“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”

1

Pavan. RDepartment of Genetics and Plant Breeding

University of Agricultural Sciences Bengaluru-65

2

3

4

5

“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 6

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?

7

Principles of plant immunity

8

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

9

PAMPs Triggered Immunity (PTI)

Effector triggered susceptibility (ETS)

Effector Triggered immunity (ETI)

Natural selection

Phases of Plant immunityPhases of Plant immunity

10

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

11

• 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

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

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)

14

Different phases of the zig-zag model

Jonathan & Jeffery, 2006 15

Model for resistance in plants

16

Defence responses post-pathogen recognition

17

Stomatal closure

Ion fluxes

Oxidative burst

Phyto-Hormone action

Induced systemic resistance

Systemic Acquired Resistance

18

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.

19

Bacteria and PAMPs Trigger Stomatal Closure in Arabidopsis20

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 21

flg22: biologically active peptide derived from flagellin

MES: Buffer

LPS: Lipopolysaccharides

22

Col-0: Wild typefls2: Flagellin receptor mutant

23

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

24

Subunit of E3 ubiquitin ligase

involvedJA signalling

25

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.

26

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

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

28

• 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

29

30

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)

31

32

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

33

34

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.

35

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

36

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

37

38

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

39

2010

2. Pyramiding and Introgressing R gene

40

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

41

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,

42

Genetic and physical maps of the genomic region

43

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.

44

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

45

(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.

46

Disadvantage of R genes …….?

Ectopic expression of R genes can

sometimes activate defence pathways in the

absence of pathogen

Reduced crop yields

Reduced Fitness

47

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.

49

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

50

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.

51

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

52

Southern blot analysis of transgenic wheat.

Fusarium head blight resistance in T2 and T3 transgenic wheat

53

54

Comparison of yield parameters between nontransgenic plants and transgenic plants

expressing the antibody fusion.

A: Single floret inoculation and B: Spray inoculation

55

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

56

57

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

58

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

59

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.

60

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.61

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

62

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.

63

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

inoculation64

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

inoculation

65

Incidence of P. infestans on transgenic tomato

plants and non-transformed plants in

dependence on the leaf insertion

66

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

67

5. Use of chemicals to induce immunity

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

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

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

70

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

71

Induced resistance to blast fungus by SA treatment. 72

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

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

In Vivo RNAi of 16D10.

Overexpression of 16D10 dsRNA in Arabidopsis. 75

76