Plant immunity towards pathogen

1. “Plant immunity: towards anPlant immunity: towards an
integrated view of plant pathogenintegrated view of plant pathogen
interaction and its implication ininteraction and its implication in
plant breedingplant breeding”
1
Pavan. R
Department of Genetics and Plant Breeding
University of Agricultural Sciences
Bengaluru-65

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6. “For each resistance gene in the host there is a
corresponding gene for avirulence in the
pathogen cnferring resistane and viceversa”
Host plant genotypePathogen
genotype R1 r2 r1 R2
Avr1, avr2 I
I
C
Cavr1, Avr2
I - incompatible - no disease
C - compatible - disease
Gene for gene hypothesis
H.H. Flor 6

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 – Stylet
Fungi – Haustoria
What is plant immunity?
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8. Principles of plant immunity
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9. 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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10. PAMPs Triggered Immunity (PTI)
Effector triggered susceptibility (ETS)
Effector Triggered immunity (ETI)
Natural selection
Phases of Plant immunityPhases of Plant immunity
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11. 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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12. • 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)
12

13. 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 13

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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15. Different phases of the zig-zag model
Jonathan & Jeffery, 2006
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16. Model for resistance in plants
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17. Defence responses post-pathogen
recognition
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18.  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

19. 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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20. Bacteria and PAMPs Trigger Stomatal Closure in Arabidopsis
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Maeli etal, 2006
(1) Stomata actively
closes as an initial
response to
both 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

21. Involvement of the FLS2 Receptor and Salicylic
Acid in PAMP Induced Stomatal Closure 21
flg22:
biologically
active peptide
derived from
flagellin
MES: Buffer
LPS:
Lipopolysaccha
rides

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Col-0: Wild type
fls2: Flagellin receptor mutant

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Salicylic Acid in PAMP Induced Stomatal Closure
eds 16-2: SA-biosynthetic mutan plant
nahG : SA-deficient transgenic plants

24. Model Depicting Bacterium- and PAMP-Induced Stomatal
Closure in the Arabidopsis Guard Cell
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Subunit of E3
ubiquitin ligase
involved
JA 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

27. 27
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

28. 4. Phyto-hormones
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29. • 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

31. 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

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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

37. 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

40. 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.

42. Late blight, caused by the oomycete pathogen Phytophthora infestans,
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43. 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 a
score of 8.

45. 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.

46. Structure of the RB gene and the deduced
RB protein.
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47. 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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48. 3. Antifungal fusion proteins to induce immunity
48
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.

50. 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.

53. 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,

56.  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
56

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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)

59. 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.

61. 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.
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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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63. 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) after
inoculation with P.
infestans.
63

64. 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
inoculation
64

65. Development of P. infestans on transgenic tomato plant
To25 (T 3 progeny) and non-transformed plant 6 days after
inoculation
65

66. Incidence of P. infestans on transgenic tomato
plants and non-transformed plants in
dependence on the leaf insertion
66

67. 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 plants
Takayoshi Iwai., et al 2008
67
5. Use of chemicals to induce immunity

68. Phenotypes of blast fungus-inoculated leaves of young and adult rice
plants. 68

69. Free SA and SAG levels in rice leaves after fungus inoculation and PBZ
treatment. 69

70. Accumulation of rice PR proteins in M. grisea-infected
leaves. 70

71. Induced
expression
of the
OsPR1a
gene in M.
grisea-
infected
leaves.
71

72. Induced resistance to blast fungus by SA treatment. 72

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
73

74. In Vitro RNAi of 16D10.
RNAi silencing of 16D10 in preparasitic M. incognita J2.
Fluorescence
microscopy
showing ingestion
of FITC in the
treated J2.
74

75. In Vivo RNAi of 16D10.
Overexpression of 16D10 dsRNA in Arabidopsis. 75

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