Cell Wall Associated Mechanisms of Disease Resistance & Susceptibility
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Cell Wall Associated Mechanisms of Disease Resistance & Susceptibility
- 1. Cell Wall Associated Mechanisms of Disease Resistance & Susceptibility Master’s Seminar (APP-600) Speaker : Pradeep Negi ID No : 50939
- 2. Contents 1. Introduction 2. Cell Wall In Plant Recognition And Signalling a. Recognition Of The Intruder b. Elicitor-binding Host Proteins c. Signal Transduction For Cell Wall Defences 3. Cell Wall In Plant Defence a. Poisoning The Apoplast b. Plant Cell Wall Strengthening c. Trafficking Of Defence Compounds 4. The Plant’s Contribution To Fungal Accommodation 5. Summary 6. Future Issues 2
- 3. 3 • Plant defence system : cell wall, waxes, hairs, antimicrobial enzymes, and secondary metabolites. Introduction
- 4. • Pathogens penetrate plant apoplast. • They possess a range of enzymatic and/or physical tools. • Nonself recognition operates. • Recognition induces apoplastic defence. • HR + cell death. 4
- 5. 5 Plant Cell Wall - Malinovsky et al. (2014)
- 6. • Pathogens circumvent recognition or suppress host defence. • Special feeding structures or intracellular accommodation. • CW is significant in basal resistance. • How plants defend themselves and how pathogens cope with such defence ? 6
- 7. 7 Arms Race between Pathogen and Host - Malinovsky et al. (2014)
- 8. Recognition of the Intruder 8 Elicitors Exogenous (non-self) PAMPs, HAMPs, NAMPs, ParAMPs, Recognised effectors Endogenous (self) Classical DAMPs, Phytocytokines Defence Response
- 9. • Recognition : Nonself activity in the apoplast. • Cutin derivatives elicit plant defence responses (Riederer et al., 1998). • Cell wall fragments have elicitor function or defence- suppressing effects (Kogel, 1992). 9
- 10. 10 Elicitors found in the apoplastic space - Mott et al. (2014)
- 11. • Nonself Molecules : Bacterial lipopolysaccharides or flagellin, fungal chitin, or cryptogein from Phytophthora etc. • LRR-like kinases detect nonself molecules (Staskawicz, 2006). • E.g. Bacterial flagellin perception in Arabidopsis (Jones et al., 2004). 11
- 12. 12 FLS2, loss of functional gene, fls2-17, leads to enhanced disease susceptibility
- 13. 13 Gain of functional gene FLS2, fls2-1, leads to decrease susceptibility
- 14. Examples : a) AvrPto, AvrPtoB, AvrE, and HopPtoM, effectors of P. syringae, suppress callose deposition (Ammouneh et al., 2006). b) Fusarium trichothecene toxins suppress wall-associated defence in wheat (Maier, 2005). • Fungi avoid chitin recognition by chitin-deacetylation (Deising, 2002). 14
- 15. Elicitor-Binding Host Proteins • Still poorly understood. • WAK are RLK and play role in recognition. • Associated with pectin and glycine-rich cell wall proteins (Kohorn, 2001). • Act as cell wall guardian. • Arabidopsis RFO1 gene, against Fusarium oxysporum, has been cloned, it codes for a WAK (Ausubel, 2005). 15
- 16. Signal Transduction for Cell Wall Defences • It is initiated by cell surface receptors. • ROS induced signalling includes MAP kinases, Ca2+ influx, activation of transcription factors (Miedema et al., 2003). • Transcription factors leads to defence gene expression (Somssich, 2002). • Ca2+ influx influence cell wall rigidity (Kogel, 2004). 16
- 17. 17 Schematic overview of some events occurring in apoplast during plant pathogen interactions - Delaunois et al. (2014)
- 18. • In defence gene expression, phytoalexin production, HR + cell death (Scheel, 1997). • Protein antioxidants control levels of ROS (Foyer, 2002). • Nitric Oxide regulates HR, defense gene expression and penetration resistance (Lamb, 2001). 18 Reactive Oxygen Species are a key player…
- 19. 19 CLSM image of localized Nitric Oxide generation in susceptible barley epidermal cells at appresorial germ tube contact site.
- 20. 20 Suppression of penetration resistance by C-PTIO treatment Penetration resistance resumed by SNP treatment
- 21. Poisoning the Apoplast a) Inhibition of cell wall degrading enzymes. b) Structural remodelling of the cell wall at sites of attempted penetration. c) Killing of potential intruders by antimicrobial means. 21
- 22. A. Inhibitors of Cell Wall Degrading Enzymes • Inhibitors such as the Xylanase Inhibtors and Polygalacturonase Inhibitor Proteins (PGIPs). • PGIPs produces OGAs that are sensed as DAMPs (Federici et al., 2006). • PGIPs : protecting function and communication function as well (Cervone, 2006). 22
- 23. B. Structural Remodelling 23 Actin filament reorganization around a CWA at 20 h after inoculation of barley with Bgh. Non-successful penetration attempt of B. graminis on barley. - Huckelhoven (2007)
- 24. • Agents : Phytoalexins and Phytoanticipins. • Antimicrobial proteins act synergistically. • Phytoalexins : penetration resistance + induced CW mechanical strengthening. E.g. Barley mlo mutants are resistant to penetration by B. graminis, as they produce p-coumaroyl-hydroxyagmatine. (Parr, 1998) 24 C. Killing Potential Intruders By Antimicrobial Means
- 25. • Cell wall–bound phenolics restrict fungal penetration. • Toxic compounds as glucosides. • Aglycons poison the pathogen. • E.g. Sorghum bicolor produces fungitoxic 3- desoxyanthocyanodin flavonoids against Colletotrichum species (Nicholson, 1990). 25
- 26. Plant Cell Wall Strengthening • Cell wall appositions. • Local deposition of chemically modified cell wall material for penetration resistance. • Lignification. • Lignified cell wall is less accessible to CWDE. • Cell wall-bound phenolics accumulation. 26
- 27. 27 Cell wall dynamics in case of necrotrophic fungal attack - Bellincampi et al. (2014)
- 28. 28 Cell wall dynamics in case of biotrophic fungal attack - Bellincampi et al. (2014)
- 29. • H 𝟐O2 in cell wall–associated defence • Peroxidase-dependent lignification + protein cross- linking in the cell wall. • Attack induces it, which reside in CWAs. 29 Continued...
- 30. • H 𝟐O2 presence in CWAs is a biochemical marker. E.g. interaction of barley with B. graminis. • Its removal enhances fungal penetration. • It helps in cross-linking of proline-rich proteins in the cell wall & makes resistant to CWDE. 30
- 31. • Speed of CW material deposition and its compaction are significant. E.g. Wild-type Arabidopsis attacked by B. graminis or Colletrotrichum truncatum builds CWAs more quickly compared to syntaxin mutant pen1 (Zimmerli et al., 2004). • Callose as a defence compound. • Reduced cellulose content by mutation of cellulose synthase CESA3, makes Arabidopsis more resistant to different PM pathogens (Cano-Delgado et al., 2003). 31
- 32. Trafficking of Defence Compounds • Requires dynamic changes in membranes and cytoskeleton. • Depends on local elicitor perception or mechanical pressure. • The cytoskeleton and the endomembrane system are dramatically rearranged. E.g. In barley, cells attacked by B. graminis, the cytoskeleton rapidly reorganizes during fungal attack. 32
- 33. • Golgi-derived vesicles are required for secretion of defence related compounds into the apoplast. • Together, transporter-mediated and Golgi-mediated secretion allocate materials for extracellular defence. 33
- 34. 34 Integration of biochemical and molecular mechanisms for cell wall associated defence - Huckelhoven (2007)
- 35. The Plant’s Contribution to Fungal Accommodation • Virulent pathogens bypass or suppress plant defence. • They suppress host responses to infection and reprogram the host. • Like when haustoria-forming or arbuscule-forming mycorrhizal fungi enter plant cells, symbiosis-related arrays starts to support pathogenesis. • Fungal hypha doesn't simply force the host cell to rebuild, but rather the host recognizes the friendly intruder. 35
- 36. Summary • Basal defense reactions to directly invading fungi often operate at the plant cell wall. • The invading pathogen is first recognized based on recognition of nonself molecules and nonself activities at the cell periphery. • Cell wall–associated defence appears to operate via inhibition of fungal cell wall– degrading enzymes, secretion of fungitoxic peptides and phytoalexins, and cell wall strengthening. 36
- 37. FUTURE ISSUES • Genetic approaches will identify several genes that are key to nonspecific disease resistance established at the cell wall. • Future genetic screenings will provide deeper insight into resistance and susceptibility to cell wall–penetrating fungal pathogens. • Genetic redundancies of defence-associated proteins challenge plant pathologists and will require sophisticated approaches to study gene function in resistance. 37
- 38. 38
Editor's Notes
- The frontline of the plant defence system consists of physical and chemical barriers such as the cell wall, waxes, hairs, antimicrobial enzymes, and secondary metabolites. If these obstacles are overcome, the pathogen is still confronted by elaborate surveillance systems in which molecular sentinels operate to activate resistance responses.
- Most pathogens penetrate plant apoplast to access intracellular nutrients. Bacterial and fungal pathogens possess a range of enzymatic and/or physical tools. In plant-microbe interactions, nonself recognition operates via perception of microbial molecules or by surveillance of host cellular intactness. On recognition, the plant induces apoplastic defence to inhibit microbial enzymes, for cell wall strengthening, or for poisoning the pathogen. The second layer of defence i.e. HR including cell death restricts pathogens in an area.
- Pathogens have evolved more sophisticated virulence strategies to circumvent recognition or to suppress host defence. Sometime they develop special feeding structures in intact host cells or become actively engulfed for intracellular accommodation. Cell wall-associated plant defence is important in basal resistance. Recent evidences has shown how plants defend themselves at their periphery and how microorganisms cope with such defence.
- During host invasion, pathogens release nonspecific exogenous and endogenous elicitors, such as oligomeric or monomeric fragments of both the cuticle and the cell wall.
- Recognition occurs because the elicitors indicate nonself activity in the apoplast. Cutin derivatives are able to elicit plant defence responses and prime plants for further responsiveness to pathogen-derived elicitors (Riederer et al., 1998). Cell wall fragments derived from enzymatic activities have elicitor function or alternative defence-suppressing effects (Kogel, 1992).
- Csp22 Bacterial cold shock protein flg22 Bacterial flagellin elf18 Bacterial Elongation Factor Tu PGN peptidoglycan, Bacterial cell wall (Gram positive) LPS Lipopolysaccharide, Bacterial cell wall (Gram negative) EIX Xylanase Fungal xylanase Pep13 Oomycete transglutaminase GE Beta Glucan Oomycete cell wall CBD2synt Oomycete cellulose-binding elicitor lectin (CBEL) AtPeps Cytosolic plant protein (HypSys) Hydroxyproline-rich Systemin glycopeptides Cytosolic plant protein Oligogalacturonides (OGs) Plant cell wall
- Plants recognize nonself activity and nonself molecules such as bacterial lipopolysaccharides or flagellin, fungal chitin, or elicitins from Phytophthora species such as cryptogein in their apoplast. Receptor-like kinases contribute to detection of conserved nonself molecules (Staskawicz, 2006). A paradigm of general nonself recognition in plants is perception of bacterial flagellin in Arabidopsis (Jones et al., 2004).
- Figure: Bacterial disease resistance is determined by flagellin perception. a, A FLS2 loss-of-function mutation, fls2-17, leads to enhanced disease susceptibility. Left: wildtype and fls2-17 mutant plants were sprayed with Pst DC3000 bacteria or with water and photographed 4 days later. Right: symptoms after 4 days in a series of leaves of decreasing age.
- B: Number of Pst DC3000 bacteria extracted from wild-type (open bars) and fls2-17 mutant plants (filled bars) 4 days after infection. Leaves were grouped by age as depicted on the left. C: A gain-of-function transgene of FLS2 leads to decreased susceptibility in the accession Wassilewskaya (Ws-0), which lacks a functional FLS2 gene. Ws-0 was stably transformed with FLS2p::FLS2-3xmyc or SIRKp::GUS as a control. Plants were sprayed with 5 x 10^8 c.f.u. ml Pst DC3000, or water.
- AvrPto, AvrPtoB, AvrE, and HopPtoM, effectors of Pseudomonas syringae, are able to suppress cell wall–associated callose (β-1,3-glucan) deposition (Ammouneh et al., 2006). Bacterial speck Pseudomonas syringae pv. tomato Fusarium trichothecene toxins contribute to suppression of wall-associated defense in wheat (Maier, 2005). Plants seem to have evolved recognition tools for nonself patterns, which are indispensable for microbial protein function. Nonspecific elicitors often derive from the fungal cell wall (Lecourieux et al., 2006). It is commonly believed that plant defence–related chitinases boost nonspecific defence by releasing elicitor-active chitin oligomers. In contrast, pathogenic fungi have evolved mechanisms to avoid chitin recognition by chitin-deacetylation (Deising, 2002). chitin deacetylase is an enzyme that catalyzes the chemical reaction chitin + H2O > chitosan + acetate Thus, the two substrates of this enzyme are chitin and H2O, whereas its two products are chitosan and acetate. This enzyme belongs to the family of hydrolases, those acting on carbon-nitrogen bonds other than peptide bonds, specifically in linear amides.
- Still poorly understood. Their association with pectin and glycine-rich cell wall proteins makes them attractive candidates for cell wall guardians that communicate with the cytoplasm (Kohorn, 2001). Recently, the Arabidopsis RFO1 gene, conferring quantitative resistance against Fusarium oxysporum, has been cloned, and it codes for a wall-associated kinase (Ausubel, 2005). WAK, wall-associated kinases are a family of protein kinases that connect the innermost portion of the cell (the cytoplasm) to the cell wall. It has been hypothesized that these kinases are important in communication from the inside of the cell (including most importantly the nucleus) to the surrounding environment including other cells. The WAKs are known to be receptor-like in nature, pointing to a possible role in cell communication.
- Signal transduction (also known as cell signaling) is the transmission of molecular signals from a cell's exterior to its interior. Signals received by cells must be transmitted effectively into the cell to ensure an appropriate response. This step is initiated by cell-surface receptors. Many transcription factors are involved in nonspecific fungal and oomycete elicitor signal transduction leading to defence gene expression (Somssich, 2002). Transcription factors are proteins involved in the process of converting, or transcribing, DNA into RNA. The function is to regulate - turn on and off - genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. GTFs are TFIIA, TFIIB, TFIID Ca2+ influx into the cytoplasm might influence cell wall rigidity due to the role of Ca2+ in noncovalent cell wall cross-linking (Kogel, 2004). Calcium ions are important for cellular signalling, as once they enter the cytosol of the cytoplasm they exert allosteric regulatory effects on many enzymes and proteins. ROS-induced signalling includes MAP kinases, Ca2+ influx, activation of transcription factors and salicylic acid accumulation (Miedema et al., 2003). A type of unstable molecule that contains oxygen and that easily reacts with other molecules in a cell. A build up of reactive oxygen species in cells may cause damage to DNA, RNA, and proteins, and may cause cell death. Reactive oxygen species are free radicals. Also called oxygen radical.. Examples include peroxides, superoxide, hydroxyl radical, and singlet oxygen. important roles in cell signalling. during times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically.[2] This may result in significant damage to cell structures. A mitogen-activated protein kinase (MAPK or MAP kinase) is a type of protein kinase that is specific to the amino acids serine and threonine (i.e., a serine/threonine-specific protein kinase). MAPKs are involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock and proinflammatory cytokines.
- In defence gene expression, phytoalexin production, and both HR and restriction of pathogen-induced cell death (Scheel, 1997).. NO regulates HR, defense gene expression, penetration resistance, and other resistance responses (Lamb, 2001). (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune . Innate immunity refers to nonspecific defense mechanisms that come into play immediately or within hours of an antigen's appearance in the body. These mechanisms include physical barriers such as skin, chemicals in the blood, and immunesystem cells that attack foreign cells in the body. The apoplast contains several lowmolecular- weight and protein antioxidants, which control levels of ROS (Foyer, 2002).
- C-PTIO > 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide SNP > Sodium nitroprusside
- There are three ways by which plants protect their cell walls from pathogenic penetration: inhibition of cell wall–degrading enzymes structural remodeling of the cell wall at sites of attempted penetration killing of potential intruders by antimicrobial means
- Plants counter the savage enzyme cocktails by producing proteinaceous inhibitors of cell wall degrading enzymes such as the xylanase inhibitors and PG inhibitor proteins (PGIPs) PGIPs are not to inhibit pectin degradation entirely, but to shift it toward producing longer OGAs (Oligo Galacturonic acid) that can be sensed as DAMPs (Federici et al., 2006). Polygalacturonase inhibiting proteins combine a cell wall–protecting function with a function in communication with the symplast by forcing fungal enzymes to release signals (Cervone, 2006).
- For Fig 1: Actin filament (green phalloidin-Alexa-488 labeling) reorganization around a CWA (autofluorescence displayed in red) at 20 h after inoculation of barley with Bgh. For Fig 2: Formation of the central CWA at 24 h after inoculation is accompanied by neighboring vesicle-like bodies. Structures are brownish stained by 3,3-diamnobenzidine for H2O2
- Plant antimicrobial agents are low molecular-weight compounds such as induced phytoalexins and constitutive phytoanticipins, respectively, as well as antimicrobial proteins. Antimicrobial proteins act synergistically to hinder fungal growth in the apoplast but are insufficient to achieve complete resistance. Phytoalexins are crucial for penetration resistance together with induced mechanical strengthening of the cell wall. E.g. barley mlo mutants, which are resistant to penetration by B. graminis, overproduce p-coumaroyl-hydroxyagmatine which has antifungal activity and inhibits haustorium formation in vivo. Cell wall– bound phenolics also accumulate locally to restrict fungal penetration. Ex chlorogenic acids, ferulic acids
- Accumulation of soluble and wall-bound indolic metabolites such as indole-3-carboxylic acid or camalexin seems important in interactions of Arabidopsis and other plant species with pathogens. Toxic compounds are often stored in plants as harmless glucosides. Hydrolytic enzymes cleave the glucosides, and the emerging aglycons poison the pathogen. A glucoside is a glycoside that is derived from glucose. Glucosides are common in plants,. aglycon, is the compound remaining after the glycosyl group on a glycoside is replaced by a hydrogen atom Sorghum bicolor produces fungitoxic 3-desoxyanthocyanodin flavonoids in a site specific manner when attacked by Colletotrichum species (Nicholson, 1990). Flavonoids are a group of plant metabolites thought to provide benefits through cell signalling pathways and antioxidant effects
- Definitive evidence for the importance of structural and chemical strengthening of the cell wall in penetration resistance is rare. Cell wall appositions are built in both compatible and incompatible interactions of plants and fungi. Cell wall apposition r cell wall material deposited at sites of microbial attack that differs in chemical and mechanical features from the normal cell wall Local deposition of chemically modified cell wall material is crucial for penetration resistance. Lignification makes the cell wall more resistant to mechanical pressure applied during penetration by fungal appressoria. Lignified cell wall is water resistant and thus less accessible to cell wall–degrading enzymes. Soluble and cell wall-bound phenolics accumulate in plant tissue challenged by fungal pathogens.
- NO and H2O2 might thus be potent messengers in cell wall–associated defence. ROS are key molecules in signaling and the biochemistry of nonspecific plant defence. H2O2 is required for peroxidase-dependent lignification and for protein cross-linking in the cell wall. Attack by fungal pathogens induces peroxidases, which reside in CWAs. The presence of H2O2 in CWAs is a biochemical marker that distinguishes non penetrated from penetrated cells in the interaction of barley with B. graminis. The importance of H2O2 for resistance to basal penetration is supported by the observation that enzymatic removal of H2O2 enhances the success of fungal penetration on leaf epidermal cells. Likewise, H2O2 contributes to cross-linking of proline-rich proteins in the cell wall, which makes it more resistant to cell wall–degrading enzymes.
- NO and H2O2 might thus be potent messengers in cell wall–associated defence. ROS are key molecules in signaling and the biochemistry of nonspecific plant defence. H2O2 is required for peroxidase-dependent lignification and for protein cross-linking in the cell wall. Attack by fungal pathogens induces peroxidases, which reside in CWAs. The presence of H2O2 in CWAs is a biochemical marker that distinguishes non penetrated from penetrated cells in the interaction of barley with B. graminis. The importance of H2O2 for resistance to basal penetration is supported by the observation that enzymatic removal of H2O2 enhances the success of fungal penetration on leaf epidermal cells. Likewise, H2O2 contributes to cross-linking of proline-rich proteins in the cell wall, which makes it more resistant to cell wall–degrading enzymes.
- In addition to the chemical nature of altered cell wall material that is deposited against an invading pathogen, the speed of deposition and of compaction might be crucial for successful defence. Wild-type Arabidopsis attacked by B. graminis or Colletrotrichum truncatum builds CWAs more quickly when compared to the syntaxin mutant pen1, which is defective in nonhost penetration resistance to B. graminis. Callose may still be a defence compound though it is not sufficient for penetration resistance. Callose is a plant polysaccharide. It is composed of glucose residues linked together through β-1,3-linkages, and is termed a β-glucan. It is thought to be manufactured at the cell wall by callose synthases and is degraded by β-1,3-glucanases. A reduced cellulose content by mutation of cellulose synthase CESA3, involved in primary cell wall formation, leads to production of lignin, and makes Arabidopsis more resistant to different powdery mildew pathogens (Cano-Delgado et al., 2003).
- Transport and secretion of defence compounds require dynamic changes in membranes and the cytoskeleton. Cytoskeleton is A microscopic network of protein filaments and tubules in the cytoplasm of many living cells, giving them shape and coherence. Cytoplasmic reorganization depends on filamentous actin and on microtubules. The direction of polarization and the amount of cell wall–associated defences may thereby depend on local elicitor perception or mechanical pressure. The cytoskeleton and the endomembrane system are dramatically rearranged and polarized when plants are attacked by fungal pathogens. An intact actin cytoskeleton is also required for penetration resistance of barley, Arabidopsis, and tobacco to leaf pathogens.
- Fungal attack also influences the endomembrane system. Golgi-derived vesicles are likely required for secretion of defence related compounds into the apoplast. Together, transporter-mediated secretion, Golgi-mediated secretion, and endosome-mediated secretion may collaborate to allocate materials for extracellular defence. In barley cells attacked by B. graminis, the cytoskeleton rapidly reorganizes from nonpolar to focused polarized patterns during fungal attack. This is particularly evident in nonhost interactions and less obvious in compatible interaction of barley and B. graminis. Transport mediated by a membrane transport protein. There are three types of mediated transport: uniport, symport, and antiport.
- Monolignols are phytochemicals acting as source materials for biosynthesis of both lignans and lignin. lignans are a large group of polyphenols found in plants. Some examples of lignans are enterolignans, enterodiol and enterolactone.
- Nonself recognition and effective defense mechanisms are common in plants interacting with most potential pathogens. Therefore, successful pathogens need to bypass or to suppress plant defense in order to be virulent. It is well established that microbial virulence factors are active in suppressing host responses to infection and in reprogramming the host When haustoria-forming or arbuscule-forming mycorrhizal fungi enter plant cells, the actin cytoskeleton and the endomembrane system reorganize and build pathogenesis-related or symbiosis-related arrays to support pathogenesis. Hence, an invading fungal hypha does not simply force the host cell to rebuild, but rather the host recognizes the friendly intruder.