated autophagy in
M.oryzae
needs bulk cycling of the whole thing in 3 cell conidium to appressorium to further differentiation,turgor generation & subsequent growth of initial stage of tissue innovation
However, infection associated autpphagy, it's mechanism is yet unknown however, it does involves cell cycle mediated regulations( e.g inhibit mitosis)
Carbon and Nitrogen starvation in
M. oryzae
O2 & N2 absence
-appresorium development occurs
-starvation stress modulates gene stress
-MPG1 hydrophobin
-Hex1 Woronin body associated gene
-MSP1 gene
-> regulated by nutritional conditions & transcriptional profiling
Nitrogen source utilization
-regulated by wide domain GATA factor encoding gene NUT1
AreA/nit-2 genes
-transcriptional activators of nitrogen source utilization pathway
ammonium absence:
-GATA proteins are activated & bind to promoter regions of genes involved in nitrogen source utilisation
ammonium presence:
-binding of GATA factor to promoter is inhibited by a transcriptional repressor NMR
-transcriptional activation inhibited
nitrogen catabolite repression (NCR) -regulated by NUT1
^nut1 mutants
-impaired to grow a variety of nitrogen sources including nitrate, nitrite, formamide, histidine, uric acid
-can use other nitrogen source (proline, glutamate, alanine)
-NUT1 gene is also a positive regulator of nitrate reductase gene
-activates expression of nitrogen-regulated genes such as MPG1
-pathogenicity
depend upon duration of biotrophic phase of infection
-Nut1 involved in regulation of the biotrophic to necrotrophic switch
-occurs during proliferation of fungus in rice tissue
NPR1 & NPR2 involved in regulating nitrogen metabolism during N/C starvation & required for pathogenicity
pathogenicity genes of NUT1 :
-MPG1, integral membrane protein PTH11, tetrahydroxynaphalene reductase T4HR, alternative oxidase AOX and neutral trehalase NTH1
-expressed during rice infection
novel gene:
SPM1 gene encodes a putative subtilisin serine protease involved in conidiation, normal appressorium development & invasive growth
^spm1 mutants do not use various secondary nitrogen sources and regulate MPG1 expression
Tps1 (trehalose-6-phosphate syntase) integrates control of glucose-6-phosphate metabolism & nitrogen source utilization
-by regulation of of oxidative pentose phosphate pathway
-NADPH is generated by glucose-6-phosphate dehydrogenase for nitrate reductase activity
Tps1 binds to NADPH - regulates trascriptional co-repressors, Nmr1, Nmr2 & Nmr3 which can bind to NADP
targeted deletion of Nmr-encoding genes suppresses the non-pathogenic phenotpe of a ^tps1 mutant
-Tps1-dependent Nmr co-repressors control the expression of virulence-associated genes
GATA factors, Asd4 is essential for rice blast disease
-requires a regulatory mechanism involving an NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional co-repressors, & balance of NADPH and NADP as the signal transducer