Plant pattern recognition receptor complexes at the plasma membrane

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Plant pattern recognition receptor complexes at the plasma membrane Jacqueline Monaghan and Cyril Zipfel A key feature of innate immunity is the ability to recognize and respond to potential pathogens in a highly sensitive and specific manner. In plants, the activation of pattern recognition receptors (PRRs) by pathogen-associated molecular patterns (PAMPs) elicits a defense programme known as PAMP-triggered immunity (PTI). Although only a handful of PAMP-PRR pairs have been defined, all known PRRs are modular transmembrane proteins containing ligand-binding ectodomains. It is becoming clear that PRRs do not act alone but rather function as part of multi-protein complexes at the plasma membrane. Recent studies describing the molecular interactions and protein modifications that occur between PRRs and their regulatory proteins have provided important mechanistic insight into how plants avoid infection and achieve immunity. Address The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK Corresponding author: Zipfel, Cyril ([email protected])

Current Opinion in Plant Biology 2012, 15:349–357 This review comes from a themed issue on Biotic interactions Edited by Pamela C Ronald and Ken Shirasu For a complete overview see the Issue and the Editorial Available online 14th June 2012 1369-5266/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pbi.2012.05.006

Introduction Plants employ a multi-layered recognition system to protect against microbial infection. One layer involves perception by surface-localized pattern recognition receptors (PRRs) of conserved molecules characteristic of entire groups of microbes (called either microbe-associated or pathogen-associated molecular patterns; MAMPs/ PAMPs), such as fungal chitin or bacterial lipopolysaccharides (LPS), peptidoglycans (PGN), quorum sensing factors, and flagellin [1]. In addition, some PRRs recognize host-derived ‘danger’ signals (damage-associated molecular patterns; DAMPs), such as plant peptides or cell wall fragments released during infection or wounding. PAMP-binding or DAMP-binding activates PRRs and triggers profound and tractable physiological changes in plant cells resulting in PAMP-triggered immunity (PTI). These include bursts of calcium and reactive www.sciencedirect.com

oxygen species (ROS), as well as the activation of mitogen-associated and calcium-dependent protein kinases (MAPKs and CDPKs), leading to massive transcriptional reprogramming [2,3]. As PTI is sufficient to halt ingress of most microbes, loss of individual PRRs leads to enhanced disease susceptibility to both adapted and non-adapted pathogens [1]. Accordingly, adapted pathogens secrete numerous effector proteins to evade and/or suppress PTI [[4]; and see review by J. Zhou and colleagues, this issue]. A second layer of immunity is mediated by intracellular receptors containing nucleotide-binding (NB) and leucine-rich repeat (LRR) domains that directly or indirectly recognize effector proteins and activate robust defence programmes often culminating in localized cell death [4]. Thus, while PTI involves the recognition of both ‘infectious self’ and ‘non-self’ molecules, effector-triggered immunity (ETI) involves the perception of molecular patterns of pathogenicity [5]. These events reflect the dynamic interplay between host and microbe, and suggest that PTI is a major initial driving force in the evolutionary arms race between plants and their pathogens. Here, we summarize recent progress in the identification of novel PRRs and on the immediate signalling events following PAMP perception in PRR complexes at the plasma membrane. We will not cover protein complexes regulating PRR biogenesis [6] or endocytosis [see review by S. Robatzek and colleagues, this issue].

PAMPs: conserved, yet under selective pressure Despite overall conservation, PAMPs are under selective pressure in adapted pathogen lineages to evade recognition. For example, although bacterial flagellin, the major component of flagella, is a potent inducer of PTI in most plants, mutations in key residues of the flg22 epitope that abolish recognition by the receptor FLS2 have been selected in several plant pathogens and symbionts [1]. Notably, this positive selection seems to be more rapid than previously thought, as modern natural isolates of Pseudomonas syringae pv. tomato (Pto) adapt to their tomato host through non-synonymous mutations in the flagellin-encoding gene fliC [7]. Thus, although PAMPs are necessary for microbial life and are therefore under strong negative selection, their immunogenic epitopes are under positive selection to evade host immune detection. These opposing evolutionary forces were recently used to identify novel candidate PAMPs from Pseudomonas and Xanthomonas species through an innovative bioinformatics approach [8]. Current Opinion in Plant Biology 2012, 15:349–357

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Plant PRRs are surface-localized receptor-like kinases or receptor-like proteins All known plant PRRs are plasma membrane-localized receptor-like kinases (RLKs) or receptor-like proteins (RLPs) with modular functional domains (Figure 1). RLKs contain an extracellular domain (ECD), a singlepass transmembrane (TM) domain, and an intracellular kinase domain. RLPs contain an ECD and a TM but have only a short cytosolic domain without an obvious signalling domain. Notably, in contrast to mammals, no intracellular NB-LRR protein recognizing a PAMP has yet been identified in plants [9]. LRR-containing RLKs and RLPs as sensors of peptidic PAMPs

The best-studied plant PRRs are the RLKs FLS2, EFR and XA21 that belong to subfamily XII of LRR-containing RLKs [1]. The flg22 epitope present in the conserved N-terminus of flagellin is recognized by most plant species [1], and the flagellin receptor FLS2 has been identified in Arabidopsis, tomato, Nicotiana benthamiana and rice [1]. Some plants, such as rice and tomato, can recognize additional domains of flagellin [7,10], although it is unclear if this recognition is also FLS2dependent. Comparatively, EFR is a Brassicaceaespecific PRR that recognizes bacterial elongation factor Tu (EF-Tu) [1]. Although XA21 was cloned 15 years ago as a locus conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo) in

rice, its matching ligand was only recently identified as the type I-secreted sulphated protein Ax21 that is conserved across Xanthomonas species and is involved in quorum sensing [11,12]. Similar to flagellin and EFTu, a minimal eliciting epitope could be defined, and the corresponding synthetic peptide AxYs21 can be used to mimic Ax21-triggered responses in XA21-expressing rice [11]. Notably, these findings revealed retrospectively that XA21 was the first PRR identified across kingdoms and preceded the identification of animal PRRs that was recognized by the 2011 Nobel Prize in Medicine. The tomato LRR-RLPs Eix1 and Eix2 are PRRs that bind fungal xylanase [13]. In addition, the tomato LRRRLP Ve1, previously known to mediate resistance to the fungus Verticillium, was recently shown to recognize Ave1, a peptide with homology to plant natriuretic peptides that is conserved in several fungi as well as the bacterium Xanthomonas axonopodis [14]. Consistently, Ve1 also mediates resistance to the fungus Fusarium oxysporum [14]. Direct binding of Ave1 to Ve1 has, however, not yet been demonstrated. FLS2 as a multi-specific PRR?

Intriguingly, Arabidopsis FLS2 was recently shown to respond to high concentrations of the shoot apical meristem (SAM) growth regulator CLV3p [15], leading to the activation of immune responses. In addition, an artificial derivative of AxYs21, AxYs21-A1, is also able to induce FLS2-dependent immune responses [16].

Figure 1

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Pattern recognition receptors with known ligands.The LRR-RLK FLS2 recognizes bacterial flagellin (or the active epitope flg22) in Arabidopsis, tomato, N. benthamiana and rice. The Brassicaceae-specific LRR-RLK EFR recognizes bacterial elongation factor Tu (or the active epitope elf18), and the related LRR-RLK XA21 binds the type-I secreted quorum-sensing peptide Ax21 (or the synthetic sulphated 17-aa peptide axYs22) in rice. The LysMRLPs LYM1 and LYM3 are the receptors for bacterial peptidoglycan (PGN), and the LysM-RLK CERK1 weakly binds PGN. Interaction has not yet been shown between LYM1, LYM3, and CERK1, but it is possible that they function as a tripartite recognition complex for PGN in Arabidopsis. The fungal PAMP xylanase is recognized by the tomato LRR-RLPs Eix1 and Eix2. Ave1 is a putative ligand for the tomato LRR-RLP Ve1, however, direct binding has not yet been shown. In rice, the LysM-RLP CEBiP binds chitin and interacts with the LysM-RLK CERK1 to signal intracellularly, while in Arabidopsis, CERK1 is the major chitin-binding protein and interaction with CEBiP-like proteins has not yet been shown. In legumes, an extracellular bglucan-binding protein (GBP) binds Phytophthora heptaglucan, however data describing how this protein accomplishes intracellular signalling is lacking; here, we hypothesize interaction with a transmembrane protein. The LRR-RLKs PEPR1 and PEPR2 bind endogenous AtPeps, and the RLK WAK1, containing an EGF-like domain, is a receptor for cell wall-derived oligogalacturonides (OGs). Current Opinion in Plant Biology 2012, 15:349–357

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Remarkably, although AxYs21-A1 and CLV3p do not share any sequence homology with flg22, mutant analyses and competition assays suggested that all three peptides share the same binding site on FLS2 [15,16]. While a biological function for the recognition of the Ax21 variant AxYs21-A1 by FLS2 still needs to be demonstrated, CLV3p perception by FLS2 was linked to anti-bacterial immunity of the SAM [15]. However, the reproducibility and/or biological significance of these extra recognition specificities for FLS2 were recently challenged, as independent investigations failed to detect induction of FLS2-mediated immune responses in Arabidopsis by naturally occurring and biologically active Ax21 and CLV3 peptides [17,18]. LysM domain-RLKs and RLPs as sensors of oligosaccharidic PAMPs

The LysM domain is a carbohydrate-binding module initially identified in bacterial lysozyme that binds PGNs. While LysM-RLKs were previously linked to recognition of rhizobial Nod factors in legumes [19], the first evidence for a role of LysM domain-containing proteins in PAMP recognition came from the identification of the LysMRLP CEBiP as a chitin-binding protein required for chitin recognition in rice [20]. CEBiP forms a chitinenhanced heteromeric complex with the LysM-RLK CERK1 [21]. Although it is unclear if CERK1 also binds chitin, it is required for chitin responsiveness in rice [21]. In Arabidopsis, however, CERK1 is the major chitinbinding protein [22,23], and is required for chitin-induced responses [24,25]. The Arabidopsis genome encodes three CEBiP paralogs, LYM1–3, but it is currently unknown if any are involved in chitin perception. By contrast, LYM1 and LYM3 were recently demonstrated to bind PGN, a bacterial N-acetyl glucosamine oligomer structurally similar to chitin, in vitro [26]. Consequently, loss of LYM1 and/or LYM2 dramatically reduces PGN responsiveness and leads to increased susceptibility to the virulent bacterium Pto DC3000 [26]. In addition to its role in chitin perception, CERK1 is also required for PGN responsiveness and weak binding of PGN to CERK1 could be detected in vitro [26]. These recent findings may explain the previously documented role of CERK1 in anti-bacterial immunity [27]. Importantly, in earlier studies, CERK1 was not required for PGN-triggered responses [28], and, unlike chitin, PGN did not induce CERK1 phosphorylation [22]. These differences may be explained by the different sources of PGN used. Therefore, CERK1 may function as part of a tripartite recognition module for bacterial PGN alongside LYM1 and LYM3; however, complex formation between LYM1 and LYM3, or between LYM1, LYM3 and CERK1 has not yet been demonstrated. In any case, it is interesting to consider CERK1 as a multi-functional LysM-RLK acting either as part of a ligand-binding complex or as a regulator of ligand-binding LysM-RLPs mediating downstream signalling. www.sciencedirect.com

Identification of the first plant PRRs for DAMPs

The Arabidopsis LRR-RLKs PEPR1 and PEPR2 recognize AtPeps, a family of peptide DAMPs that may amplify PTI signalling [29,30]. An innovative chimeric receptor approach using the intracellular region of EFR revealed that the EGF-like domain-containing RLK WAK1 is a PRR for oligogalacturonides, which are released from plant cell walls during fungal infections or wounding [31]. The relationship between DAMPinduced and PAMP-induced signalling in plant immunity is likely to be deciphered in the coming years. Additional PRRs

Several putative PRRs have been uncovered in multiple plant species [32] for which more mechanistic insight is required. For example, the LRR-RLPs AtRLP52, AtRLP30 and AtRLP51/SNC2, as well as the unique RLK SNC4, play a role in immunity and may represent PRRs with currently unknown ligands [33–35]. The barley kinase RPG1, which localizes to both the cytosolic and membrane compartments, interacts with two Puccinia graminis f.sp. tritici proteins in vitro and confers resistance to stem rust [36–38]. In legumes, an extracellular bglucan-binding protein (GBP) binds Phytophthora cellwall derived hepataglucan [39], however data describing how this protein accomplishes intracellular signalling is lacking. Notably, the LRR-RLP Cf proteins that confer resistance to Cladisporium fulvum in tomato may be reclassified as PRRs, however this is still an open debate [40].

The LRR-RLK BAK1 is a master positive regulator of innate immunity mediated by LRR-RLK and LRR-RLP PRRs BAK1/SERK3 is a small LRR-RLK belonging to a family of five related SERK proteins. BAK1 was initially identified as an interactor and positive regulator of the brassinosteroid (BR) receptor BRI1, but also rapidly forms ligand-induced complexes with FLS2 and EFR [1,41,42] (Figure 2A). BAK1 is not required for ligand binding [1,41], and therefore functions as a signal enhancer, not as a co-receptor. In addition to BAK1, other SERKs are recruited into the FLS2 and EFR complexes in a ligand-dependent manner but with unequal affinities, correlating with the contrasting importance of BAK1 for FLS2-mediated and EFR-mediated responses [42]. BAK1 also interacts with the LRR-RLKs PEPR1 and PEPR2 in yeast two-hybrid assays [43]; however in vivo interaction has not yet been demonstrated. BAK1 and its closest paralog BKK1/SERK4 are the major regulators of FLS2-mediated, EFR-mediated, and PEPR1/2mediated signalling as the double-mutant bak1-5 bkk1-1 is nearly insensitive to flg22, elf18 and AtPep1 [42,44]. Interestingly, a rice BAK1 ortholog associates with XA21 and positively regulates XA21-mediated resistance to Xoo [P. Ronald, personal communication] (Figure 2B). The Current Opinion in Plant Biology 2012, 15:349–357

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Plasma membrane-localized pattern recognition receptor complexes. (A) The FLS2 complex. In Arabidopsis, FLS2 interacts constitutively with BIK1 and related PBLs, as well as with SCD1. BAK1 interacts constitutively with PUB12 and PUB13 and also may associate with BIK1. Upon flg22 binding, BAK1 heteromerizes with FLS2 and trans-phosphorylates BIK1 (and other PBLs) and PUB12/13. BIK1 then trans-phosphorylates BAK1 and FLS2, resulting in a signalling-competent FLS2 complex. PUB12/13 then polyubiquitinate FLS2 resulting in its degradation and attenuation of signalling. Phosphorylation is annotated as black circles labelled ‘P’, ubiquitination is annotated as grey circles labelled ‘U’, and arrows indicate the direction of post-translational modifications. (B) The XA21 complex. In rice, XA21 has been shown to constitutively interact with the ATPase XB24, the E3-ligase XB3, the WRKY transcription factor XB10, the PP2C XB15, the thiamine pyrophosphokinase ROX1, the NOL1/NOL2/sun protein ROX2, and the nudC-like nuclear migration factor ROX3. XB24 keeps XA21 in an inactive state by promoting (denoted by dotted arrows) auto-phosphorylation at specific residues. Ligand binding results in XB24 dissociation, XA21 phosphorylation at distinct residue(s), and the activation of immune signalling. XB15 then dephosphorylates XA21 to attenuate signalling. In vivo interaction data is lacking for proteins coloured in grey, phosphorylation is annotated as black circles labelled ‘P’, and arrows indicate the direction of post-translational modifications. In (A) and (B), the stoichiometry of receptors and their interactors is not represented, nor is potential cellular or temporal specificity.

tomato BAK1 constitutively interacts with Eix1, but not with the signalling-competent receptor Eix2, in yeast-two hybrid and split-YFP assays in N. benthamiana, suggesting a complex BAK1-dependent regulation of Eix2 by Eix1 in response to ethylene-induced xylanase [45]. Additionally, the importance of BAK1 in several plant species for responses triggered by other PAMPs, such as bacterial Current Opinion in Plant Biology 2012, 15:349–357

LPS and PGN, and oomycete INF1, suggests that BAK1 may form ligand-induced complexes with additional PRRs [46,47]. Intriguingly, PGN responses seem to depend on both CERK1 and BAK1 in Arabidopsis [26,46], which is in contrast to the strict BAK1-independency of CERK1mediated chitin responses in Arabidopsis [27,46,48]. The BAK1-dependence of Ve1-mediated resistance to www.sciencedirect.com

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Verticillium in tomato and Arabidopsis further highlights the role of Ve1 as a PRR [14,49,50].

dissociation and activation/deactivation of downstream substrates resulting in specific responses.

A clear conclusion about the role of BAK1 in immunity is normally hampered by pleiotropic phenotypes of bak1 mutants linked to hypo-responsiveness to BR and increased cell death. However, the recent identification of the novel allele bak1-5 that is unaffected in BR responses and cell death control revealed that BAK1 is important for resistance to obligate biotrophic and hemibiotrophic pathogens [42,44]. Notably, BAK1 and BKK1 contribute to resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa), and the insensitivity of bak1-5 bkk1-1 plants to extracts from Hpainfected Arabidopsis tissues reveals that BAK1 may be involved in signalling downstream of unknown PRRs recognizing uncharacterized PAMPs or DAMPs released during Hpa infection [42].

Plants must manage trade-offs between normal growth and defence programmes. Given the positive role of BAK1 in both BR-mediated growth and PTI, it was long hypothesized that BAK1 may be involved in balancing these two pathways. Accordingly, a unidirectional antagonism between BR and PTI responses can be observed whereby BRI1 activation results in decreased PTI responses, both genetically [57] and upon exogenous application of BR [58]. The antagonism triggered by BRI1 overexpression can be alleviated upon overexpression of BAK1 [57], suggesting that BRI1 may titrate BAK1 away from PRRs. However, in native conditions, BAK1 is not rate-limiting between the two pathways, and the antagonism between the BR and PTI pathways does not correlate with a decreased amount of BAK1 in FLS2 complexes, or with decreased phosphorylation of FLS2 or the downstream substrate BIK1 [58]. These recent findings highlight a finely tuned regulation between BR-mediated growth and PTI that probably involves both BAK1-dependent and BAK1-independent mechanisms.

The mechanisms underlying complex formation between BAK1 and PRRs, as well as the molecular events occurring within BAK1-PRR complexes are still largely unknown. The almost instantaneous ligand-induced heteromerization between FLS2 and BAK1 [51] suggest that they exist in close proximity at the plasma membrane in a loose pre-formed complex (Figure 2A). Subsequent to ligand-induced stabilization of the FLS2–BAK1 complex, phosphorylation of both proteins occurs [51]; however, the order of events, the identities of phosphorylated residues, and their importance for downstream signalling remain largely unknown. Studies on phosphorylation events in the paradigmatic BRI1–BAK1 complex following BR perception suggest that BAK1 acts as an enhancer of BRI1 kinase activity and may contribute to the recruitment of specific downstream substrates by phosphorylation of residues in the intracellular juxtamembrane and C-terminal regions of BRI1 [41]. Notably, while FLS2 and EFR are non-RD kinases, BRI1 is an RD kinase [52], and major differences in the properties of the association between these kinases and BAK1 have been noted. For example, the kinase activities of EFR, FLS2, or BAK1 are not required for their ligand-dependent heteromerization [44,51], while the kinase activities of both BRI1 and BAK1 are required for their association [41]. In addition, while C-terminal tagged BAK1 fusions are functional in BR signalling, they do not complement the PAMP hypo-responsiveness of null bak1 mutants [53], further revealing important mechanistic differences between the BRI1–BAK1 and FLS2/EFR–BAK1 complexes. The recent identification and characterization of novel BAK1 alleles, bak1-5 [44] and BAK1elg [54], as well as BAK1 phosphosites [55,56], revealed that the function of BAK1 in BR signalling, PTI, and cell death control can be mechanistically uncoupled in a phosphorylation-dependent manner. A key challenge for the coming years is to decipher how ligand-induced phosphorylation in BAK1 complexes leads to the recruitment/ www.sciencedirect.com

The cytoplasmic kinase BIK1 is a direct substrate of multiple PRR complexes and is a positive regulator of PTI Recent mechanistic insight into early PRR signalling events has come from studies describing the role of a family of cytoplasmic kinases acting at the plasma membrane [59,60]. BIK1 and the paralogous proteins PBS1, PBL1, and PBL2 associate constitutively with FLS2 and potentially with BAK1 [59,60]. Following flg22 perception, BAK1 trans-phosphorylates BIK1 which then phosphorylates both FLS2 and BAK1 and partially dissociates from the FLS2 complex [59,60] (Figure 2A). BIK1 is also phosphorylated following elf18 treatment [59] and interacts with EFR and CERK1 in un-elicited protoplasts [60], suggesting that BIK1 represents a common node downstream of both BAK1-dependent and BAK1-independent PRR complexes. Accordingly, loss-of-function bik1 and pbl mutants are compromised in several PTI responses triggered by flg22, elf18 or chitin, as well as basal immunity to the non-virulent bacterium Pto DC3000 hrcC- [59,60]. Interestingly, BIK1 is dispensable for flg22-induced MAPK activation [61]. This latter finding reveals signal branching at the level of PRR complexes, as previously indicated by the characterization of ER quality control mutants affecting EFR biogenesis [62]. BIK1 is also involved in ethylene signalling and resistance to the necrotrophic fungus Botrytis cinerea [63], further adding to the complexity of studying the role of BIK1 in PTI. Consistent with its significance in plant immunity, BIK1 is a direct target of the Pseudomonas syringae pv. phaseolicola cysteine protease AvrPphB [60] and the Xanthomonas campestris pv. Current Opinion in Plant Biology 2012, 15:349–357

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campestris uridylyl transferase AvrAC [61], leading to BIK1 cleavage or kinase activity inhibition, respectively.

Role of phosphorylation and ubiquitination in the negative regulation of PRRs Constitutive or hyper-activation of immune responses are detrimental to plant growth. Therefore, PRRs and their immediate downstream substrates must be tightly regulated before and after ligand perception. While some progress has been made, the mechanisms underlying the attenuation of PRR activation remain largely unknown. Before ligand binding, XA21 is kept in an inactive state through association with the ATPase XB24, which promotes XA21 auto-phosphorylation at specific residues [64] (Figure 2B). Binding of Ax21 to XA21 results in the dissociation of XB24 and the activation of XA21 [64]. Following activation, the POL-type protein phosphatase 2C (PP2C) XB15 associates with XA21 leading to its dephosphorylation and inactivation [65] (Figure 2B). In Arabidopsis, the PP2C KAPP interacts with FLS2 and negatively regulates flg22 responses [66]; however, the specificity of KAPP is questionable since it was also reported to interact with many RLKs [67]. Notably, we have identified novel phosphatases that regulate FLS2/ EFR complexes pre-ligand and post-ligand binding [C. Segonzac, R. Niebergall, C. Zipfel, unpublished data]. Recently, an elegant study revealed the function of two E3ubiquitin ligases, PUB12 and PUB13, as negative regulators of FLS2 post-ligand perception [68]. PUB12 and PUB13 exist in a constitutive complex with BAK1 and are recruited into the FLS2 complex alongside BAK1 upon ligandinduced heteromerization (Figure 2A). PUB12/13 are phosphorylated by BAK1 and poly-ubiquitinate FLS2 leading to degradation of FLS2 (Figure 2A). Given that FLS2 is endocytosed after flg22 perception [69], it will be interesting to test if PUB12/13-mediated ubiquitination is required for FLS2 internalization. Consistent with their role in postligand FLS2 degradation, loss of PUB12/13 results in heightened flg22-induced responses and enhanced resistance to Pto DC3000 [68]. Whether a similar mechanism attenuates signalling mediated by other BAK1-interacting PRRs, such as EFR, remains to be determined. Previously, additional PUBs, PUB22/23/24 belonging to a separate clade from PUB12/13, were also reported as negative regulators of PTI responses [70], but the mechanisms underlying this regulation are still unknown.

NOL2/sun family member ROX2 [72], and the nudClike nuclear migration factor ROX3 [72] (Figure 2B). In addition, XA21 interacts with the E3-ligase XB3 in vivo and is required for full activation of XA21-mediated immunity [73] (Figure 2B). In Arabidopsis, the DENN domain protein SCD1 associates constitutively with FLS2 in vivo and is required for a subset of FLS2mediated and EFR-mediated responses [74] (Figure 2A).

Outlook The study of PTI in plants is a rapidly expanding field, which highlights the importance of this layer of immunity and mirrors the identification of numerous pathogenderived virulence effectors that suppress PTI signalling. Currently, our knowledge of PRRs and downstream signalling components is limited. We need to uncover the full repertoire of PAMP-PRR pairs governing PTI to understand how plants perceive pathogenic non-self and defend against infection. In particular, the identification of novel family-specific PAMP-PRR pairs would enable the pyramiding of PRRs across plant families to engineer broad-spectrum immunity, as recently demonstrated for EFR and Ve1 [49,75]. Following PAMP perception at the plasma membrane, we need to understand the very early molecular events that initiate distinct signalling branches ultimately leading to PTI, and how PTI signalling is tightly controlled to avoid ‘autoimmune’ disorders. In addition, the molecular interconnection between PTI and ETI, which represent two layers of plant innate immunity, needs to be deciphered. Intriguingly, recent results suggest that FLS2 associates with several components of ETI including NB-LRR proteins themselves and the important immune regulator RIN4 [76]. Lastly, it is important to understand how PTI signalling is connected to hormonal pathways involved in growth and development.

Acknowledgements We thank Pamela Ronald and Jian-Min Zhou for sharing results before publication and apologize to colleagues whose work could not be covered because of space limitations. All members of the Zipfel laboratory are acknowledged for stimulating discussions and helpful comments. Work in the Zipfel laboratory is funded by The Gatsby Charitable Foundation, the UK Biotechnology and Biological Sciences Research Council, ERA-NET Plant Genomics, and the Two Blades Foundation. JM is funded by a LongTerm Fellowship from the European Molecular Biology Organization.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest

Other components of PRR complexes Several complementary approaches have identified additional PRR-associated proteins as regulators of PTI for which more mechanistic studies are needed. Yeast two-hybrid screens for XA21 interactors identified several regulators of XA21-mediated immunity in rice including the WRKY transcription factor XB10/OsWRKY62 [71], the thiamine pyrophosphokinase ROX1 [72], the NOL1/ Current Opinion in Plant Biology 2012, 15:349–357

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Maekawa T, Kufer TA, Schulze-Lefert P: NLR functions in plant and animal immune systems: so far and yet so close. Nat Immunol 2011, 12:817-826.

10. Takai R, Isogai A, Takayama S, Che FS: Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice. Mol Plant Microbe Interact 2008, 21:1635-1642. 11. Lee SW, Han SW, Sririyanum M, Park CJ, Seo YS, Ronald PC: A type I-secreted, sulfated peptide triggers XA21-mediated innate immunity. Science 2009, 326:850-853. 12. Han SW, Sriariyanun M, Lee SW, Sharma M, Bahar O, Bower Z,  Ronald PC: Small protein-mediated quorum sensing in a Gramnegative bacterium. PLoS ONE 2011, 6:e29192. The XA21 ligand, Ax21, was previously shown to be a type-I secreted peptide [11] with a predicted role in bacterial quorum sensing (QS). In this paper, the authors present strong evidence supporting Ax21 as a bone fide QS factor that is required for biofilm formation, cell aggregation, cell motility, and virulence. Notably, Ax21 is the first QS peptide shown to activate immune responses in plants or animals.

Competition assays between flg22 and Ax21 furthermore suggest that both ligands bind FLS2 at the same site. 17. Mueller K, Chinchilla D, Albert M, Jehle AK, Kalbacher H, Boller T, Felix G: Inadvertent cross-contamination as a risk for work  with synthetic peptides: flg22 as an example of a pirate peptide in commercial peptide preparations. Plant Cell 2012, in press. In response to [15,16], this commentary shows that, unlike flg22, newly synthesized and naturally occurring CLV3p and AxYs21 peptides do not bind or activate FLS2. The authors also show that flg22 is capable of activating FLS2-dependent immune responses at very low concentrations, whereas CLV3p and AxYs21 do not activate immunity even at extremely high concentrations. 18. Segonzac C, Nimchuk Z, Beck M, Tarr PT, Robatzek S, Meyerowitz EM, Zipfel C: The shoot apical meristem regulatory  peptide CLV3 does not activate innate immunity. Plant Cell 2012, in press. Also in response to [15], this commentary shows that independently synthesized and biologically active CLV3p does not activate FLS2mediated immune responses in Arabidopsis. The authors furthermore show that inducible expression of CLV3 in transgenic Arabidopsis plants does not result in the induction of immune marker genes. Live confocal microscopy analysis of Arabidopsis SAMs inoculated with Pto reveals that SAMs do not get infected by this bacterium, even in clv3 mutant plants. 19. Popp C, Ott T: Regulation of signal transduction and bacterial infection during root nodule symbiosis. Curr Opin Plant Biol 2011, 14:458-467. 20. Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto-Tomiyama C, Dohmae N, Takio K, Minami E, Shibuya N: Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc Natl Acad Sci USA 2006, 103:11086-11091. 21. Shimizu T, Nakano T, Takamizawa D, Desaki Y, Ishii-Minami N, Nishizawa Y, Minami E, Okada K, Yamane H, Kaku H, Shibuya N:  Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice. Plant J 2010, 64:204-214. Although the LysM-RLP CEBiP was previously identified as the chitin receptor in rice [20], it was unclear how intracellular signalling was achieved. In this paper, the LysM-RLK CERK1, an ortholog of which was previously shown to be required for chitin signalling in Arabidopsis [24], is shown to interact with CEBiP, and the interaction is enhanced after chitin treatment. This suggests that CEBiP and CERK1 function as a heteromeric receptor complex for chitin perception in rice. 22. Petutschnig EK, Jones AM, Serazetdinova L, Lipka U, Lipka V: The lysin motif receptor-like kinase (LysM-RLK) CERK1 is a major chitin-binding protein in Arabidopsis thaliana and subject to chitin-induced phosphorylation. J Biol Chem 2010, 285:28902-28911.

13. Ron M, Avni A: The receptor for the fungal elicitor ethyleneinducing xylanase is a member of a resistance-like gene family in tomato. Plant Cell 2004, 16:1604-1615.

23. Iizasa E, Mitsutomi M, Nagano Y: Direct binding of a plant LysM receptor-like kinase, LysM RLK1/CERK1, to chitin in vitro. J Biol Chem 2009, 285:2996-3004.

14. de Jonge R, Peter van Esse H, Maruthachalam K, Bolton MD,  Santhanam P, Saber MK, Zhang Z, Usami T, Lievens B, Subbarao KV, Thomma BP: Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing. Proc Natl Acad Sci USA 2012, 109:5110-5115. Ave1 is defined as a protein from Verticillium that activates Ve1-dependent immunity in tobacco and tomato and is required for fungal virulence. Interestingly, Ave1 is conserved in several fungal and bacterial pathogens. Given its similarity to plant natriuretic peptides, Ave1 may have been acquired through horizontal gene transfer from a host plant.

24. Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H, Shibuya N: CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci USA 2007, 104:19613-19618.

15. Lee H, Chah OK, Sheen J: Stem-cell-triggered immunity  through CLV3p-FLS2 signalling. Nature 2011, 473:376-379. The authors demonstrate that high concentrations of the shoot apical meristem (SAM) regulatory peptide CLV3p can activate immune responses in Arabidopsis through binding and activation of FLS2, suggesting that FLS2 is a multi-specific receptor contributing to immunity in the SAM. 16. Danna CH, Millet YA, Koller T, Han SW, Bent AF, Ronald PC,  Ausubel FM: The Arabidopsis flagellin receptor FLS2 mediates the perception of Xanthomonas Ax21 secreted peptides. Proc Natl Acad Sci USA 2011, 108:9286-9291. The authors show that high concentrations of Ax21-related peptides activate immune responses in Arabidopsis seedlings in an FLS2-dependent manner, suggesting that FLS2 functions beyond flagellin perception. www.sciencedirect.com

25. Wan J, Zhang XC, Neece D, Ramonell KM, Clough S, Kim SY, Stacey MG, Stacey G: A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis. Plant Cell 2008, 20:471-481. 26. Willmann R, Lajunen HM, Erbs G, Newman MA, Kolb D, Tsuda K,  Katagiri F, Fliegmann J, Bono JJ, Cullimore JV et al.: Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. Proc Natl Acad Sci USA 2011, 108:19824-19829. The authors report the identification of the LysM-RLPs LYM1 and LYM3 as PGN-receptors in Arabidopsis and show that the LysM-RLK CERK1 weakly binds PGN in vitro. Accordingly, LYM1, LYM2, and CERK1 are required for PGN-triggered gene induction. This work provides evidence to explain an earlier observation [27,28] that CERK1 is required for bacterial immunity in addition to its role in chitin perception. 27. Gimenez-Ibanez S, Hann DR, Ntoukakis V, Petutschnig E, Lipka V, Rathjen JP: AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants. Curr Biol 2009, 19:423-429. Current Opinion in Plant Biology 2012, 15:349–357

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28. Gimenez-Ibanez S, Ntoukakis V, Rathjen JP: The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis. Plant Signal Behav 2009, 4:539-541. 29. Krol E, Mentzel T, Chinchilla D, Boller T, Felix G, Kemmerling B, Postel S, Arents M, Jeworutzki E, Al-Rasheid KA et al.: Perception of the Arabidopsis danger signal peptide 1 involves the pattern recognition receptor AtPEPR1 and its close homologue AtPEPR2. J Biol Chem 2010, 285:13471-13479. 30. Yamaguchi Y, Huffaker A, Bryan AC, Tax FE, Ryan CA: PEPR2 is a second receptor for the Pep1 and Pep2 peptides and contributes to defense responses in Arabidopsis. Plant Cell 2010, 22:508-522. 31. Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G: A domain  swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci USA 2010, 107:9452-9457. An innovative chimera strategy with EFR was used to identify Arabidopsis WAK1 as a receptor for cell wall-derived oligogalacturonides. 32. Schwessinger B, Ronald PC: Plant innate immunity: perception of conserved microbial signatures. Annu Rev Plant Biol 2012, 63:451-482. 33. Wang G, Ellendorff U, Kemp B, Mansfield JW, Forsyth A, Mitchell K, Bastas K, Liu CM, Woods-Tor A, Zipfel C et al.: A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis. Plant Physiol 2008, 147:503-517. 34. Zhang Y, Yang Y, Fang B, Gannon P, Ding P, Li X: Arabidopsis snc2-1D activates receptor-like protein-mediated immunity transduced through WRKY70. Plant Cell 2010, 22:3153-3163. 35. Bi D, Cheng YT, Li X, Zhang Y: Activation of plant immune responses by a gain-of-function mutation in an atypical receptor-like kinase. Plant Physiol 2010, 153:1771-1779. 36. Nirmala J, Brueggeman R, Maier C, Clay C, Rostoks N, Kannangara CG, von Wettstein D, Steffenson BJ, Kleinhofs A: Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1. Proc Natl Acad Sci USA 2006, 103:7518-7523. 37. Nirmala J, Drader T, Lawrence PK, Yin C, Hulbert S, Steber CM, Steffenson BJ, Szabo LJ, von Wettstein D, Kleinhofs A: Concerted action of two avirulent spore effectors activates Reaction to Puccinia graminis 1 (Rpg1)-mediated cereal stem rust resistance. Proc Natl Acad Sci USA 2011, 108:14676-14681. 38. Nirmala J, Drader T, Chen X, Steffenson B, Kleinhofs A: Stem rust spores elicit rapid RPG1 phosphorylation. Mol Plant Microbe Interact 2010, 23:1635-1642.

leucine-rich repeat receptor kinase BAK1 is implicated in Arabidopsis development and immunity. Eur J Cell Biol 2009, 89:169-174. 44. Schwessinger B, Roux M, Kadota Y, Ntoukakis V, Sklenar J,  Jones A, Zipfel C: Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genet 2011, 7:e1002046. This work describes the isolation and characterization of the novel bak1-5 allele, which is specifically impaired in immunity but remains competent in BR signalling and cell death control, revealing that the role of BAK1 in different pathways can be mechanistically uncoupled. In addition, comparative studies revealed key differences in the association properties of the RD kinase BRI1 or the non-RD kinases FLS2/EFR with BAK1. 45. Bar M, Sharfman M, Ron M, Avni A: BAK1 is required for the attenuation of ethylene-inducing xylanase (Eix)-induced defense responses by the decoy receptor LeEix1. Plant J 2010, 63:791-800. 46. Shan L, He P, Li J, Heese A, Peck SC, Nurnberger T, Martin GB, Sheen J: Bacterial effectors target the common signaling partner BAK1 to disrupt multiple MAMP receptor-signaling complexes and impede plant immunity. Cell Host Microbe 2008, 4:17-27. 47. Chaparro-Garcia A, Wilkinson RC, Gimenez-Ibanez S, Findlay K, Coffey MD, Zipfel C, Rathjen JP, Kamoun S, Schornack S: The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana. PLoS ONE 2011, 6:e16608. 48. Heese A, Hann DR, Gimenez-Ibanez S, Jones AM, He K, Li J, Schroeder JI, Peck SC, Rathjen JP: The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci USA 2007, 104:12217-12222. 49. Fradin EF, Abd-El-Haliem A, Masini L, van den Berg GC,  Joosten MH, Thomma BP: Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis. Plant Physiol 2011, 156:2255-2265. This work describes the first successful transfer of an RLP between plant families, and shows that tomato Ve1 confers Verticillium resistance in Arabidopsis. 50. Fradin EF, Zhang Z, Juarez Ayala JC, Castroverde CD, Nazar RN, Robb J, Liu CM, Thomma BP: Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1. Plant Physiol 2009, 150:320-332.

40. Thomma BP, Nurnberger T, Joosten MH: Of PAMPs and effectors: the blurred PTI-ETI dichotomy. Plant Cell 2011, 23:4-15.

51. Schulze B, Mentzel T, Jehle AK, Mueller K, Beeler S, Boller T,  Felix G, Chinchilla D: Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1. J Biol Chem 2010, 285:9444-9451. Complex formation and phosphorylation between FLS2 and BAK1 is shown to occur within seconds of flg22 treatment. Rapid heteromerization between BAK1 and EFR or PEPR1 is furthermore shown to occur rapidly following ligand treatment. The kinase activities of FLS2 and BAK1 are not required for their heteromerization, providing further evidence for mechanistic differences between BAK1-BRI1 and BAK1-PRR complexes.

41. Clouse SD: Brassinosteroid signal transduction: from receptor kinase activation to transcriptional networks regulating plant development. Plant Cell 2011, 23:1219-1230.

52. Dardick C, Ronald P: Plant and animal pathogen recognition receptors signal through non-RD kinases. PLoS Pathog 2006, 2:e2.

42. Roux M, Schwessinger B, Albrecht C, Chinchilla D, Jones A,  Holton N, Malinovsky FG, Tor M, de Vries S, Zipfel C: The arabidopsis leucine-rich repeat receptor-like kinases BAK1/ SERK3 and BKK1/SERK4 are required for innate immunity to Hemibiotrophic and Biotrophic pathogens. Plant Cell 2011, 23:2440-2455. Using co-immunoprecipitation and mass spectrometry analyses, the authors demonstrate that EFR and FLS2 undergo ligand-induced heteromerization in planta with several SERK-family LRR-RLKs including BAK1 and its closest paralog BKK1. Taking advantage of the newly isolated bak1-5 allele [44], they furthermore define cooperative roles for BAK1 and BKK1 as major regulators for several PAMP and DAMP pathways as well as immunity against hemi-biotrophic and biotrophic pathogens.

53. Ntoukakis V, Schwessinger B, Segonzac C, Zipfel C: Cautionary  notes on the use of C-terminal BAK1 fusion proteins for functional studies. Plant Cell 2011, 23:3871-3878. In this commentary, the authors show that C-terminal tagged BAK1 fusion proteins do not function equally in BR-signalling and PTI, further revealing key mechanistic differences between the BRI1–BAK1 and FLS2/EFR– BAK1 complexes.

39. Fliegmann J, Mithofer A, Wanner G, Ebel J: An ancient enzyme domain hidden in the putative beta-glucan elicitor receptor of soybean may play an active part in the perception of pathogen-associated molecular patterns during broad host resistance. J Biol Chem 2004, 279:1132-1140.

43. Postel S, Kufner I, Beuter C, Mazzotta S, Schwedt A, Borlotti A, Halter T, Kemmerling B, Nurnberger T: The multifunctional Current Opinion in Plant Biology 2012, 15:349–357

54. Jaillais Y, Belkhadir Y, Balsemao-Pires E, Dangl JL, Chory J:  Extracellular leucine-rich repeats as a platform for receptor/ coreceptor complex formation. Proc Natl Acad Sci USA 2011, 108:8503-8507. Careful analysis of the previously isolated gain-of-function bak1elg allele reveals the role of extracellular LRRs in determining binding specificity with receptor partners. Importantly, D122 is defined as a key residue in BAK1 modulating differential interaction between BRI1 and FLS2. www.sciencedirect.com

Plant pattern recognition receptor complexes at the plasma membrane Monaghan and Zipfel 357

55. Oh MH, Wang X, Wu X, Zhao Y, Clouse SD, Huber SC:  Autophosphorylation of Tyr-610 in the receptor kinase BAK1 plays a role in brassinosteroid signaling and basal defense gene expression. Proc Natl Acad Sci USA 2010, 107:17827-17832. Y610 is defined as an autophosphorylated residue on BAK1. Site-directed mutagenesis of this phosphosite resulted in impaired BR signalling, steady-state expression of defence genes, and resistance against Pto hrcC-, revealing the previously unknown role of tyrosine phosphorylation in BAK1 function. 56. Wang X, Kota U, He K, Blackburn K, Li J, Goshe MB, Huber SC, Clouse SD: Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling. Dev Cell 2008, 15:220-235. 57. Belkhadir Y, Jaillais Y, Epple P, Balsemao-Pires E, Dangl JL,  Chory J: Brassinosteroids modulate the efficiency of plant immune responses to microbe-associated molecular patterns. Proc Natl Acad Sci USA 2011, 109:297-302. This study reports the unidirectional inhibition of PTI by BR and provides genetic evidence supporting a model whereby overexpression of BRI1 titrates BAK1 away from FLS2. 58. Albrecht C, Boutrot F, Segonzac C, Schwessinger B, Gimenez Ibanez S, Chinchilla D, Rathjen JP, de Vries SC, Zipfel C: Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1. Proc Natl Acad Sci USA 2011, 109:303-308. As in [57], this study reports the unidirectional inhibition of PTI by BR, but provides evidence that BAK1 is not rate-limiting between FLS2-complexes and BRI1-complexes in native conditions. 59. Lu D, Wu S, Gao X, Zhang Y, Shan L, He P: A receptor-like  cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proc Natl Acad Sci USA 2010, 107:496-501. This study reports the role of the cytoplasmic kinase BIK1 as a major substrate of PRR complexes and a positive regulator of PTI. 60. Zhang J, Li W, Xiang T, Liu Z, Laluk K, Ding X, Zou Y, Gao M,  Zhang X, Chen S et al.: Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe 2010, 7:290-301. This study demonstrates the role of BIKI and related PBL proteins as positive regulators of immunity interacting with PRR complexes that are cleaved by the Pseudomonas effector AvrPphB. 61. Feng F, Yang F, Rong W, Wu X, Zhang J, Chen S, He C, Zhou JM: A  Xanthomonas uridine 50 -monophosphate transferase inhibits plant immune kinases. Nature 2012, 485:114-118. The Xanthomonas campestris pv. campestris effector AvrAC is identified as a uridylyl transferase that directly targets the cytoplasmic kinases BIK1, PBL1, and RIPK in Arabidopsis and inhibits PTI and RPM1mediated ETI. Uridylation masks conserved phosphorylation sites in BIK1 and RIPK, thus reducing their kinase activity and inhibiting signalling. In addition, BIK1 and PBL1 are shown to be dispensable for flg22induced MAPK activation, further suggesting an early branch point in PTI signalling. 62. Lu X, Tintor N, Mentzel T, Kombrink E, Boller T, Robatzek S, Schulze-Lefert P, Saijo Y: Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidase II allele. Proc Natl Acad Sci USA 2009, 106:22522-22527. 63. Laluk K, Luo H, Chai M, Dhawan R, Lai Z, Mengiste T: Biochemical and genetic requirements for function of the immune response regulator BOTRYTIS-INDUCED KINASE1 in plant growth, ethylene signaling, and PAMP-triggered immunity in Arabidopsis. Plant Cell 2011, 23:2831-2849. 64. Chen X, Chern M, Canlas PE, Ruan D, Jiang C, Ronald PC: An  ATPase promotes autophosphorylation of the pattern recognition receptor XA21 and inhibits XA21-mediated immunity. Proc Natl Acad Sci USA 2010, 107:8029-8034. The ATPase XB24 interacts with XA21 in planta, and dissociates from the complex after infection with Xanthomonas oryzae pv. oryzae. As autophosphorylation of XA21 is enhanced by co-expression of XB24, and genetic

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evidence suggests a negative role of XB24 in XA21-mediated immunity, a model is proposed whereby XB24 keeps XA21 in an ‘inactive state’ before infection. 65. Park CJ, Peng Y, Chen X, Dardick C, Ruan D, Bart R, Canlas PE, Ronald PC: Rice XB15, a protein phosphatase 2C, negatively regulates cell death and XA21-mediated innate immunity. PLoS Biol 2008, 6:e231. 66. Gomez-Gomez L, Bauer Z, Boller T: Both the extracellular leucine-rich repeat domain and the kinase activity of FSL2 are required for flagellin binding and signaling in Arabidopsis. Plant Cell 2001, 13:1155-1163. 67. Ding Z, Wang H, Liang X, Morris ER, Gallazzi F, Pandit S, Skolnick J, Walker JC, Van Doren SR: Phosphoprotein and phosphopeptide interactions with the FHA domain from Arabidopsis kinase-associated protein phosphatase. Biochemistry 2007, 46:2684-2696. 68. Lu D, Lin W, Gao X, Wu S, Cheng C, Avila J, Hesse A,  Devarenne TP, He P, Shan L: Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity. Science 2011, 332:1439-1442. This study shows that the paralogous E3 ligases PUB12 and PUB13 associate with ligand-bound FLS2 through their interaction with BAK1. Furthermore, PUB12 and PUB13 poly-ubiquitinate FLS2 to promote flagellin-induced FLS2 degradation and attenuation of immune responses. 69. Robatzek S, Chinchilla D, Boller T: Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev 2006, 20:537-542. 70. Trujillo M, Ichimura K, Casais C, Shirasu K: Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis. Curr Biol 2008, 18:1396-1401. 71. Peng Y, Bartley LE, Chen X, Dardick C, Chern M, Ruan R, Canlas PE, Ronald PC: OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice. Mol Plant 2008, 1:446-458. 72. Lee I, Seo YS, Coltrane D, Hwang S, Oh T, Marcotte EM,  Ronald PC: Genetic dissection of the biotic stress response using a genome-scale gene network for rice. Proc Natl Acad Sci USA 2011, 108:18548-18553. RiceNet, a genome-scale functional gene network database for monocotyledous species, is presented and experimentally validated through analysis of genes predicted to be involved in XA21-mediated immunity. In this way, three new XA21-binding proteins are identified (ROX1, ROX2, and ROX3) and functionally confirmed as regulators of XA21-mediated immunity in rice. RiceNet therefore represents a powerful tool to aid in the prediction of gene function in monocots. 73. Wang YS, Pi LY, Chen X, Chakrabarty PK, Jiang J, De Leon AL, Liu GZ, Li L, Benny U, Oard J et al.: Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance. Plant Cell 2006, 18:3635-3646. 74. Korasick DA, McMichael C, Walker KA, Anderson JC, Bednarek SY, Heese A: Novel functions of Stomatal Cytokinesis-Defective 1 (SCD1) in innate immune responses against bacteria. J Biol Chem 2010, 285:23342-23350. 75. Lacombe S, Rougon-Cardoso A, Sherwood E, Peeters N,  Dahlbeck D, van Esse HP, Smoker M, Rallapalli G, Thomma BP, Staskawicz B et al.: Interfamily transfer of a plant patternrecognition receptor confers broad-spectrum bacterial resistance. Nat Biotechnol 2010, 28:365-369. Interfamily transfer of the Brassicaceae-specific receptor EFR into Solanaceae species confers broad-spectrum bacterial resistance, demonstrating that PTI signalling pathways are conserved across plants, and that interfamily transfer of PRRs can be used to confer broad-spectrum disease resistance. 76. Qi Y, Tsuda K, Glazebrook J, Katagiri F: Physical association of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) immune receptors in Arabidopsis. Mol Plant Pathol 2011, 12:702-708.

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