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Section 1Plant Immune Response Pathways

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1 Innate Immunity: Pattern Recognition in PlantsDelphine Chinchilla and Thomas Boller

1.1 Pattern Recognition through MAMPs (Microbe-AssociatedMolecular Patterns)

Classic work attempted to define and characterize the so-called “elicitors,”pathogen-derived molecules that would elicit a defense response in plants(Darvill and Albersheim, 1984; Boller, 1995). In the case of oomycetes andfungi, these “elicitors” turned out to be characteristic microbial structuresderived from their cell walls, such as the heptaglucan epitope of Phytophthoramegasperma (see Darvill and Albersheim, 1984) and chitin fragments (Felixet al., 1993), or from microbial membranes, such as arachidonic acid (Preisigand Kuc, 1985) and ergosterol (Granado et al., 1995). Thus, plants appearedto perceive microbes through common patterns that were not specificallyassociated with pathogens (Boller, 1995). However, although these elicitorswere able to induce a vigorous defense response, their importance for actualplant–pathogen interactions remained elusive.

The appreciation of these “general elicitors” changed when a similar prin-ciple was described in the field of (human) immunology: In the evolutionarilyancient “innate immunity,” a group of receptors named “pattern recognitionreceptors (PRRs)” was found to recognize conserved molecular patterns of mi-crobes that are essential for their survival, the so-called “pathogen-associatedmolecular patterns (PAMPs)” (Medzhitov and Janeway, 2000, Janeway andMedzhitov, 2002). Interestingly, both plants (Gomez-Gomez and Boller, 2000)and animals (Hayashi et al., 2001) were found to possess specific PRRs forbacterial flagellin, namely flagellin sensing 2 (FLS2) and Toll-like receptor 5(TLR5). This highlighted the similarities of plant and animal innate immunity(Asai et al., 2002), particularly because it appeared that the two PRRs hadarisen by convergent evolution rather than from a primeval eukaryotic PRR(see Ausubel, 2005; Boller and Felix, 2009). As well as illustrated by thePRRs for flagellin, it is apparent that the molecular patterns recognized arecharacteristic of whole classes of microbes, independent of whether they arepathogenic or not, and therefore should more precisely be called “microbe-associated molecular patterns (MAMPs),” a term we will use throughout this

Effectors in Plant–Microbe Interactions, First Edition. Edited by Francis Martin and Sophien Kamoun.C© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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4 PLANT IMMUNE RESPONSE PATHWAYS

chapter (see also Mackey and McFall, 2006; Boller and Felix, 2009; Bollerand He, 2009). In fact, well-adapted pathogens might alter and camouflage themolecular patterns that lead to recognition by the PRRs, as illustrated by thechanges in the flagellin genes of some plant pathogenic bacteria, such that truepathogens may no longer present the MAMP in question (Felix et al., 1999;Pfund et al., 2004; Sun et al., 2006).

The variety of MAMPs is large, as summarized in Table 1.1. Typically, fora given class of microbes, a given plant species can perceive several differentMAMPs. This redundancy guarantees a robust recognition of the microbe.

While MAMPs are generally characteristic of a whole class of microor-ganisms, many of these MAMPs are not perceived in a general way by mostplants, but only by a few of them, e.g., by most members of an order orfamily (reviewed in Boller and Felix, 2009). For example, recognition of theactive epitope of elongation factor-Tu (EF-Tu), called elf18, is restricted to theBrassicaceae family (Kunze et al., 2004, Zipfel et al., 2006) and bacterial coldshock protein (CSP) is active only in Solanaceae (Felix and Boller, 2003).From an evolutionary point of view, it is probable that perception of EF-Tuand CSP are more recent systems than the perception of flagellin, which iscommon to numerous plant species (Albert et al., 2010a). There may be anadvantage for a given host plant, in terms of coevolution, to recognize a MAMPthat most other plants do not.

1.2 Some Classical MAMP-Receptor Pairs

1.2.1 MAMP Receptors: PRRs

In animals, PRRs can be separated into surface-located receptors, called Toll-like receptors (TLRs) and intracellular receptors of the NOD-like family(Takeuchi and Akira, 2010). In plants, the PRRs identified so far are all locatedat the plasma membrane (Zipfel, 2008). There is currently no example of in-tracellular recognition of a MAMP in terms of the above definition, althoughplants possess specialized intracellular receptors of the NOD-like family toperceive effectors (see Chapter 2).

Most plant PRRs described so far belong to the class of receptor-like kinase(RLKs) (Shiu and Bleecker 2001; Shiu et al., 2004): these proteins containan ectodomain probably acting as the binding site for the respective ligand,followed by a single pass transmembrane domain and a cytoplasmic proteinkinase domain, which is likely to function in intracellular signal transduc-tion. Many RLKs are induced by biotic stresses, including MAMP treatments(Zipfel et al., 2004, 2006; Kemmerling et al., 2007), and some were shown tobe dispensable for plant development and thus are good candidates as PRRs(Lehti-Shiu et al., 2009).

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INNATE IMMUNITY: PATTERN RECOGNITION IN PLANTS 7

Three typical leucine-rich repeat (LRR)-RLKs acting as PRRs (which canbe referred as LRR-receptor kinases) are FLS2 and EFR, the Arabidopsisreceptors for flagellin (Gomez-Gomez and Boller, 2000) and EF-Tu (Zipfelet al., 2006), respectively, and Xa21, an RLK of rice that has long been consid-ered an unusual “resistance gene” product (Song et al., 1995) but was recentlyshown to perceive ax21 (Lee et al., 2009), a peptide universally conservedin Xanthomonas oryzae that should be considered as a MAMP (reviewed byRonald and Beutler, 2010; see also Table 1.1 for further examples).

Other functional PRRs are members of a second class of receptor proteinscalled receptor-like proteins (RLPs). These proteins have a similar structureto RLKs but lack kinase domains; instead, these proteins often exhibit ashort cytoplasmic domain with no signaling signature. This suggests differentmolecular mechanisms of receptor activation than those controlling RKs; theyprobably have to interact with other membrane proteins to transmit the signalacross the membrane (Wang et al., 2008a). RLPs have a similar structure tothe animal TLRs, but in contrast to TLRs that are encoded by 10–12 differentgenes in mammals (Leulier and Lemaitre, 2008), RLPs expanded into a largerfamily, with 57 members in the Arabidopsis genome (Wang et al., 2008a). Theapparent expansion of the families of RLKs and RLPs may indicate that inevolution these receptors have become one of the preferred systems for nonself-perception in plants. To date, two RLPs have been identified as PRRs forindividual MAMPs: (1) the receptor for the fungal MAMP xylanase (ethylene(ET)-inducing xylanase, EIX) in tomato, named LeEIX (Ron and Avni, 2004);(2) and the chitin-binding site CEBiP (chitin elicitor binding protein) in rice(Kaku et al., 2006).

PRRs are often identified on the basis of genetics, and only for a smallnumber, biochemical evidence has been provided to demonstrate direct inter-action between the receptor and its respective ligand (Table 1.1). However,technical advances in methods, such as affinity chromatography, chemicalcross-linking, and immunoprecipitation, have allowed the unequivocal iden-tification of PRR–MAMP interactions and will hopefully shape the future ofPRR characterizations (Chinchilla et al., 2006; Kaku et al., 2006; Shinya et al.,2010).

1.2.2 Flagellin Perception in Arabidopsis through FLS2: A Paradigm forMAMP Recognition in Plants

Flagellin, the best-characterized MAMP in plants, was serendipitously iden-tified as the active elicitor in a “harpin” preparation from the plant pathogenPseudomonas syringae (Felix et al., 1999). Flagellin is the main buildingblock of the flagellum, which allows bacteria to “swim”; typically, a flag-ellum consists of 10,000 monomers of flagellin. Due to its essential role in

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bacterial motility, and also due to its abundance and surface exposure, flagellinrepresents a perfect “molecular pattern” to detect the approach of potentiallypathogenic bacteria. The “epitope” of flagellin perceived by plants is locatedat the N-terminus of the protein and is strongly conserved in bacteria (Felixet al., 1999). Its effect can be mimicked by a synthetic peptide called flg22.Most higher plants, including both gymnosperms and angiosperms, share thecapacity to perceive flg22 as a MAMP (Albert et al., 2010a).

Among responses typical for MAMPs (see also next Section 1.3), aprolonged treatment with flg22 induces a growth arrest in seedlings. Usingthis growth defect as a readout, a genetic screen was conducted in Arabidopsisthat allowed the identification of FLS2 as a gene essential for flagellinresponses (Gomez-Gomez and Boller, 2000). Using a biochemical approachwith radiolabeled flg22, it was shown that these mutants were not onlyimpaired in flg22 signaling but also in flg22 binding (Gomez-Gomez et al.,2001). Both Arabidopsis and tomato perceive flg22, but interestingly thesetwo species show different specificities of binding and responses to derivativesof flg22 (Meindl et al., 2000; Bauer et al., 2001). Transfer of ArabidopsisFLS2 into tomato cells (having an endogenous flagellin receptor) inducednew recognition specificities representative of the flagellin receptor fromArabidopsis indicating that AtFLS2 is the “bona fide” receptor for bacterialflagellin, controlling binding of ligand and activation of responses (Chinchillaet al., 2006). This was corroborated by cross-linking experiments with labeledflg22, followed by immunoprecipitation with antibodies specific for FLS2,which showed unequivocally that FLS2 interacts directly with its flg22 ligand(Chinchilla et al., 2006). Several orthologs of FLS2 were identified in tomato(LeFLS2; Robatzek et al., 2007), Nicotiana benthamiana (NbFLS2; Hann andRathjen, 2007), and rice (OsFLS2; Takai et al., 2008). In rice, responses toflg22 are weak, but OsFLS2 is able to functionally complement Arabidopsisfls2 mutants, confirming that it is indeed a flagellin receptor (Takai et al., 2008).Comparison of orthologs of FLS2 indicated a highly conserved structure forthis protein, including a large ectodomain of 28 LRRs, except for OsFLS2 lack-ing LRR3, and other conserved domains at the N- and C-terminus, indicatingin particular that the large LRR domain is of functional relevance (Boller andFelix 2009).

It remains an open question where the exact binding site of flg22 lies withinthe LRR domain of FLS2. A random mutagenesis approach using the de-fined individual LRR domains of AtFLS2 indicated that LRR9 to LRR15 playan important role for FLS2 function (Dunning et al., 2007). Protein crystal-lography would seem a method of choice to delineate the ligand–receptorinteraction; however, up to now, attempts to functionally express the extracel-lular flg22-binding site of FLS2 in heterologous systems were unsuccessful,perhaps because of its high degree of glycosylation in vivo (see Chinchillaet al., 2006).

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The analysis of chimeric receptors is another approach to understand howthe large ectodomains of the PRRs function in ligand binding and receptoractivation. This is exemplified by a study where the ectodomain of the brassi-nolide receptor BRI1, a typical LRR-RK (Li and Chory, 1997) was fused tothe cytoplasmic domain of the rice PRR Xa21 (Song et al., 1995). Rice cellsexpressing this chimeric receptor were able to induce defense responses af-ter application of exogenous brassinolides (He et al., 2000). More recently, arole of the wall-associated kinase 1 (WAK1) in the perception of oligogalac-turonides (damage-associated molecular patterns (DAMPs) released from theplant cell wall, which activate the plant-immune response) was demonstratedusing chimeric receptors between the ectodomain of WAK1 and the proteinkinase domain of EF-Tu receptor (EFR) (Brutus et al., 2010). In this work, as aproof of concept, functional chimeric receptors inducing an immune responsewere also constructed between the ectodomain of FLS2 and the protein kinasedomain of EFR (Brutus et al., 2010). A refined analysis employing chimeraswithin the extracellular LRR domains of EFR and FLS2 allowed mapping ofsubdomains relevant for ligand binding and receptor activation in EFR (Albertet al., 2010b). Work is in progress to map such domains in FLS2, using theorthologs of the flagellin receptor from Arabidopsis and tomato, which showdistinct specificities for ligand binding and responses (Robatzek et al., 2007).

1.2.3 EFR: An Evolutionarily Young but Efficient PRR PerceivingBacterial EF-Tu

A protein able to induce defense responses was isolated from an extract of anEscherichia coli strain mutated for the flagellin synthesis gene FliC; it turnedout to be the bacterial EF-Tu (Kunze et al., 2004). This new MAMP plays acrucial role in protein synthesis and belongs to the most abundant and mosthighly conserved bacterial proteins. Peptides representing the N-terminus ofbacterial EF-Tu, namely elf18 and elf26, require N-terminal acetylation forfull activity—a typical modification of bacterial but not of eukaryotic EF-Tu.Interestingly, elf18 and elf26 are recognized as elicitors only by plants fromthe Brassicaceae family. No activity could be measured in any other plantfamilies tested to date (Kunze et al., 2004; Zipfel et al., 2006; Albert et al.,2010a). This indicates that evolution has shaped the recognition specificityof EFR—structurally a close relative of Xa21 (Boller and Felix, 2009)—onlyafter the emergence of Brassicaceae, about 40 million years ago.

Interestingly, elf18 induces very similar responses as flg22, particularly withregard to altered gene expression (Zipfel et al., 2006). The observation thattreatment with flg22 or elf18 induced transcript accumulation of FLS2 led tothe hypothesis that RLKs induced by MAMPs are potential PRRs. Along thishypothesis, and focusing on the class of LRR-RLKs, Zipfel and colleagues

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established a collection of knock out mutants of Arabidopsis affected in theLRR-RLKs induced by flg22. This collection was screened for responsivenessto elf18, which allowed the identification of the EF-Tu receptor, called “EFR”(Zipfel et al., 2006). The EFR protein has a similar structure as FLS2: bothbelong to the LRR-RLK subfamily XII and the ectodomain of EFR consists of21 LRRs. Transfer of the EFR gene to heterologous plants “blind” for elf18,such as N. benthamiana or tomato, resulted in responsiveness to this elicitor,demonstrating EFR is the receptor for EF-Tu, and also in an enhanced resis-tance to several bacterial pathogens, demonstrating the biological relevance ofMAMP perception in disease resistance (Zipfel et al., 2006; Lacombe et al.,2010).

Recent genetic screens aiming at identifying new regulators of elf18 signal-ing allowed the identification of an element of the secretory pathway importantfor maturation of the EFR receptor, namely the ER quality control system (ER-QC; Nekrasov et al., 2009; Li et al., 2009b; Saijo et al., 2009; Lu et al., 2009;Haweker et al., 2010). Intriguingly, neither FLS2 nor the RLK CERK1 (chitinelicitor receptor kinase1) involved in chitin signaling (see also Section 1.2.6)seem to be affected by these mutations.

1.2.4 A newly Recognized MAMP–PRR Pair: The Rice LRR-RK Xa21Recognizes ax21 from Xoo

The receptor kinase Xa21 is among the first receptor kinases cloned in plants(Song et al., 1995). It was shown to provide rice cultivars with considerableresistance to X. oryzae pv oryzae (Xoo), a pathogen causing bacterial blight.The Xa21 gene was for a long time considered to be a resistance gene involvedin race specific resistance but now it is considered a PRR (Park et al., 2010;Ronald and Beutler, 2010).

This is because the ligand of this receptor was recently identified as thesulfated peptide axYS22, more commonly called “ax21”, for activating Xa21immunity (Lee et al., 2009). This microbial molecule appears to be conservedin all Xanthomonas species and may play a role in quorum sensing (Lee et al.,2006); thus, it resembles a MAMP rather than an “avirulence” determinant.

Some Xanthomonas strains evade ax21 recognition by avoiding sulfation ofthe secreted peptide, a modification that appears to be crucial for recognitionby Xa21 (da Silva et al., 2004). In general, posttranslational modifications ofMAMPs, such as acetylation and sulfation, seem to emerge as an importantway to modulate MAMP recognition in plants (Kunze et al., 2004; Lee et al.,2009).

Numerous genetic, molecular, and biochemical studies have been con-ducted on the Xa21 immunity (reviews: Park et al., 2010; Ronald and Beutler,2010). Among them is the finding that a kinase dead version of Xa21 is still

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partially active (Liu et al., 2002). Instead, activation of the receptor complexvia phosphorylation may be controlled by an unknown kinase (Park et al.,2010). In addition, several interactors of Xa21 were identified by diverse ap-proaches (Park et al., 2010). In particular, an ATPase called XB24 (for Xa21binding 24) was shown to interact with Xa21 to promote its autophospho-rylation (Chen et al., 2010). In plants, silencing of XB24 enhances Xa21immunity; thus, activation of Xa21 following ax21 perception may be the re-sult of the inactivation or the release of XB24 from the receptor complex. Asdiscussed in Section 1.2.3, Xa21 accumulation seems to be regulated by ER-QC and ER-associated degradation systems, as indicated by the identificationof several regulators of these pathways in the Xa21 receptor complex (Parket al., 2010).

1.2.5 BAK1—A Positive Regulator of PRRs

Our understanding of PRR activation and signal transduction made an impor-tant step forward with the identification of a second RLK involved in flagellinsignaling, called BAK1 (Chinchilla et al., 2007; Heese et al., 2007).

The BRI1-associated kinase 1 protein was first identified as a coreceptorfor the brassinosteroid receptor BRI1 in Arabidopsis (Li et al., 2002; Namand Li, 2002). Surprisingly, BAK1 seems to be shared by several signalingpathways controlling developmental as well as defense responses (reviewedin Chinchilla et al., 2009; see also Table 1.1). Consistent with a role in plantimmunity, plants depleted for BAK1 show reduced responses to flg22 andexhibited more symptoms to virulent bacteria (Chinchilla et al., 2007; Heeseet al., 2007; Kemmerling et al., 2007).

BAK1 is localized at the plasma membrane of plant cells (Li et al., 2002;Nam and Li, 2002) and further biochemical analysis demonstrated that BAK1interacts with FLS2 in a flg22-dependent manner (Chinchilla et al., 2007;Heese et al., 2007): this oligomerization process is extremely quick, occurringwithin seconds of elicitation with flg22 (Schulze et al., 2010). However, BAK1seems to be dispensable for flg22 binding (Chinchilla et al., 2007). Using an invivo phospholabeling approach, phosphorylation events were detected in bothBAK1 and FLS2 very rapidly after flg22 perception (∼15s; Schulze et al.,2010). These data indicate that the flagellin receptor is activated in a similarway as animal tyrosine receptor kinases (Lemmon and Schlessinger, 2010)or the RK BRI1 in Arabidopsis (Wang et al., 2008b). In the latter model, theligand-binding RK BRI1 is activated after BL perception, which promotes itsassociation with BAK1. In this heterocomplex, BRI1 and BAK1 phosphorylateeach other in “trans” to amplify the BL signal. In absence of BAK1, BRI1 canstill exhibit some kinase activity and BL signaling is functional but at a lowerlevel (Wang et al., 2008b).

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Recently, the kinase activity of BAK1 was shown to be essential for flg22signaling, although not for its heteromerization with FLS2 (Schulze et al.,2010). Consistently, treatment of Arabidopsis cells with a general kinaseinhibitor did not affect the FLS2–BAK1 complex formation. This is in contrastto the BRI1–BAK1 complex, which requires BRI1 kinase activity for itsformation or stability (Wang et al., 2008b). It remains to be tested if FLS2kinase activity is required for signaling and if this activity is promoted byBAK1 in vivo.

Several studies support a role of BAK1 as a central regulator of PRRs (seeTable 1.1). For example, activation of EFR also involves BAK1 (Chinchillaet al., 2007; Schulze et al., 2010). Furthermore, BAK1 seems to play a role inDAMP signaling since it is phosphorylated upon perception of Pep1 (Huffakeret al., 2006) in Arabidopsis and interacts with the Pep1 receptors, PEPR1 andPEPR2 (Yamaguchi et al., 2006, 2010), and two other LRR-RKs (Schulzeet al., 2010; Postel et al., 2010). Since BAK1 appears to regulate several LRR-RKs, it will be interesting to test if OsBAK1, the homolog of BAK1 recentlyidentified in rice (Li et al., 2009a), is involved in Xa21-mediated immunity.The role of BAK1 in PRR regulation is not restricted to LRR-RKs; a recentreport indicated a role of BAK1 in regulation of xylanase perception controlledby the RLP LeEIX1 and LeEIX2 (Bar et al., 2010). Interestingly, chitinperception, which involves a different class of receptor kinase (LysM RKs),does not require BAK1 (Shan et al., 2008; Schulze et al., 2010). Finally, BAK1also plays a role in resistance to fungi and oomycetes, because plants depletedfor BAK1 were more susceptible to several of these pathogens, includingVerticillium, Alternaria, and Hyaloperonospora parasitica (see Chinchillaet al., 2009).

BAK1 is a member of a small gene family of five members, called theSERKs, initially defined as “somatic embryogenesis-receptor kinases” (seeBoller and Felix 2009; Chinchilla et al., 2009). Two of them, BAK1 (SERK3)and SERK4, share a particularly high homology on the level of amino acidsequences. Intriguingly, bak1(serk3) serk4 double mutants of Arabidopsis arelethal at the seedling stage, displaying constitutive defense-gene expression,callose deposition, reactive oxygen species (ROS) accumulation, and sponta-neous cell death even under sterile growing conditions (He et al., 2007). Thus,BAK1 and its SERK4 homolog must be involved in cell death control as well(see Chinchilla et al., 2009).

1.2.6 Chitin Perception in Plants: A New Scenario for Molecular Eventsof MAMP Perception

Chitin is a polymer of N-acetylglucosamine found in fungal cell walls, insectexoskeletons, and crustacean shells, but not in plants. Plants do not have chitin

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but instead possess chitinases that degrade chitin. It was hypothesized thatplant chitinases can degrade chitin in the cell wall of the invading fungusand release short fragments of chitin (chito-oligosaccharides) that can act asMAMPs (reviewed in Boller, 1995).

Similar to flg22, chitin oligosaccharides are active in a wide range of plants,including both dicots (Felix et al., 1993) and monocots (Shibuya and Minami,2001). Much effort has been made since many years to identify and character-ize high affinity binding sites for chitin oligomers in membrane fractions ofdiverse plant species such as rice (Shibuya et al., 1993) and tomato (Baureithelet al., 1994). But the molecular identity of the first chitin-binding protein wasdiscovered only recently with the isolation of CEBiP from extracts of riceculture cells using chitin affinity chromatography (Kaku et al., 2006).

The CEBiP from rice possesses two “lysine motifs” (LysM) in itsectodomain. In legumes, LysM motifs were identified in the plant receptorkinases involved in the recognition of Nod factors, lipo-chitooligosaccharidessecreted by symbiotic rhizobium bacteria to establish the nitrogen-fixing nod-ule symbiosis (Limpens et al., 2003; Madsen et al., 2003; Radutoiu et al.,2003). In Lotus japonicus, NFR1 and NFR5 are the candidate Nod Factorreceptors. L. japonicus (Lj) and Lotus filicaulis (Lf) associate with differentsymbiotic strains of Rhizobium due to different Nod factor recognition speci-ficities. By domain swapping of LysM motifs from LjNFR1/5 and LfNFR1/5,it was shown that the second LysM domain of NFR5 is involved in this recogni-tion process; thus, this domain appears to be capable of binding carbohydratemolecules in a highly specific way (Radutoiu et al., 2007) and may be in-volved, remarkably, in recognition of both friends and foes in plants (Knoggeand Scheel, 2006).

Since CEBiP does not contain a cytoplasmic signaling domain, in contrastto LysM-domain receptor kinases such as NFR1 and NFR5, it is likely that itcooperates with other proteins to transmit the signal from the plasma mem-brane to the cytoplasm. One LysM RK was characterized as the CERK1 inArabidopsis and more recently in rice: plants affected in cerk1 expression areunable to respond to chitin, indicating that this LysM RK is involved in chitinperception and/or signaling (Miya et al., 2007; Wan et al., 2008; Shimizu et al.,2010). Interestingly, in rice, OsCERK1 can form heteromers with CEBiP invivo, in a ligand-dependent manner (Shimizu et al., 2010). But does OsCERK1contribute to ligand binding? The main band cross-linked to labeled chitin wasfound to be CEBiP, this cross-linking signal was not affected in knockdownlines of OsCERK1, indicating that CEBiP is the major molecule that bindschitin oligosaccharides on the rice cell surface (Miya et al., 2007; Shimizuet al., 2010). In contrast, recent studies in Arabidopsis report the capacity ofAtCERK1 to bind chitin (Iizasa et al., 2010; Petutschnig et al., 2010). How-ever, some other data speak against CERK1 as a binding site specific for chitin:notably AtCERK1 was shown to be targeted by a bacterial effector and thus

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might be involved in signaling of a bacterial MAMP as well (Gimenez-Ibanezet al., 2009).

Although the role of CERK1 in ligand binding remains elusive, it exhibits aclear kinase activity in vitro (Miya et al., 2007) and this kinase activity is essen-tial for activation of early defense responses in Arabidopsis (Petutschnig et al.,2010). Moreover, similar to other RKs, such as FLS2 and BAK1, CERK1 isphosphorylated in vivo in its cytoplasmic domain, as shown by proteomic anal-ysis of CERK1 in chitin-treated cells of Arabidopsis (Petutschnig et al., 2010).

Currently, it is difficult to draw a clear model for chitin perception basedon the divergent findings in Arabidopsis and rice. CERK1 and CEBiP areclearly essential for chitin signaling, and it is tempting to imagine that thesemembrane proteins collaborate to form a functional receptor for chitin inrice. But in contrast to the BRI1–BAK1 and FLS2–BAK1 models whereactivation of receptors seems to occur via transphosphorylation events betweenthe kinase domains of both RKs in the cytoplasm, it is more difficult to forecastthe molecular mechanisms controlling activation of chitin receptor elements.Since CERK1 can form homodimers (Shimizu et al., 2010), it is possible thatchitin perception activates CERK1 via transphosphorylation events withinthese homodimers. Alternatively, another unknown protein (kinase) present inthe chitin receptor complex may activate CERK1 in response to ligand binding.More work needs to be done to clarify the mechanism of chitin signaling, afundamental and very interesting example of MAMP perception.

1.3 Physiological Responses and Signaling Events Induced by Elicitors

Recognition of MAMPs through PRRs leads to a number of responses, in awell-ordered temporal pattern, and culminates in a state of “PTI” (originallydefined as PAMP-triggered immunity, but now better redefined as “pattern-triggered immunity”). In the following, an overview is provided on the ele-ments of PTI.

1.3.1 Immediate Early Responses

Ion Fluxes. Among the earliest and most easily recordable physiologicalresponses to MAMPs is an alkalinization of the growth medium due to changesof ion fluxes across the plasma membrane in plant cell cultures (Boller, 1995;Boller and Felix, 2009). This response starts after a lag phase of ∼0.5–2.0minutes and is certainly the easiest readout to identify new MAMPs frommicrobial extracts.

Rapid changes in ions include increased influx of H+ and Ca2+ and aconcomitant efflux of K+; and an efflux of anions, in particular of nitrate

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(Wendehenne et al., 2002; Jeworutzki et al., 2010). These ion fluxes lead tomembrane depolarization as recorded, for example, in electrophysiologicalstudies with soybean cells challenged with the fungal MAMP, heptaglucan(Mithofer et al., 2005) and more recently with mesophyll cells and root hairsfrom Arabidopsis treated with flg22 and elf18 (Jeworutzki et al., 2010).

In eukaryotes, the Ca2+ ion is a ubiquitous intracellular second messengerinvolved in numerous signaling pathways regulating developmental as well asdefense processes. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt)couple a large array of signals and responses in plants, although the way thisresponse activates specific targets and responses remains unclear. Specificityof [Ca2+]cyt may be due to the time course of [Ca2+]cyt variations and thelocation of the [Ca2+]cyt increase (Garcia-Brugger et al., 2006).

Different Ca2+ signatures (varying with amplitude, frequency, time, andlocation) have been associated with diverse MAMPs (Lecourieux et al., 2005;Gust et al., 2007; Aslam et al., 2008; Aslam et al., 2009; Jeworutzki et al.,2010), and these signatures may potentially be decoded by distinct calciumsensors.

Calcium sensors perceive changes in [Ca2+]cyt that directly binds to theEF-hand motif of these proteins to modulate their activity. Best evidencefor a role of calcium sensors in PTI is based on a recent study on calcium-dependent protein kinases (CDPKs) (Boudsocq et al., 2010), but other sensorssuch as calmodulins and calmodulin-binding proteins may also contribute tothis regulation (Reddy and Reddy 2004). Using a functional genomic screenand genome-wide gene expression profiling, specific CDPKs, CPK4, CPK5,CPK6, and CPK11, were shown to control ROS production and expressionof a subset of genes induced by MAMPs in Arabidopsis (Boudsocq et al.,2010). Moreover, multiple knockout mutants cpk5/cpk6 and cpk5/cpk6/cpk11are affected in disease resistance to P. syringae. It is still unknown how theseCDPKs are regulated, when they are activated, and how they regulate plantdefense responses.

Production of Reactive Oxygen Species (ROS). A characteristic defense re-sponse of plants is the rapid and robust production of ROS by host cells, areaction also known as “oxidative burst” (review: Boller and Felix, 2009). Thisresponse occurs after a lag phase of ∼2 minutes in Arabidopsis plants and istransient. ROS are highly toxic intermediates comprising reduced forms ofoxygen, such as the superoxide anion and hydrogen peroxide.

The sources of ROS can be diverse: In Arabidopsis, a membrane localizedNADPH oxidase called AtRbohD (for respiratory burst oxidase homolog D)appears to be responsible for all flg22-induced ROS produced in the apoplast(Nuhse et al., 2007; Mersmann et al., 2010). In other systems, peroxidasesmay have a role in apoplastic ROS generation (Torres, 2010).

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Regulation of NADPH oxidase-dependent ROS production probably in-volves Ca2+ ions, which may bind to the two EF hands motifs present in theN-terminal region of the protein, thereby regulating these enzymes. In addi-tion, NADPH oxidases and therefore ROS generation appear to be regulatedby phosphorylation (Benschop et al., 2007; Nuhse et al., 2007; Ogasawaraet al., 2008). Two phosphosites were identified in AtRbohD as a result offlg22 perception in Arabidopsis cells that were shown to be essential for ROSproduction (Nuhse et al., 2007). In potato, the production of ROS by thepotato NADPH oxidase B appears to be regulated by phosphorylation throughtwo CDPKs, namely StCDPK4 and StCDPK5 (Kobayashi et al., 2007). Con-sistently, multiple mutants affected in CDPK genes showed impairment inflg22-induced ROS production (Boudsocq et al., 2010).

The role of ROS in disease resistance is not yet well understood. ROScan contribute to defense either directly as an antibiotic agent, or indirectlyby promoting oxidative cross-linking in the cell wall (Apel and Hirt, 2004).Furthermore, it may also be involved in the closure of stomata, a defensemechanism restricting bacterial entrance (Melotto et al., 2006; see also Section1.3.5).

Activation of More Cytoplasmic Kinases: MAPK. Mitogen-activated proteinkinase (MAPK) cascades are highly conserved modules in all eukaryoteswhere they transfer information from sensors to cellular responses. Activationof MAPK cascades involves a phosphorelay mechanism composed of MAPKkinase kinases (MAPKKK), MAPK kinases (MAPKK), and MAPKs.

In plants, MAPK cascades play an important role in signaling in response tobiotic and abiotic stresses (Colcombet and Hirt, 2008; Pitzschke et al., 2009).

Two distinct MAPK cascades regulate PTI in Arabidopsis. A first MAPKmodule MEKK1-MKK4/MKK5-MPK3/MPK6 was originally proposed to beresponsible for flg22 signal transduction (Nuhse et al., 2000; Asai et al.,2002). However, more recent work demonstrated that although mekk1 mutantswere compromised in activation of MPK4, MPK3, and MPK6, responses toflg22 were not affected (Ichimura et al., 2006; Suarez-Rodriguez et al., 2007).Thus, the MEKK protein activating the MKK4/5-MPK3/MPK6 cascade forpositive regulation of defense responses remains unknown. MEKK1 forms asecond cascade with MKK1/2-MPK4. This cascade is thought to negativelyregulate immunity because loss-of-function mutations in these kinases lead toconstitutive activation of defenses and a dwarf phenotype associated with mu-tants accumulating salicylic acid (SA) and other defense-related compounds(Ichimura et al., 2006; Suarez-Rodriguez et al., 2007; reviewed by Pitzschkeet al., 2009). However, the mechanisms by which MPK4 regulates immunityare largely unknown.

Importantly, the activation mechanism for neither of the two MAPK mod-ules is known to date. Is it possible that PRRs at the plasma membrane directly

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activate top-level member of the cascades (the MAPKKK, also called MEKK)directly (occurring ∼1–2 minutes after MAMP perception)? Or, is activationmediated by other yet unidentified kinases?

Activation of Membrane Kinases: BIK1. The membrane-anchored kinaseBIK1 was recently identified as a new component involved in early stepsof MAMP signaling (Lu et al., 2010; Zhang et al., 2010). Botrytis-induced Ki-nase1 was originally identified as an Arabidopsis gene that is transcriptionallyregulated by pathogen or elicitor treatment (Veronese et al., 2006). More recentstudies showed that BIK1 is phosphorylated upon flg22 treatment (as observedby mobility shift in Western blot analysis) (Lu et al., 2010; Zhang et al., 2010).This modification of BIK1 peaks at ∼5–10 minutes after treatment and can thusbe distinguished from the phosphorylation events between FLS2 and BAK1which were observed ∼15 seconds after elicitation (Schulze et al., 2010). In-terestingly, BIK1 interacts with the BAK1-FLS2 complex in nonstimulatedcells and seems to be released from this complex upon 10 minutes of flagellintreatment (Lu et al., 2010; Zhang et al., 2010). In vitro, BIK1 can phospho-rylate BAK1 and FLS2 and may be a direct substrate for BAK1, suggesting arole of BIK1 in the regulation of the flagellin receptor and/or signaling.

Consistently, Arabidopsis bik1 mutants are impaired in some responses(ROS production and callose) induced by flg22 and elf18, but also chitin (incontrast to bak1) (Lu et al., 2010; Zhang et al., 2010). Since bik1 mutantsshowed high SA content, bik1 sid2 double mutants, which exhibit normalaccumulation of SA, were generated to study the effect of BIK1 in respectto bacterial growth. As expected, these mutants had a defect in flg22-inducedresistance to P. syringae (Zhang et al., 2010)

Overall, these studies report on BIK1 as an interesting and new signalingelement of PTI although further studies are necessary to get a more compre-hensive view on its role in MAMP signaling and immunity.

1.3.2 Hormone Changes in Response to MAMPs with a Focuson Ethylene Signaling

The three major plant hormones associated with MAMP perception and bioticstress are SA, jasmonic acid (JA), and ET, but other plant hormones may playa role in the defense response of plants as well (reviewed in Bari and Jones,2009). Here, we briefly summarize recent advances in our understanding ofthe role of ET.

ET accumulation is a well-known response to MAMP treatment andis a consequence of the phosphorylation-dependent activation of 1-aminocyclopropane-1-carboxylate (ACC) synthases (reviewed in Boller andFelix, 2009). Interestingly, the MAMP-induced MAPKs MPK3 and MPK6

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phosphorylate ACC synthases ACS2 and ACS6 (Liu and Zhang, 2004; Hanet al., 2010) as well as EIN3, a transcription factor involved in the ET re-sponse, leading to its stabilization (Yoo et al., 2008). But how is ET involvedin broad-spectrum resistance? Two recent reports have shown that ET is in-volved in the regulation of FLS2 gene expression, thereby rendering the plantsmore sensitive to flg22 perception (Mersmann et al., 2010; Boutrot et al.,2010). Consistently, mutants of the well-known ET regulator EIN2 or the ETreceptor ETR1 were nearly insensitive to flg22 and FLS2 transcript levels werestrongly reduced. Chromatin immunoprecipitation assays revealed that EIN3is able to bind the FLS2 promoter (Boutrot et al., 2010). On the basis of this,a simple model was proposed in which ET signaling would control the FLS2pathway by a positive feedback mechanism (Boutrot et al., 2010). Accordingto this model, plants would maintain a constant pool of FLS2 levels even in theabsence of MAMPs due to endogenous ET levels. Upon activation by flg22,FLS2 undergoes endocytosis, which leads to a reduction of the amount ofFLS2 at the plasma membrane (Robatzek et al., 2006). However, at the sametime, flg22 treatment will activate ACC synthases through phosphorylationby MAPKs, which leads to EIN3 accumulation in the nucleus and therebyinduction of FLS2 expression (Chen et al., 2009). Thus, FLS2 level would beregulated by a positive feedback loop driven by ET. The expression of BAK1is not affected (Mersmann et al., 2010); it remains an open question whetherother PRRs are regulated by ET.

1.3.3 Responses at the Level of Gene Expression

Modification of Gene Expression in Response to MAMP Treatments. In Ara-bidopsis, about a thousand genes are upregulated ∼30–60 minutes afterMAMP treatment, as revealed by several transcriptome analyses (Zipfel et al.,2004, 2006; Gust et al., 2007; Wan et al., 2008). Among them are manygenes involved in perception and signaling of MAMPs, including PRRs them-selves (Navarro et al., 2004), and thus increase the “awareness” for potentialpathogens. Other MAMP-induced genes encode enzymes involved in pro-tein degradation, cell wall modification, secondary metabolite biosynthesis,and vesicle trafficking, which may help to arrest, directly or indirectly, theinvading microbes (Navarro et al., 2004).

There is a clear overlap between the genes upregulated after flg22, elf18, andchitin treatments, albeit small differences exist (Zipfel et al., 2006; Wan et al.,2008). These data suggest that the signaling pathways triggered by differentMAMPs converge. By contrast, only a small number of downregulated genesare common to chitin and flg22/elf18 responses (Wan et al., 2008).

These considerable transcriptional changes in defense genes appear to bemediated by two types of kinases already described above, MAPKs and CDPKs

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(Asai et al., 2002; Boudsocq et al., 2010). Transcriptome profile comparisonsuggests that MAPKs and CDPKs are two convergence points of signaling trig-gered by most MAMPs (Boudsocq et al., 2010). Using a functional screen anddiverse MAMP marker genes, this study revealed that CDPK and MAPK cas-cades act differentially in four MAMP-mediated regulatory programs (CDPKspecific/MAPK specific/MAPK dominant and synergistic pathways) to con-trol early genes involved in PTI. In addition, two transcription factors fromthe WRKY family, WRKY22 and WRKY29, were shown to act downstreamof MPK3/MPK6 activation in response to flg22 (Asai et al., 2002).

Role of Silencing in Regulation of PTI. RNA silencing is an inducible defensepathway that uses small interfering RNAs (siRNAs) to specifically target andinactivate invading nucleic acids as a defense against viruses (Ruiz-Ferrer andVoinnet, 2009). Interestingly, there are also endogenous small RNAs that act inreprogramming gene expression in response to pathogen attack. For example,the microRNA miR393 is induced by flg22 in Arabidopsis and negativelyregulates auxin signaling by targeting auxin receptors (Navarro et al., 2006).Repression of auxin signaling restricts P. syringae growth, implicating auxinin disease susceptibility and miRNA-mediated suppression of auxin signalingin resistance.

Consistent with a main role of small RNAs in immunity, several componentsof the silencing machinery were shown to be essential for pathogen resistance(Agorio and Vera, 2007; Navarro et al., 2008). Indeed, AGO1, a main compo-nent in the generation of small RNAs, seems to be required for some MAMPresponses (Li et al., 2010). Analysis of AGO1-bound small RNAs led to theidentification of a number of miRNAs up/downregulated by flg22 treatment;for some of them, a role in PTI was confirmed (Li et al., 2010). Future work willshow to what extent small RNAs regulate PTI, and what their target genes are.

1.3.4 Final Outcome of the Response: HR or no HR?

The hypersensitive response (HR) is a form of rapid cell death that may restrictpathogen growth and is often associated with specific resistance (Jones andDangl, 2006). Most MAMPs do not induce HR in plants; exceptions are theoomycete elicitins in tobacco (Takemoto et al., 2005) or fungal xylanase intomato (Ron and Avni, 2004). In addition, recent studies have revealed thatthe prototypic MAMP flagellin can also induce HR in plant cells (Naito et al.,2008). Full-length flagellin from P. syringae pv tabaci 6605 induces an HR inArabidopsis, in contrast to the classic flg22 from Pseudomonas aeruginosa;this is due to the presence of a single aspartate residue within the core regionof the flg22 epitope (Naito et al., 2008). Interestingly, this residue is alsoimportant for bacterial virulence. Moreover, although flg22 does not induce

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cell death in wild type cells from rice, it induces HR in transgenic rice cellsoverexpressing OsFLS2 (Takai et al., 2008). Thus, the absence or presence ofplant cell death should not be used as reliable criterion to distinguish betweenMAMPs and effectors.

1.3.5 Stomatal Closure

A main step in disease resistance is the closure of stomata, the pores formedby guard cells in the plant epidermis (reviewed in Zeng et al., 2010). Stomataare believed to be the main point of entry for pathogenic microbes and inparticular for bacteria, which cannot penetrate the plant epidermis directlyin the same way as some fungi and oomycetes. Thus, stomatal closure is animportant response to MAMP perception (Melotto et al., 2006).

A recent study addressed the question of the contribution of different PRRs(FLS2 and EFR) in stomata closure and demonstrated that FLS2 plays adecisive role during the interaction of P. syringae DC3000 and Arabidopsis(Zeng and He, 2010). The signaling pathway through which activation of FLS2in guard cells leads to stomatal closure is discussed in Chapter 2 in the contextof effectors.

1.3.6 The Role of Vesicle Trafficking in PTI

Striking changes in compartmentalization of plant cells can be observed uponpathogen attacks, which involve vesicle secretion and endocytosis processes(Frei dit Frey and Robatzek, 2009).

The role of secretion was especially well described in the case of fungalinfection, which causes a number of organelles to aggregate in infected cellsbeneath pathogen entry sites. This may contribute to the establishment of aphysical barrier against pathogens, and to secretion of antimicrobial com-pounds (reviewed in Bednarek et al., 2010). The importance of secretion innonhost resistance was emphasized by the study of penetration (pen) mutantsof Arabidopsis (reviewed in Bednarek et al., 2010). These pen mutants, whichare unable to stop the ingress of the nonhost powdery mildew fungi Blumeriagraminis f sp hordei, are affected in genes important for secretion. PEN1, alsocalled SYP121 (syntaxin of plants 121), encodes a plasma membrane localizedsyntaxin protein from the superfamily of SNARE, known to mediate mem-brane fusion events (Collins et al., 2003). PEN1 forms a ternary complex withan adaptor called SNAP33 and several vesicle-associated membrane proteins,including VAMP721/722 (Kwon et al., 2008). All these proteins become con-centrated at the pathogen entry concomitantly to the formation of papillae, astructure that serves as physical barrier against penetration of pathogens (Kwonet al., 2008; Meyer et al., 2009). This suggests a focal delivery of cargo con-trolled by the SNARE complex, possibly by exocytosis, at infection sites to

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restrict pathogen entry by locally reinforcing the plant cell wall and/or di-rectly secreting antimicrobial compounds. The molecular mechanisms con-trolling secretion by SNARE complexes are not well known, and they mayinvolve phosphorylation of key regulators. Differential phosphorylation wasreported for Arabidopsis PEN1/SYP121 and SYP122 and PEN3 and for to-bacco Nt Syp121 in response to flg22 (Nuhse et al., 2003, 2007; Benschopet al., 2007).

Examples for antimicrobial compounds secreted are the “pathogen-relatedproteins,” also called “PR” (van Loon et al., 2006). Downregulation of theexpression of the tobacco syntaxin SYP132 correlates with a decrease in PR1accumulation in the apoplast in response to bacterial infection, suggesting adirect role of SYP132 in PR1 secretion (Kalde et al., 2007). How many of thesecreted PR proteins are translocated into the apoplast by different SNAREcomplexes remains to be tested.

Another cargo class may include the cell wall reinforcing molecules, namelycell wall precursors (such as callose) and/or enzymes for cell wall synthesis.Callose is a �-1,3 glucan polymer and has been observed to accumulate inplant cell walls in papillae formed at the site of pathogen contact, but also moregenerally in response to diverse MAMPs (Gomez-Gomez et al., 1999; Clayet al., 2009. Callose deposition induced by flg22 is controlled by the PMR4gene (powdery mildew-resistant 4) encoding a plasma membrane localizedcallose synthase (Nishimura et al., 2003). Interestingly, pen1 mutants andvamp721/722 silenced plants show a delayed deposition of callose in responseto fungal infection (Kwon et al., 2008), suggesting their role in the secretionof callose.

Trafficking from the plasma membrane to the cytoplasm, a process com-monly referred to as endocytosis has also been observed in response toMAMPs. The best example is the endocytosis of the FLS2 receptor: a func-tional FLS2-GFP fusion protein was shown to be internalized in small vesiclesin Arabidopsis tissues and this process requires activation of the receptor by itsactive ligand (Robatzek et al., 2006). A complete disappearance of the FLS2-GFP signal is observed after 30 minutes of treatment with flg22, and thisprocess is blocked by inhibitors of proteasome and endocytosis; this suggeststhat endocytosis of FLS2 is required for degradation of activated receptors(Robatzek et al., 2006). Thus, PRR endocytosis in plants may be involved inattenuation of MAMP signaling as suggested for TLR4, a receptor involvedin lipopolysaccharide (LPS) recognition in animals (Husebye et al., 2006).

1.3.7 Conclusion

As reviewed in this chapter, a number of MAMPs and their receptors arewell known, as are the downstream events of MAMP signaling (summarizedin Fig. 1.1). However, we currently miss the link of PRR activation, which

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occurs almost instantaneously, to downstream signaling, which starts after alag phase of 1 minute. Second messengers such as Ca2+, ROS, ET, SA areclearly involved, but it remains an open question how these pathways areinterconnected and to what extent they contribute to PTI.

An emerging concept is the convergence of signaling pathways early afterMAMP perception, which is supported by the common responses induced bydifferent MAMPs. However, the localization and timing of these responsesover the different plant tissues has not been well studied, and such analysesmay still reveal some significant differences (Zipfel and Robatzek, 2010). Forexample, a prolonged flg22 treatment induces a strong arrest of root and shootof Arabidopsis seedlings, while only the shoot is affected by elf18 (Gomez-Gomez and Boller, 2000; Zipfel et al., 2006). This observation suggests thatdifferent PRRs may exhibit different tissue-specificities. Since different classesof pathogens infect different parts of the plant, it is easy to imagine thebenefit for plants to express its different PRRs specifically in different tissues.Thus, recognition of different MAMPs may play different roles during theinfection process depending on the type of invading pathogens. The fact thatsome PRRs occur in a single plant family or even in some genera within afamily (such as EFR in Brassicaceae and Xa21 in some rice cultivars) showsthat MAMP perception is shaped in plants by a dynamic coevolution withtheir microbial environment (Boller and Felix, 2009). The identification ofadditional MAMPs and their respective PRRs will help to fully understandthese dynamic relationships.

1.4 The Biological Relevance of PTI

Despite tremendous progress made in the field of MAMP perception in plants,we still do not understand the key changes responsible for pathogen growtharrest. For a long time, MAMP perception in plants was underappreciated be-cause of the lack of genetic evidence demonstrating that PTI contributes finallyto disease resistance. But in the last decade, several studies demonstrated thesignificance of PTI in plant disease by analyzing mutations in PRRs, whichoften compromise overall plant resistance to pathogens.

A first demonstration was obtained using Arabidopsis plants lacking func-tional FLS2 which were infected with the pathogen P. syringae pv. tomatoDC3000: while fls2 mutant plants are as susceptible as the wild-type whenbacteria are infiltrated into leaves, they are more susceptible to this pathogenwhen bacteria were sprayed onto the leaf surface (Zipfel et al., 2004). Thus,flagellin perception restricts bacterial invasion, probably at an early step andcontributes to the plant’s disease resistance. A logical explanation for this phe-notype was provided by a later study that showed that the FLS2-mediatedresistance to DC3000 is largely attributed to MAMP-induced guard cell

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closure, which limits bacterial entry into the leaf tissue (Melotto et al., 2006).Flagellin perception increases resistance to both host and nonhost bacteriaas demonstrated in N. benthamiana (Zipfel et al., 2004; Hann and Rathjen,2007).

Plants lacking the EFR PRR are more susceptible to Agrobacterium tume-faciens (Zipfel et al., 2006). Interestingly, cerk1 mutants exhibit enhancedsusceptibility to fungal pathogens (Miya et al., 2007; Wan et al., 2008) andto bacteria (Gimenez-Ibanez et al., 2009). In the case of Xa21, this rice geneclearly increases plant resistance against the bacterial pathogen X. oryzae(reviewed in Park et al., 2010; Ronald and Beutler, 2010).

Overall, although PTI is activated and contributes to resistance even ina compatible reaction, it clearly constitutes an important aspect of nonhostresistance, which renders most plants resistant to the majority of potentialpathogens they encounter (Dodds and Rathjen, 2010).

It is actually not so rare that disease susceptibility phenotypes appear verysubtle or conditional or just not detectable in mutants affected in PTI regula-tors, although typical defense responses are clearly impaired in these plants.As we have seen in the former sections, many studies support the idea thatMAMP perception systems are indeed functionally highly redundant, thus, itis rather expected that impairment of a single PRR does not affect the fitness ofplants. Indeed, some natural mutants of FLS2 were found including the Ara-bidopsis ecotypes Ws-0 (Zipfel et al., 2004) and Cvi-0 (Dunning et al., 2007).These ecotypes probably survived because they have several PRRs signalingbacterial infection: Arabidopsis can perceive bacteria also via the detection ofEF-Tu, peptidoglycans, LPS, and probably other unknown MAMPs (Bollerand Felix, 2009).

The importance of PTI in plant immunity is underlined by the fact that manypathogens developed molecules called “effectors” that suppress PTI signalingin order to be able to invade plant tissues (Boller and He, 2009; see Fig. 1.1).Indeed, as will become clear from the next sections, the presence of sucheffectors in pathogenic/adapted bacteria further complicated investigation onthe contribution of PTI for disease resistance (see Fig. 1.1).

Now that we have a better understanding of the role of PTI in plant immunity,can we use this knowledge to improve crop resistance? We believe yes: First,MAMPs are clearly essential for survival and lifestyle of a given class ofmicrobes and cannot be mutated easily without compromising fitness, makingthem good targets for a defense strategy. Second, while many specific PRRsappear to have evolved only recently in one plant family or genus, the signalingpathways appear to be conserved even between monocots and dicots. Thus,there is good probability that a given plant, engineered to express new PRRsfrom other plant families, may display a strong successful defense responseto its pathogens. In fact, transgenic expression of EFR in N. benthamiana andtomato plants, two members of the Solanaceae that are normally “blind” to

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bacterial EF-Tu plants, led to an increased disease resistance to a range ofphytopathogenic bacteria (Lacombe et al., 2010).

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