11. WHY TO STUDY THE H-P
INTERACTION AND GENETICS
OF BLAST DISEASE RESISTANCE
IN RICE ???
12. ‘‘ ALL FACTS MUST BE DISCLOSED TO DERIVE
AN EFFECTIVE MANAGEMENT STRATEGY TO
FIGHT BACK RICE BLAST DISEASE’’
13. NEED FOR LISTENING MY PPT
Study of disease development.
we can understand the mechanism and
molecules contribute to virulence and the
capacity to cause damage in the host.
we study the recognition
phenomenon responsible for success or failure
of infection.
14. Soong Ying-Shin in China (1637)
Pyricularia oryzae (Cavara, 1891) in Italy: P. grisea (Rosman et
al., 1990)
Blast: damage it causes
India: McRae (1922) in Thanjavur (Tanjore) delta of South India
in 1918, epidemics occurred in 1919.
54 races have been identified so far in India
History, Geographical distribution
15. DRR, Production Oriented Survey, 1994 - 2006
Rice Blast incidence in different states of India during 1994 - 06
21. South India
DRR, Hyderabad
Nellore, Andhra Pradesh
Ambasamuddram, Tamilnadu
Coimbatore, Tamilnadu
Gudalur, Tamilnadu
Mandya, Karnataka
Ponnampet, Karnataka
Pattambi, Kerala
East and Central India
Chiplima, Orissa
Rewa, Madhya Pradesh
Jagadalpur, Madhya Pradesh
Wangbal, Manipur
Hazaribagh, Jharkhand
Ghagharghat, Uttar Pradesh
North and west India
Lonavala, Maharashtra
Malan, Gujarat
Hot spot locations for blast infection
Ram et al., 2007, Current Science 92: 224-230.
22. Reaction types of rice seedlings induced by
P. oryzae (Goto et al., 1967)
23. 0 No lesions on leaves. High resistant
1. Pinhead size and brown lesions on leaves. Resistant
2. Lesions size slightly bigger than that of scale 1. Resistant
3. Round to oval grey lesions with brown margins, lesion diameter
is about 1–2 mm. Moderately resistant
4. Typical spindle-shape lesion, 1–2 cm long, lesions were usually
localized between two veins. Moderately resistant
5. Typical spindle-shape lesion, the lesions area is ≤10% of the leaf
area. Susceptible
6. Typical spindle-shape lesion, the lesions area is 11% –25% of
the leaf area. Susceptible
7. Typical spindle- shape lesion, the lesions area is 26%–50% of
the leaf area. Susceptible
8. Typical spindle- shape lesion, the lesions area is 51%–75% of
the leaf area. High susceptible
9. Typical spindle- shape lesion, the lesions area is >75% of the
leaf area. High susceptible
Standard for resistance identification against rice seedling blast disease
SES, IRRI. 1996
24. First fungal pathogen of an economically important
International Rice Blast Genome Consortium: USA, UK, France
and Korea.
40 Mb distributed among 7 chromosomes
11,109 genes: 1 gene every 3.5 kb in the rice blast genome
Availability of rice genome sequence complemented by
pathogen sequence, having the way for carryout some insightful
experiments on fungal biology and molecular host-pathogen
interactions (Roy and Chattoo, 2005).
Molecular basis of disease development
Rice blast fungus sequenced
http://www.riceblast.org
27. Genetic changes through heterokaryosis, Parasexual
recombination, aneuploid.
Unstability of avirulence (AVR) genes of the fungal pathogen,
AVR-Pita, AVR-TSUY and PWL2 leads to virulence changes at
molecular level.
Valent and Chumley, (1994): M. grisea race containing AVR-Pita
gained virulence through mutations (Deletions, point mutations
and insertion of Pot3 in the AVR gene.
Pi-b gene and AVR-Pi-b: Originally found in indica cv.
introgressed into japonica background. Retrotransposon (Osr1)
mediated insertional mutation – Resistance breakdown (Jwa and
Lee, 2001).
Precise mechanism of resistance breakdown to be elucidated.
Reasons for blast resistance-breakdown in rice
28. PATHOGEN DEFENCE IN PLANTS
• Two types of plant resistance response to
potential pathogens :
• Non-host resistance response (frequent)
• Racecultivar- specific host resistance
response
(comparatively rare).
29. BASIC INCOMPATIBILITY
(Basic Resistance)
PATHOGEN
No genes expressed
needed For pathogenecity,
hence No colonization of
the plant feasible
No interaction
( or only interaction
towardsexpression of “active”
basic resistance)
PLANT
Non susceptible
to pathogen attack,
unfit for becoming
parasitized
“FIRST LEVEL” of
pathogen defense
heterologous non-host
(Prell & Day,2000)
30. Inducible plant defense reactions: active basic resistance and race specific
resistance
PLANTPLANT PATHOGENPATHOGEN
active basic resistance,
pathogenecity genes,
adapted to one single or
several plant species
Pathogenecity factors
triggering of defense reactions,
effective against
different pathogens
host plant resistance,
avirulence gene
produces the “signal”
recognized by the
resistant host plant
Avirulence factor
(specific elicitor)
non- host plant resistance, mechanism
pathogen non specific, and determined by
many different genes
SENSOR
SIGNAL
(general elicitor)
signal recognition leads to race
specific resistance,
Only to pathogen races with a corresponding
avirulence gene, expressed as HR
“gene for gene” recognition leading to
conditional expression of resistance
SENSOR
the resistance gene originates from
mutation, effective only against
particular pathogen races
race –specific resistance factor (receptor)
SIGNAL
(Specific elicitor)
(Prell & Day,2000)
32. PERCEPTION
• RECOGNIZATION
• It may take place directly or indirectly.
• Gene-for-gene relationship between host and
pathogen for triggering race- specific resistance.
– Direct interaction models
– Indirect interaction models
33.
34.
35. Four models have been proposed to
demonstrate the nature of recognition
reaction and the expression of the defense
reaction:
–The Elicitor- Receptor model
–The Dimer Model
–The Ion Channel defense model
–The Suppressor- receptor model.
36. • This hypothesis involve the two gene group
system of plant genes,
– where one gene act as a sensor pathogen
recognition.
– Second group of several genes express the
plant defence reactions
• However, this model does not explain how the
recognition by the plant turns on expression of the
plant dense genes.
Elicitor-Receptor Model
(Albersheim et al., 1981)
38. New defence mechanism are established in
the presence of compatibility
New defence mechanism are established in
the presence of compatibility
After specific recognition at the cell
membrane additional reaction chains release
the expression of defence genes in the cell
nucleus.
After specific recognition at the cell
membrane additional reaction chains release
the expression of defence genes in the cell
nucleus.
E-R
E-R
39. • Given by Ellingboe 1982.
• HERE Avr product and R-gene product form a dimer
that is , single gene in the host and single gene in
pathogen form a product made up of two gene
products.
• Directly blocking the genes leading to the
establishment of basic compatibility
• Re-activated the basic resistence
• Dimer acts as a genetic regulator at the level of
transcription.
41. • Regulate metabolic activities by either
activating or blocking gene expression,
permanently or transiently.
•Trans-proteins located at the cell surface,
function as ion channels, and triggering race
specific resistance.
•Not purely pathogen recognition but also for the
transduction of signals involved in pathogen
defence.
ION CHANNEL MODELGabriel,1988
43. • Another reason for proposing this model was plants
are subjected to stress such as chemical wounding or
infection or due to pathogen toxin or effectors, loss
of electrolytes from the affected cell or tissue.
• first response is loss of k+ ions, electrolytes and
membrane depolarization results from recognition
event associated with the pathogen attack that ends
in hypersensitive response.
44. SUPRESSOR RECEPTOR MODEL
• Model was extended by Bushnell and
Rowell (1981) and Heath (1982).
• In order to colonize a particular plant the
homologous pathogen has to produce specific
suppressor to block the action of general elicitor
i.e. pathogen blocks secondarily its own elicitor of
basic resistance.
• Model was extended by Bushnell and
Rowell (1981) and Heath (1982).
• In order to colonize a particular plant the
homologous pathogen has to produce specific
suppressor to block the action of general elicitor
i.e. pathogen blocks secondarily its own elicitor of
basic resistance.
47. Magnaporthe grisea (M. oryzae) INFECT host plant
3-celled conidia
• Release mucilage from the spore tip immediately upon hydration and contact with a host
surface . Spore Tip Mucilage (STM) – Hamer et al. 1988
• Produce germ - 30 minutes of contact with host surface (from the apical and basal cells )
• Start of appressorium differentiation - "hook" formation - this marks the onset of
appressorium differentiation
•Appressorial walls are thicker, multilayered and melanized compared to vegetative hyphae
(no melanin at point of contact with host - called the "appressorium pore“)
•"pore ring" - where the penetration peg develops to push through the cuticle
• After penetration of cuticle and cell wall of host - penetration peg branches into many
hyphae.
Magnaporthe grisea (M. oryzae) INFECT host plant
3-celled conidia
• Release mucilage from the spore tip immediately upon hydration and contact with a host
surface . Spore Tip Mucilage (STM) – Hamer et al. 1988
• Produce germ - 30 minutes of contact with host surface (from the apical and basal cells )
• Start of appressorium differentiation - "hook" formation - this marks the onset of
appressorium differentiation
•Appressorial walls are thicker, multilayered and melanized compared to vegetative hyphae
(no melanin at point of contact with host - called the "appressorium pore“)
•"pore ring" - where the penetration peg develops to push through the cuticle
• After penetration of cuticle and cell wall of host - penetration peg branches into many
hyphae.
48. Pathogens after perceiving plant signals may
transduce the signals using heterotrimeric guanine
nucleotide (G) proteins.
G-proteins act as transducers and signal amplifiers.
It composed of alpha, beta and gamma subunits
48
G - Proteins
49. G proteins are known to regulate many fundamental
events in fungi such as pathogenecity and virulence.
G proteins are important intermediates in a number of
signal pathways involving protein kinases.
G protein interacts with adenylate cyclase to result in
cAMP (cyclin adenosine monophosphate) generation
and protein kinase A activation
52. 52
Genes involved in appressorium formation
in M. oryzae
MPG1 - first identified as a differentially
expressed gene during infection of rice
(Talbot et al. 1993).
MPG1 encodes a class I hydrophobin -
play a role in surface perception.
Exogenous addition of cAMP causes
Mpg1 mutants to form appressoria.
53. 53
• Mpg1 plays a role in adhesion to the host leaf
surface, spore germination and appressorium
formation.
• It provides an adaptor surface between the
hydrophilic surface of the germ tube and the
hydrophobic rice leaf.
(Dean, Annu. Rev. Phytopthol.
1997).
54. 54
Calcium / Calmodulin Dependent Signaling
• Cytoplasmic calcium plays a role in germination,
hyphal tip growth and appressorial development.
• Cytosol calcium plays an important role in hyphal
tip growth and appressorial development in many
organisms.
Calcium signal transduction pathway
55. 55
IP3 in turn causes the release of endogenous calcium
from intercellular calcium stores.
Intercellular calcium result in the activation of
calmodulin, receptor protein.
Calmodulin gene expression is dependent on spore
attachment to the host surface and appressorium
formation
56. cAMP: Cyclic adenosine mono phosphate)
The adenylate cyclase gene MAC1 used for penetration
into the host.
Adenylate cyclase may be a downstream target of the
activated G-protein and exogenous cAMP.
Gα subunit protein encoded by the MAGB in M. grisea
stimulate adenylate cyclase activates cAMP mediated
signalling.
Adenylate cyclase mediated cAMP signalling pathway
operates downstream of G-protein signalling system.
56
57. 57
• cAMP act as part of an early signalling event in
appressorium formation.
• On addition of cAMP to germinating conidia
appressorium formation was observed even in
hydrophilic surfaces.
• In fungi, cAMP has been shown to be an important
regulator of development.
58. MAPK signaling pathways play a pivotal role in
sensing extracellular signals and relaying the signals
to control gene expression.
The best-characterized MAPKs are MPK3, MPK4
and MPK6, all of which are activated by a diversity of
stimuli including abiotic stresses, pathogens and
oxidative stress.
MAPK cascade
58
59. PAMP: (Pathogen-Associated Molecular Pattern)
• PAMP-triggered activation of MAPK cascades.
• Plants have adapted the capacity to recognize
pathogens through PAMPs via cell surface
located pathogen-recognition receptors.
• The activation of these receptors induces PAMP-
triggered immunity.
59
60.
61. RESPONSE
Programmed cell death (PCD)
PCD is a process aimed at eliminating redundant or harmful cells during the life
cycle of multicellular organisms.
It is essential to the development and maintenance of multicellular organisms.
PCD is responsible for
the removal of excess cells in the developing nervous system, or
activated in defence against infected or mutated cells, preventing further
proliferation of a pathogen or disease
Specific biochemical and morphological features
Condensation of the nucleus and cytoplasm,
Fragmentation of genomic DNA into large (50 to 300 kb) and subsequently small
(200 bp) nucleosomal fragments (DNA laddering)
Fragmentation of the cell into membrane-confined vesicles (apoptotic bodies).
The cell death process displaying such features is called apoptosis.
Hoeberichts and
62. Programmed cell-death ( PCD)
• 1964 Lockshin & Williams : PCD concept was used - respect to insect
tissue development
• PCD has been the subject of increasing attention and research efforts.
• 2002 Nobel prize in Physiology or Medicine
Sydney Brenner(UK)
H.Robert Horvitz(US) and
John E.Sulston(UK)
• Death of a cell in any form, mediated by an intracellular program.
• PCD is carried out in a regulated process which generally confers
advantage during an organism's life cycle.
• PCD serves fundamental functions during tissue development.
64. Sites of PCD in Vascular Plants
Pennell and Lamb, 1997
Deletion of cells with temporary functions
suspensor cells in embryos and of aleurone
cells in seeds
Deletion of unwanted cells
stamen primordia cells in unisexual flowers
and root cap cells
Deletion of cells during sculpting of the plant
body
leaf cells during leaf lobing
Deletion of cells during cell specialization in
TEs (cell death in xylem tracheary elements )(TEs)
Deletion of cells during plant interactions with
pathogens
cells in an HR and cells in uninfected leaves
in response to HR-derived signals
65. Model of plant programmed cell
death
Hoeberichts and Woltering, 2002
66. Plant –Pathogen Interactions are dynamic
Much communication between the host and the
parasite
Outcome reflects the attempts of the host and the
parasite to adapt to defenses and virulence factors
respectively
Robust Resistance to Pathogen
Specifically recognize the pathogen
Rapidly induce a variety of potential defenses
Often interaction results in plant cell death
Greenberg,J.T. , 1997
67. Spatial Uncoupling of Mitosis and Cytokinesis during
Appressorium-Mediated Plant Infection by the Rice
Blast
Fungus Magnaporthe oryzae
Saunders, D. G. O et al., 2010
The Plant Cell, (22): 2417–2428
68. • The appressorium development in M. oryzae involves spatial
uncoupling of mitosis and cytokinesis.
The cell division
appressorium differentiation
Initially by formation of a heteromeric septin ring complex before the
onset of mitosis.
Mitosis in the fungal germ tube
Followed by long-distance nuclear migration
Rapid formation of an actomyosin contractile ring in the neck of the
developing appressorium, at a position previously marked by the
septin complex.
• The appressorium development in M. oryzae involves spatial
uncoupling of mitosis and cytokinesis.
The cell division
appressorium differentiation
Initially by formation of a heteromeric septin ring complex before the
onset of mitosis.
Mitosis in the fungal germ tube
Followed by long-distance nuclear migration
Rapid formation of an actomyosin contractile ring in the neck of the
developing appressorium, at a position previously marked by the
septin complex.
69. When considered together, these results indicate that
SEP1 (SEPTATION-ASSOCIATED1), is essential for
determining the position and frequency of cell division
sites in M. oryzae
Conditional mutation of Sep1 a
key spatial regulator of cytokinesis and nuclear division,
is sufficient to prevent rice blast disease.
70. 70
Fig: Spatial Uncoupling of Mitosis and Cytokinesis in the Rice Blast Fungus M.
oryzae.
Mitosis occurring during appressorium development by M. oryzae. Conidial
suspensions of the M. oryzae H1: RFP strain were prepared and the lipophilic stain
DiOC6 used to stain the nuclear envelope. A differential interference contrast (DIC)
image of the whole germ tube and developing appressorium in the left panel.
71. 71
Fig: Micrographs of M. oryzae strain Guy-11, ΔcpkA, Δ pmk1, and Δmst12
mutants expressing H1:RFP, and tpmA:GFP gene fusions incubated on cover
slips to allow appressorium development. Septation was spatially separated
from the site of nuclear division only in Guy-11 and the Δ mst12 strains.
72. 72
Fig: Septin Ring Formation Occurs Prior to Mitosis during Appressorium
Development by M. oryzae.
Laser confocal microscopy shows septin ring formation at the base of the
germ tube proximal to the conidium, at the base of incipient appressoria,
dual localization to both of these positions, and the dispersal of septin
complexes during appressorium maturation. Arrows indicate the positions of
septin ring structures.
73. 73
Cell Cycle–Mediated Regulation of Plant
Infection by the Rice Blast Fungus
(Saunders, D.G.O et al., 2010).
Plant Cell (22): 497–507
74. 74
• Entry into S-phase is critical for regulating initiation of
appressorium development.
• In mitosis, the swollen germ tube tip always developed
prior to mitosis, providing the destination for one of the
daughter nuclei, which rapidly migrates into the germ
tube apex
75. 75
Fig: Microtubule Dynamics and Nuclear Division in M. oryzae during Germination and
Appressorium development. (A) nuclear division during appressorium development in
M. oryzae. The grg(p):H1:RFP and ccg1(p):bm1:sGFP gene fusion vectors were
introduced into the M. oryzae wild-type strain Guy-11. Nuclei (red) and microtubules
(green) were observed during conidial germination and appressorium development in M.
oryzae.
76. 76
Fig: Model for the Regulation of Infection Structure Development by Cell Cycle
Progression in M. oryzae.
Arresting S-phase with HU or nim1I327E
mutation prevents germ tubes from swelling and
initiating appressorium formation. Entry into mitosis is both necessary and sufficient for
appressoria to fully differentiate in the phenotypes of
the nimAE37G
and nimAE37G
bim1F1763*
mutants. Mitotic progression and exit are not necessary
for appressoria to mature but may regulate repolarization and subsequent disease
development.
77. 77
Fig: B) Astral microtubules were observed during mitotic spindle elongation and the
concurrent rapid separation of chromosomes.
(C) Continued expansion in diameter of the incipient appressorium during mitotic
division.
78. Effectors and Effector Delivery in
Magnaporthe oryzae
78
(Zhang and Xu, 2014)
PLoS Pathog. 10: e1003826.
79. 79
• pathogenic micro-organisms secrete effector proteins
into host tissue to suppress immunity and support
pathogen growth.
• The rice blast fungus M. oryzae possesses two distinct
secretion systems to target effectors during plant
infection.
• Apoplastic effectors accumulate extracellularly at the
host–pathogen interface.
80. 80
• Actively secreted via the conventional secretory
process in filamentous fungi.
• cytoplasmic effectors delivered inside rice cells are
secreted by a different pathway involving the exocyst
complex.
• BIC (biotrophic interfacial complex) have been
hypothesized as the site of translocation of
cytoplasmic effectors into the rice cytoplasm
81. 81
Fig: Localization of M. oryzae effectors during plant infection. A. Cytoplasmic
effectors (♦) are secreted into the biotrophic interfacial complex (BIC) before being
translocated into plant cells. B. Apoplastic effectors (◊) are secreted into the space
between the fungal cell wall and extra-invasive-hyphal membrane (EIHM). Like IH,
effector proteins may move from cell to cell via plasmodesmata. AP, appressorium.
82. 82
Nucleo cytoplasmic tansport
pathways
signaling complexes
Fig: Integrative Model of M. oryzae Differentiation on Leaves and Roots.
PLS1-dependent signalling complexes are also active during both leaf and root colonization.
MPG1 and PTH11 constitute upstream sensors involved in appressorium morphogenesis.
GAS1, GAS2, CPKA-mediated turgor generation and melanin production are required for
appressorium-mediated penetration
83. 83
Common Genetic Pathways
Regulate Organ-Specific Infection-
Related Development in the Rice
Blast Fungus
Sara L. Tucker,a,1 Maria I. Besi,a,1 Rita
Galhano,a Marina Franceschetti,a Stephan
Goetz,a Steven Lenhert,b Anne Osbourn,c
and Ane Sesmaa,
a Department of Disease and Stress
Biology, United Kingdom
84. 84
• Here, we characterize an infection-related development in M. oryzae
produced on hydrophilic polystyrene (PHIL-PS) and on roots.
• We show that fungal spores develop preinvasive hyphae (pre-IH) from
hyphopodia (root penetration structures) or germ tubes and that pre-IH
also enter root cells.
• Changes in fungal cell wall structure accompanying pre-IH are seen on
both artificial and root surfaces. Using characterized mutants, we show
that the PMK1 (for pathogenicity mitogen-activated protein kinase 1)
pathway is required for pre-IH development.
• Twenty mutants with altered pre-IH differentiation on PHIL-PS identified
from an insertional library of 2885 M. oryzae T-DNA transformants were
found to be defective in pathogenicity.
• The phenotypic analysis of these mutants revealed that appressorium,
hyphopodium, and pre-IH formation are genetically linked fungal
developmental processes.
• We further characterized one of these mutants, M1373, which lacked
the M. oryzae ortholog of exportin-5/Msn5p (EXP5).
• Mutants lacking EXP5 were much less virulent on roots, suggesting an
important involvement of proteins and/or RNAs transported by EXP5
during M. oryzae root infe
85. CONCLUSION:
•Plant-breeding strategies that can integrate multigene traits
that are more durable.
• Biotechnology solutions to rice blast could also introduce
broad spectrum resistance genes, or pyramids of several
genes, directly into high-cropping commercial rice
cultivars.
•Allow the intervention and rapid deployment of plant
defence signalling within the plant using rice-expressed
transgenes.