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Pathogen associated molecularpatterns Ashwin jayale 1 Id no PALB 1222
Contents• Introduction• Basal plant defenses• Signal transduction• Mechanism of PAMPs• Case study• Conclusion 2
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 3 constitute a (pathogen-specific) PAMP.
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 4 PAMP.
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. 6
How do pathogens enter the apoplast? Fungi Bacteriapenetration peg 7 Illustrated glossary of plant pathology www.apsnet.org/
Strategies used by bacterial pathogens 8Abramovitch et al. Nature Reviews Molecular Cell Biology 7, 601–611 (August 2006) | doi:10.1038/nrm1984
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 9
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. 10
Signaltransduction events PAMPS (Pathogen-Associated Molecular Patterns) oligosaccharides, lipids, polypeptides (flagellin), glycoproteins, etc… 11 Espinosa, Avelina & Alfano Disabling surveillance: bacterial type III secretion system effectors that suppress innate immunity. Cellular Microbiology 6 (11), 1027-1040.
Induced basal defenses (Innate immunity)Antimicrobial compounds, Defense proteins Bacterium Signal transduction cascade Secretory pathway Hormones (Salicylic acid, Golgi jasmonic acid, Nucleus ethylene) RN DN A A ER nsla tra , 12 tio n Antimicrobial New proteins nu compounds cle us
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
Plant pathogenic bacteria secrete proteins called “virulence effectors” directly into the host cellBacteria use a sophisticated “injection” apparatus, called a Type III SecretionSystem, to deliver virulence effector proteins directly in the cytoplasm of the host cell.Bacterial type III effectors disable host surveillance by suppressing innate immunity. 14 Espinosa & Alfano Cellular Microbiology 6 (11), 1027-1040.
Bacterial virulence effectors suppress host innate immunity BacteriumAntimicrobial compounds, Defense proteins Signal transduction cascade Secretory pathway Hormones (Salicylic acid, Golgi jasmonic acid, Nucleus ethylene) RN DN A A ER nsla tra , tio 16 n Antimicrobial New proteins nu cle compounds us
Some of the available tools for dissectingplant-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) 17
PAMP PERCEPTION AS A KEY COMPONENT OF DISEASERESISTANCE• 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 18 or to suppress defense responses through secreted virulence effectors.
So what’s the difference betweenMAMPs and AVR/Effector genes andbetween MAMP receptors and R-genes? 20
• 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. 21
From Brent and Mackey , Ann 22Rev Phtyopath, 2007 45:399-436
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 24 PEPR1 and PEPR2 are responsible for the detection of the peptidic DAMP AtPep1 (Krol et al., 2010; Yamaguchi et al., 2010).
CHALLENGES AND FUTUREDIRECTIONS• 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 25 broad-spectrum disease resistance in the field.
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 27 might act through interaction with the cell wall.
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 Isolation Aequorin-transformed BY-2 tobacco cells were grown as described by Pauly et al. (2001).• Construction of Recombinant Agrobacterium tumefaciens Binary PVX Vectors 28
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. 29
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 30 indispensable for proper receptor function.