Pathogen associated molecular patterns

Ashwin jayaleId no PALB 1222 1

Contents

• Introduction • Basal plant defenses • Signal transduction• Mechanism of PAMPs• Case study• Conclusion

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Introduction

• Pathogen-associated molecular patterns, or PAMPs, are

molecules associated with groups of pathogens, that are recognized

by cells of the innate immune system.

• The term "PAMP" has been criticized on the grounds that most

microbes, not only pathogens, express the molecules detected; the

term microbe-associated molecular patternor MAMP, has

therefore been proposed.

• A virulence signal capable of binding to a pathogen receptor, in

combination with a MAMP, has been proposed as one way to

constitute a (pathogen-specific) PAMP.3

Cont..

• immunology frequently treats the terms "PAMP" and "MAMP"

interchangeably, considering their recognition to be the first step in plant

immunity, PTI (PAMP-triggered immunity), a relatively weak immune

response that occurs when the host plant does not also recognize

pathogenic effectors which damage it or modulate its immune response.

• These molecules can be referred to as small molecular motifs conserved

within a class of microbes. They are recognized by Toll-like receptors

 (TLRs) and other pattern recognition receptors(PRRs) in both plants and

animals.

• Bacterial Lipopolysaccharide (LPS), an endotoxin found on the bacterial

cell membrane of a bacterium, is considered to be the prototypical

PAMP. 4

Plant basal defenses

1. Pre-existing

2. Induced

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Pre-existing defenses: the first barrier

• Physical barriers involve properties of the plant surface, that is, the cuticle, stomata and cell walls.

• Chemical barriers include compounds, such as phytoanticipins that have antimicrobial activity, and defensins, which interfere with pathogen nutrition and retard their development.

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How do pathogens enter the apoplast?

Illustrated glossary of plant pathology www.apsnet.org/

penetration peg

Fungi Bacteria

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Abramovitch et al. Nature Reviews Molecular Cell Biology 7, 601–611 (August 2006) | doi:10.1038/nrm1984

Strategies used by bacterial pathogens

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Pathogen-induced responses:It’s a race!!!!

“As soon as a plant has recognized an attacking pathogen, the race is on. The plant attempts to prevent infection and to minimize potential damage, the pathogen attempts to gain access to nutrients for growth and reproduction.”

Schmelzer, 2002

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The plant basal defense system

• A relatively recent concept

• Plants can recognize certain broadly-conserved molecules

associated with pathogens- PAMPs (pathogen-associated

molecular patterns)

• Also known as MAMPs (microbe-associated…)

• Examples include flagellin, elongation factor-Tu,etc.

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Espinosa, Avelina & AlfanoDisabling surveillance: bacterial type III secretion system effectors that suppress innate immunity.Cellular Microbiology 6 (11), 1027-1040.

PAMPS(Pathogen-Associated Molecular Patterns)

oligosaccharides, lipids, polypeptides (flagellin), glycoproteins, etc…

Signal transduction

events

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Signal transduction cascade

Antimicrobialcompounds

Hormones(Salicylic acid, jasmonic acid,

ethylene)

Induced basal defenses (Innate immunity)

New proteins

Bacterium

DNA

RNA

nucleus

ER,translation

Nucleus

Secretory pathway

Golgi

Antimicrobial compounds,Defense proteins

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Output of Induced basal defenses Recognition events (elicitors, receptors)

• Signal transduction cascades• MAP kinases, phosphorylation cascades

• Chemical changes:• Synthesis of NO, ROSs, signaling molecules (SA, JA, Ethylene), etc…

• Gene expression changes (transcriptional regulation)

• Synthesis of antimicrobial compounds and proteins (phytoalexins, PR proteins)

• Cytoskeletal rearrangements, vesicle trafficking, secretion

• Morphological changes (organelle redistribution, cell wall modifications) 13

Espinosa & AlfanoCellular Microbiology 6 (11), 1027-1040.

Plant pathogenic bacteria secrete proteins called

“virulence effectors” directly into the host cell

Bacteria use a sophisticated “injection” apparatus, called a Type III Secretion

System, to deliver virulence effector proteins directly in the cytoplasm of the host cell.

Bacterial type III effectors disable host surveillance by suppressing innate immunity.

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Signal transduction cascade

Bacterium

Antimicrobialcompounds

Hormones(Salicylic acid, jasmonic acid,

ethylene)

New proteins

DNA

RNA

nucleus

ER,translation

Nucleus

Secretory pathway

Golgi

Antimicrobial compounds,Defense proteins

Bacterial virulence effectors suppress host innate immunity

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Some of the available tools for dissecting plant-pathogen interactions:

- Pathogenesis Assays (assessing symptom development and pathogen multiplication in the host)

- Microarrays (analysis of global gene expression in the host plant)

- Genetic transformation (expression of any given plant or pathogen gene in the host plant)

- Gene knock-out(both in plant and pathogen)

- Fluorescent protein tagging and microscopy (allows visualization of protein localization and cellular dynamics)

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PAMP PERCEPTION AS A KEY COMPONENT OF DISEASE RESISTANCE

• With the identified PRRs(encoded PAMP receptors, or pattern-recognition

receptors (PRRs) it finally became feasible to address the importance of

PAMP perception in plants. Treatment with PAMPs induces local and

systemic resistances to several unrelated virulent pathogens. For example,

flg22 treatment induced resistance to the bacterium Pseudomonas

syringae pv tomato DC3000, as well as to the fungus Botrytis

cinerea (Zipfel, 2009).

• The best manifestation that recognition of PAMPs is key to plant immunity

is the fact that pathogens must suppress this level of resistance to cause

disease.

• As a result, pathogenic microbes evolved mechanisms to avoid recognition

or to suppress defense responses through secreted virulence effectors.

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PAMP Minimal motif Origin Sensitive plants

Flagellin Flg22 Gram-negative bacteria

Tomato,Arabidopsis thaliana

Elongation factor (EF-Tu)

Elf18 Gram-negative bacteria

Arabidopsis thaliana, otherBrassicaceae

Transglutaminase Pep-13 motif Oomycetes (Phytophthoraspp.)

Parsley, potato, grapevine, tobacco, N. benthamiana

Xylanase TKLGE pentapeptide

Fungi (Trichoderma spp.)

Tobacco, tomato

Cold shock protein RNP-1 motif Gram-negative bacteria, gram-positive bacteria

Solanaceae

Cellulose-binding elicitor lectin (CBEL)

Conserved cellulose binding domain

Oomycetes (Phytophthoraspp.)

Tobacco,Arabidopsis thaliana

Table 1. Pathogen-associated molecular patterns recognized by plants.

So what’s the difference between MAMPs and AVR/Effector genes and between MAMP receptors and R-genes?

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• MAMPs are molecules that are highly conserved and found in a wide

range of microbes, pathogens and non-pathogens alike. They do not

necessarily play a direct role in pathogenesis.

• Avr/Effector genes are generally specific to a few species of plant

pathogens and play a role in pathogenesis. Often are exported into host

cells or into the apoplast.

• Avr/Effector genes often supress MAMP-induced defences (alternatively

they may supress other types of defence response.

• MAMP-receptors recognise MAMPs, often (always?) by direct interaction.

They are (usually) conserved within a species.

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From Brent and Mackey , Ann Rev Phtyopath, 2007 45:399-436

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Molecular components of PAMP-triggered immunity and their interactions. PAMP, DAMP, PRR, regulatory RK, cytoplasmic kinase (K), phosphatase (PP), CDPKs, MAPKs, 1-aminocyclopropane-1-carboxylate synthase (ACS), WRKY transcription factors (WRKY), reactive oxygen species (ROS), ethylene (ET), and SA; black arrows indicate downstream interactions, dashed arrows possible amplifications.

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EMERGING CONCEPTS IN PAMP-TRIGGERED IMMUNITY

• PAMPs trigger early responses (seconds to minutes; e.g. ion fluxes, oxidative

burst), intermediate responses (minutes to hours; e.g. MAPK/CDPK activation,

ethylene production, stomatal closure, transcriptional reprogramming), and late

responses (hours to days; e.g. salicylic acid [SA] accumulation, callose deposition).

• Intriguingly, many of these responses include the production of molecules that

potentially can act as second messengers (calcium, reactive oxygen species,

ethylene, SA) and we may predict roles for each of the signaling pathways in

PAMP-triggered immunity .

• Like in animals, host endogenous molecules released upon wounding and infection,

called damage-associated molecular patterns (DAMPs), are capable of inducing

immune reactions in plants .

• Only recently, the first plant DAMP receptor has been identified. The LRR-RKs

PEPR1 and PEPR2 are responsible for the detection of the peptidic DAMP AtPep1

(Krol et al., 2010; Yamaguchi et al., 2010).

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CHALLENGES AND FUTURE DIRECTIONS

• Not every microbe displays all PAMPs and not every plant recognizes all PAMPs.

For example, flg22 is detected by most plant species, but some pathogens evade

recognition through mutation of key residues

• In addition, EF-Tu is only sensed by Brassicaceae, and recognition of Ax21 is

restricted to specific rice cultivars. However, EF-Tu perception can be transferred

across plant families and importantly confers resistance to bacteria belonging to

several classes, indicating that all necessary components downstream of EFR are

conserved (Lacombe et al., 2010).

• These examples suggest that there is a dynamic evolution in the display of PAMPs

by microbes and in the recognition of PAMPs by plants (Boller and Felix, 2009),

• Novel PRRs will provide useful tools for engineering sustainable quantitative

broad-spectrum disease resistance in the field.25

Case studyCellulose Binding Domains of a PhytophthoraCell Wall Protein Are Novel

Pathogen-Associated Molecular PatternsElodie Gaulina,1, Nani Draméa, Claude Lafittea, Trudy Torto-Alalibob, Yves

Martinezc, Carine Ameline-Torregrosaa, Moustafa Khatiba, Honoré Mazarguild, François Villalba-Mateosa,2, Sophien Kamounb, Christian Mazarsa, Bernard Dumasa, Arnaud Bottina, Marie-Thérèse Esquerré-Tugayéa and Martina Rickauera

The Plant Cell 18:1766-1777 (2006)© 2006 American Society of Plant Biologists

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Introduction

• CBEL production in planta induced necrosis. Site-directed mutagenesis

on aromatic amino acid residues located within the CBDs as well as leaf

infiltration assays using mutated and truncated recombinant proteins

confirmed the importance of intact CBDs to induce defense responses.

• Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic

peptides showed that the CBDs of CBEL are essential and sufficient to

stimulate defense responses. Moreover, CBEL elicits a transient variation

of cytosolic calcium levels in tobacco cells but not in protoplasts.

• These results define CBDs as a novel class of molecular patterns in

oomycetes that are targeted by the innate immune system of plants and

might act through interaction with the cell wall.27

Material and methods• Plant Material

Tobacco (Nicotiana tabacum) plants were grown on vermiculite in a growth chamber at 75% hygrometry, with a photoperiod of 12 h of light at 25°C and 12 h of dark at 22°C. Plant leaves were infiltrated 7 weeks after seed germination.

• BY-2 Cell Suspension Culture and Protoplast IsolationAequorin-transformed BY-2 tobacco cells were grown as described by Pauly et al. (2001).

• Construction of Recombinant Agrobacterium tumefaciens Binary PVX Vectors

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RESULTS

• RESULTS• Elicitor Activity of CBEL Produced in E. coli or in Planta

To study the relationship between the structure of CBEL and its elicitor activity, we set up its expression in two heterologous systems.

• the various protein elicitors isolated from oomycetes, CBEL has the unique property to bind to cellulose and to plant cell walls, consistent with the modular structure of the protein comprising two CBDs.

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Conclusion

• Perception systems on the host cell surface form the first line of defense in animals and plants.

• Innate immunity in both systems is based on the perception of similar pathogen-associatedmolecular patterns by pattern recognition receptors.

• Inactivation of these surface receptors leads to loss of immunity.

• This supports the significance of PAMP-triggered defenses in plant immunity in general.

• Pattern recognition receptor complex formation is often initiated upon ligand binding and was shown to be indispensable for proper receptor function. 30

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