Plant immune system
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Transcript of Plant immune system
Doctoral Seminar on
“Plant immunity: towards an Plant immunity: towards an integrated view of plant pathogen integrated view of plant pathogen interaction and its implication in interaction and its implication in
plant breedingplant breeding”
H G Kencharaddi 2nd Ph d
Welcome
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Seminar on
“Plant immunity: towards an Plant immunity: towards an integrated view of plant pathogen integrated view of plant pathogen interaction and its implication in interaction and its implication in
plant breedingplant breeding”2
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“For each resistance gene in the host there is a corresponding gene for avirulence in the
pathogen cnferring resistane and viceversa” Host plant genotype
Pat
hoge
nge
noty
peR1 r2 r1 R2
Avr1, avr2 I
I
C
Cavr1, Avr2
I - incompatible - no diseaseC - compatible - disease
Gene for gene hypothesis
H.H. Flor 7
It is a state of defense against infectious pathogens
Pathogens are like Bacteria, Fungi, Virus, Nematode, Oomycetes etc.
Mode of entry of pathogen depend on type of pathogen
Bacteria – stomata, hydathodes and wounds Nematode – StyletFungi – Haustoria
What is plant immunity?
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Principles of plant immunity
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Forms of plant resistance
Antipathy- Lack of interest of pests or pathogens in a plant. Ex- Resistance of Arabidopsis to insects - Glucosinolate contents
Hindrance- Lack of pathogen’s ability to parasitize the plant because of certain plants features
Ex-higher levels of calcium - macerating pathogens through strengthening the cell walls
(Datnoff et al. 2007)
Defence- Based on the plant innate immune system
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PAMPs Triggered Immunity (PTI)
Effector triggered susceptibility (ETS)
Effector Triggered immunity (ETI)
Phases of Plant immunityPhases of Plant immunity
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PAMP Triggered Immunity (PTI)PAMP (Pathogen Associated Molecular Pattern)
The molecules of pathogens, conserved across larger group of pathogens
Highly indispensable to the pathogens, required for their survivality
These molecules do not exist in host
Ex. Flagellin, EF-Tu, lipid, chitin, protein molecules of
pathogens
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• Plasma membrane-localized receptors that
recognize the presence of PAMP’s in the
extracellular environment.
• Located in plasma membrane
• Ex. FLS2, ERF, CEBiP, etc
PRR (Pattern recognition receptor)
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ETS (Effector Triggered Susceptibility)ETS (Effector Triggered Susceptibility)
Effector are any regulatory molecules secreted
by pathogens
Modifies host protein to establish their growth
Effector perform three main functionsEffector perform three main functions
Structural role: Ex. Fungi, secret extra haustorial molecule
Nutrient leakage: Ex. P. syringae HopM effector protein
Pathogenicity: Ex. HopA1 dephosphorylates MAP kinase results in inhibition of PTI 14
The plant defence response elicited by effector recognition.
The effector molecules are recognized by R protein
Four major classes of R genes
NB-LRR (nucleotide binding leucine rich repeat) genes
Ser/Thr kinases
Receptor-like kinases (RLKs)
Receptor-like proteins (RLPs)
Effector triggered immunity (ETI)
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Different phases of the zig-zag model
Jonathan & Jeffery, 2006 16
Model for resistance in plants
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Defence responses post-pathogen recognition
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Stomatal closure
Ion fluxes
Oxidative burst
Phyto-Hormone action
Induced systemic resistance
Systemic Acquired Resistance
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Defence Mechanism of plant toward off pathogens
1. Stomatal closure1. Stomatal closure
Stomata are natural opening through pathogen can
easly enter into apoplast
Stomatal closure is part of a plant innate immune
response to restrict bacterial invasion.
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Bacteria and PAMPs Trigger Stomatal Closure in Arabidopsis21
Maeli etal, 2006
(1) Stomata actively closes as an initial response toboth plant and human pathogenic bacteria,
(2) Pst DC3000 has evolved a mechanism to reopen stomata 3 hr after incubation
(3) Inoculum concentration 1x 107
cfu/ml 1hr-closure & 3hr-Reopen
Involvement of the FLS2 Receptor and Salicylic Acid in PAMP Induced Stomatal Closure 22
flg22: biologically active peptide derived from flagellin
MES: Buffer
LPS: Lipopolysaccharides
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Col-0: Wild typefls2: Flagellin receptor mutant
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Salicylic Acid in PAMP Induced Stomatal Closure
eds 16-2: SA-biosynthetic mutan plantnahG : SA-deficient transgenic plants
Model Depicting Bacterium- and PAMP-Induced Stomatal Closure in the Arabidopsis Guard Cell
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Subunit of E3 ubiquitin ligase
involvedJA signalling
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2. Ion fluxes Membrane permeability changes rapidly leading to
a loss of cellular electrolytes such as K+ and an
uptake of H+.
At the same time, there is often an influx of Ca2+,
a key intracellular signal in plants that is involved
in the activation of enzymes and gene expression.
The experimental blocking of Ca2+ transport across
membranes in inoculated bean cells also inhibits
gene activation and subsequent defence responses.
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3. Oxidative burst
It is a rapid, transient, production of huge
amounts of reactive oxygen species (ROS)
Produced from membrane localized NADPH
oxidase (Nuhse et al, 2007)
JA/SA pathway activated, finally PCD
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Abbreviations used : AC, adenylate cyclase ; CWP, cell-wall-bound peroxidase ;E, elicitor; Er: receptor; G, GTP-binding protein(s); PLase A and PLase C, phopholipases A and C; R, reductant.
Schematic representation of major hypotheses describing the possible origin of ROS building the oxidative burst
4. Phyto-hormones
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• Rapid death of cells in the local region surrounding an infection.
• Restrict the growth and spread of pathogens to other
parts of the plant.
• Favor growth of pathogens with a necrotrophic lifestyle
5. Hypersensitive response
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Biotrophic: pathogens propagate in living plant tissue and generally do not cause necrosis as a result of infection.
Necrotrophic: pathogens actively induce necrosis in infected tissues, often through the production of toxins.
Hemibiotroph: An organism that is parasitic in living tissue for some time and then continues to live in dead tissue
It is secondary resistance response
Because, once plant defense responses are
activated at the site of infection, a systemic defense
response is triggered in distal plant parts to protect
these undamaged tissues against subsequent
invasion by the pathogen.
Long-lasting and broad-spectrum induced disease
resistance
Act non-specifically through out the plant and
reduce disease severity
6. Systemic Acquired Resistance(SAR)
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SAR signal is a generated with in 4hr of
inoculation
SA could be detected in phloem of leaf 8hr after
inoculation
Increased level of SA in phloem of leaf above
the incubated leaf
Expression of SAR occurs with in 24hr after
inoculation
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PR proteins (PRP) Proteins produced in plants when it is attacked by
pathogen, they are antimicrobial/viral/ insecticidal
Its extremely acidic/ basic in nature, therefore it is
highly soluble an highly reactive.
Crosslink the molecules of cell wall and acts as
barricade by accumulation of lignin which helps the
cell wall to protrude as papillae.
Gives alarming signals to neighbouring cells
It present in both resistant and susceptible plant, but
concentration is differs. When there is infection its
concentration increases and viceversa.
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PR proteins
Plants in which PRP detected
Function
PR1 Rice, barley, maize, tomato, tobacco
Plant cell wall thickening, resistance to the spread of the pathogen on the apoplast
PR 2 Rice, barley, maize, tomato, tobacco, potato, pepper, bean, Brassica, sugar beet
β-1-3-glucanase
PR3 Rice, maize, tomato, pepper, sugar beet, rape seed
Chitinase
PR 4 Tomato, tobacco, rubber tree
Chitinase
PR5 Rice, wheat, barley, oats, tomato, tobacco, potato
Alternation of fungal memnrane
PR6 barley, tomato, tobacco
Proteinase inhibitor
PR7 Tomato Endoproteinase
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PR proteins
Plants in which PRP detected
Function
PR8 Cucumber Chitinase
PR9 Tomato, rice, tobacco, wheat
Peroxidase
PR10 Potato, asperagus, pea, bean, rice
Ribonucleases
PR11 Tobacco Chitinase
PR12 Arabidopsis, pea, Defensin
PR13 Barley Thionin
PR14 Barley Lipid transfer proteins
PR15 Barley Germin like oxalate oxidase
PR16 Barley and wheat Germin like proteins without oxalate oxidase
PR17 Wheat, barley, tobacco Peptidase
Breeding and biotechnological strategies used to induce resistance (Immunity ) in plants
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1. Manipulating PAMP/MAMP receptors to induce immunity
PTI activation is based upon the recognition of
microbial surface structures (PAMPs/MAMPs), such
as bacterial flagellin, bacterial elongation factor EF-
Tu or fungal chitin.
For example, Arabidopsis FLS2 and EFR are plasma
membrane receptor kinases that sense flagellin or
EF-Tu through binding to their leucine-rich repeat
(LRR) ectodomains
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2010
2. Pyramiding and Introgressing R gene
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2003, PNAS
Late blight, caused by the oomycete pathogen
Phytophthora infestans, is the most devastating
potato disease in the world
The wild diploid potato species Solanum
bulbocastanumis highly resistant to all known
races of P. infestans
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Cloning of the major resistance gene RB in S.
bulbocastanum by using a map-based approach in
combination with a long-range (LR)-PCR strategy.
A cluster of four resistance genes of the CC-NBSLRR
(coiled coil–nucleotide binding site–Leu-rich repeat)
class was found within the genetically mapped RB
region.
Transgenic plants containing a LR-PCR product of
one of these four genes displayed broad spectrum
late blight resistance.
Late blight, caused by the oomycete pathogen Phytophthora infestans,
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Genetic and physical maps of the genomic region
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BAC clones from the RB haplotype (filled boxes) and BAC clones from the rb haplotype (open boxes). Both 177O13 and CB3A14 contain one truncated and four complete RGAs. The direction of transcription of each gene(an arrow). The 3.6-kb deletion region between RGA2 and RGA-tris marked.
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Late blight screening of transgenic plants by using isolate US930287
Plants were scored as resistant (R) if the resistance score was >7.0 (< 25% infection) and plants were scored as susceptible was <6.9 (>25% infection). † Of the 14 resistant plants, nine plants had a score of 7 and five plants had ascore of 8.
Complementation analysis of putative RB genes
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(A–C) Transgenic Katahdin plants- RGA1-PCR,RGA2-PCR, and RGA4-PCR, respectively. (D) Control Katahdin plant. (E) Katahdin plant that was not inoculated. (F–I) Transgenic Katahdin plants containing constructs RGA1-BAC, RGA2-BAC,RGA3-BAC, andRGA4-BAC, respectively.
Structure of the RB gene and the deduced RB protein.
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Disadvantage of R genes …….?
Ectopic expression of R genes can
sometimes activate defence pathways in the
absence of pathogen
Reduced crop yields
Reduced Fitness
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3. Antifungal fusion proteins to induce immunity
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Fusarium head blight (FHB) or scab of wheat is a devastating disease in warm and humid regions at wheat-flowering periods worldwide.
Expression of pathogen-specific antibodies in plants has been proposed as a strategy for crop protection.
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An antibody fusion protein comprising a Fusarium-
specific recombinant antibody derived from chicken
and an antifungal peptide from Aspergillus giganteus
was expressed in wheat as a method for protecting
plants against FHB pathogens.
Plants expressing the antibody fusion displayed a
very significantly enhanced resistance in T2 and T3
generations upon single-floret inoculation with the
macroconidia of Fusarium asiaticum, the
predominant species causing FHB in China, indicating
a type II resistance.
Structure of AG-D2 fusion construct
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An antifungal peptide sequence from Aspergillus giganteus (AG) and a single-chain Fv (scFv) antibody coding region from chicken.
Connected by a sequence encoding a 10-amino-acid glycine-serine linker.
The AG-scFv fusion construct was inserted into the plant expression vector pAHC25 using EcoRI and SacI sites.
Ubi-Pro, maize ubiquitin promoter; UT: 5′ untranslated region of the petunia chalcone synthase gene; LP, leader peptide sequence; c-myc, c-myc epitope tag; His6, histidine 6 tag; Nos-T-Nos terminator.
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Integration and expression of AG-scFv fusion gene in transgenic wheat.
A, T3 transgenic wheat lines 2, and To detect the presence of the AG-scFv fusion gene with primers AGP1 and scFvP2.
B, RNA extracted from leaves of the plants in A was used in a RT- PCR assay to analyze expression of the AG-scFvfusion gene with the same set of primers in A.
C, Proteins extracted from leaves in A were fractionated by electrophoresis on a 12% SDS-PAGE and then subjected to immunoblot analysis with an antibody against the Histidine 6 tag
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Southern blot analysis of transgenic wheat.
Fusarium head blight resistance in T2 and T3 transgenic wheat
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Comparison of yield parameters between nontransgenic plants and transgenic plants
expressing the antibody fusion.
A: Single floret inoculation and B: Spray inoculation
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FHB-susceptible cv. Bobwhite,
FHB-resistant cv. Sumai3 at 21 days postinoculation with the conidia of Fusarium asiaticum.
A, Spikes of a single floret (indicated by an arrow) inoculated with the conidia of F. asiaticum.
B, Spikes by spray inoculation with the conidia of F. asiaticum.
C, Grains from a spike of a single-floret inoculation in A.
Phenotype of representative spikes and grains from T3 transgenic wheat line 2,
Phytoalexins are antimicrobial and often antioxidative
substances synthesized de novo by plants that accumulate
rapidly at areas of pathogen infection
They are broad spectrum inhibitors and are chemically
diverse with plant species.
Phytoalexins tend to fall into several classes including
terpenoids, glycosteroids and alkaloids
4. Use of phytoalexins to induce immunity
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1997
Stilbene synthase occurs in several plant species and
synthesizes the stilbene phytoalexin transresveratrol
Transfer of two genes from grapevine (Vitis Šinifera)
coding for stilbene synthase genes (vst1 and vst2 ) to
tomato by means of Agrobacterium tumefaciens
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The accumulation of the phytoalexin trans-
resveratrol, the product of stilbene synthase, for
resistance tomato to Phytophthora infestans (Late
blight of tomato).
Accumulation of resveratrol occurred after
inoculation with Botrytis cinerea (Gray mould in
tomato) and Alternaria solani (Early blight in
tomato)
Southern blot analysis of transgenic tomato plants of the T 3 progeny
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Southern blot analysis of transgenic tomato plants of the T3
progeny from regenerant To25 (lane 1±4), To42 (lane 5±8), and
transgenic oilseed rape as a positive control (lane c). Genomic DNA
was isolated from leaves and digested with EcoRI that generates
two fragments of 3.4 kb and 4.9 kb representing the two
transferred stilbene synthase genes.
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Northern blot analysis showing the transient accumulation of stilbene synthase mRNA in leaves
Northern blot analysis showing the transient accumulation
of stilbene synthase mRNA in leaves of a transgenic tomato
plant of the T3 progeny from To25 after inoculation with
P.infestans. No specific mRNA was detectable immediately
after inoculation.*Leaves were treated with tap water only.
Resveratrol (stilbenoid, a type of natural phenol, and a
phytoalexin) accumulation in leaves of a transgenic
tomato plant from the T2 progeny of regenerant To25
after inoculation with P. infestans and B. cinerea.62
Resveratrol contents of leaves of transgenic tomato plants
from T3 progeny of To25 4 days after inoculation with B.
cinerea, A. solani, and P. infestans
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Disease symptoms on leaves of a transgenic tomato plant from the T3 progeny of To25(right) and non-transformed tomato plant (left) 4 days (upper) and 6 days (lower) afterinoculation with P. infestans.
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Biological testing of transgenic tomato plants from progenies T2,
T3, and T4 of regenerant To25 and To42 for an increased
resistance to A. solani, B. cinerea, and P. infestans 4 days after
inoculation65
Development of P. infestans on transgenic tomato plant To25 (T 3 progeny) and non-transformed plant 6 days after
inoculation
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Incidence of P. infestans on transgenic tomato
plants and non-transformed plants in
dependence on the leaf insertion
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Probenazole (PBZ) is the active ingredient
of Oryzemate
Protection of rice plants from Magnaporthe
grisea (blast fungus)
PBZ pre-treatment increased accumulation
of SA and PR proteins in the eighth leaves
of adult plantsTakayoshi Iwai., et al 2008
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5. Use of chemicals to induce immunity
Phenotypes of blast fungus-inoculated leaves of young and adult rice plants. 69
Free SA and SAG levels in rice leaves after fungus inoculation and PBZ treatment. 70
Accumulation of rice PR proteins in M. grisea-infected leaves.
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Induced expression of the OsPR1a gene in M. grisea-infected leaves.
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Induced resistance to blast fungus by SA treatment. 73
6. RNAi-mediated silencing of pathogen’s genes
Parasitism genes expressed in esophageal gland
cells mediate infection and parasitism of plants by
root-knot nematodes (RKN).
Parasitism gene 16D10 encodes a conserved RKN
secretory peptide
Used in vitro and in vivo RNA interference to induce
immunity
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In Vitro RNAi of 16D10.
RNAi silencing of 16D10 in preparasitic M. incognita J2.
Fluorescence microscopy showing ingestion of FITC in the treated J2.
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In Vivo RNAi of 16D10.
Overexpression of 16D10 dsRNA in Arabidopsis. 76
Conclusion
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