Plant PRRs and the Activation of Innate Immune Signaling

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1 Plant PRRs and the Activation of Innate Immune Signaling Alberto P. Macho 1 and Cyril Zipfel 1, * 1 The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK *Correspondence: cyril.zipfel@tsl.ac.uk Despite being sessile organisms constantly exposed to potential pathogens and pests, plants are surprisingly resilient to infections. Plants can detect invaders via the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Plant PRRs are surface-localized receptor-like kinases, which comprise a ligand-binding ectodomain and an intracellular kinase domain, or receptor-like proteins, which do not exhibit any known intracellular signaling domain. In this review, we summarize recent discoveries that shed light on the molecular mechanisms underlying ligand perception and subsequent activation of plant PRRs. Notably, plant PRRs appear as central components of multiprotein complexes at the plasma membrane that contain additional transmembrane and cytosolic kinases required for the initiation and specificity of immune signaling. PRR complexes are under tight control by protein phosphatases, E3 ligases, and other regulatory proteins, illustrating the exquisite and complex regulation of these molecular machines whose proper activation underlines a crucial layer of plant immunity. Introduction The perception of environmental signals and the ability to respond accordingly are essential for organisms to survive. To defend themselves against potential pathogenic microbes or pests, plants rely only on innate immunity and lack specialized immune cells. Plant innate immunity employs a two-tier perception system (Dodds and Rathjen, 2010). The first layer is mediated by surface-localized pattern recognition receptors (PRRs) leading to PRR-triggered immunity (PTI). The second layer involves intracellular immune receptors, most often of the NODlike receptor (NLR) type, which directly or indirectly recognize virulence effectors secreted within host cells by pathogens, thereby inducing effector-triggered immunity (ETI). PTI relies on the perception of specific molecular patterns that are interpreted by the plant cell as danger signals. These danger signals can be either infectious non-self determinants, such as microbe- or pathogen-associated molecular patterns (MAMPs/ PAMPs), or self molecules (damage-associated molecular patterns, DAMPs) that are released upon pathogen perception or pathogen-induced cell damage (Boller and Felix, 2009). PTI comprises a wide array of responses, from cell to organism level, aimed at hampering pathogen replication and disease progression. Early cellular PTI events include the rapid generation of reactive oxygen species (ROS), the activation of mitogen-activated protein kinases (MAPKs), and the expression of immunerelated genes (Boller and Felix, 2009). PTI is sufficient to ward off most microbes, and the best demonstration of the biological relevance of PTI is the necessity for adapted successful pathogens to evade or to actively suppress this first layer of immunity in order to cause disease (Dodds and Rathjen, 2010; Dou and Zhou, 2012). Additionally, plants defective in PRRs or PTI signaling components are often more susceptible to both adapted and nonadapted pathogens (Hann and Rathjen, 2007; Lu et al., 2010; Miya et al., 2007; Roux et al., 2011; Shi et al., 2013; Tintor et al., 2013; Veronese et al., 2006; Wan et al., 2008; Zhang et al., 2010; Zipfel et al., 2006; Zipfel et al., 2004). At the cellular level, the awareness of external threats and the efficient integration of such information for an appropriate response often require robust and adaptable molecular systems. Plant immunity has become an interesting biological system to study perception of and response to biotic stresses, uncovering complex regulatory mechanisms governing signaling initiation. Several factors may account for the need of complexity in the initiation of plant immune signaling: (1) plants are sessile organisms, and as such need to mount a prompt response in situ upon menace by potential pathogens; (2) unlike animals, plants lack specialized immune cells, and therefore cellular responses to different stimuli (whether internal or external, and of biotic or abiotic nature) have to be integrated simultaneously at the cellular, tissue, and organ levels to ensure adaptation and survival; (3) immune responses are demanding in cellular resources, requiring a tight control of the initiation, timing, and amplitude of immune signaling. In recent years, it became apparent that PRRs exist within intricate protein complexes at the plasma membrane, resembling supramolecular structures, where numerous regulators are required to achieve initiation and fine-tuning of immune responses. The aim of this review is to discuss the multilayered molecular mechanisms that allow PRR complexes to perceive extracellular danger and in turn trigger intracellular immune signaling. Plant PRRs as Surface-Localized Ligand-Binding Receptors In contrast to mammals, which employ both intracellular and surface-localized PRRs to perceive PAMPs and DAMPs, all plant PRRs known so far are surface localized. Plant PRRs are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs). RLKs are composed of an ectodomain potentially involved in Molecular Cell 54, April 24, 2014 ª2014 Elsevier Inc. 263

2 Figure 1. Examples of Ligand Perception Mediated by Heterodimerization, Homodimerization, or Multimerization of Plant PRRs (A) In Arabidopsis, long chitin oligomers act as bivalent ligands, leading to the homodimerization of AtCERK1 and the generation of an active receptor complex. (B) In Arabidopsis, the flg22 peptide is perceived by the extracellular LRRs of FLS2 and causes the immediate formation of a stable heterodimer with the coreceptor BAK1. (C) In rice, a multimeric receptor formed by dimers of OsCEBiP and OsCERK1 mediates chitin binding. ligand binding, a single-pass transmembrane domain, and an intracellular kinase domain. RLPs have a similar structural organization but lack the intracellular kinase domain. PRR ectodomains can contain leucine-rich repeats (LRRs), lysine motifs (LysMs), lectin motifs, or epidermal growth factor (EGF)-like domains. LRR-type PRRs bind to proteins or peptides, such as bacterial flagellin, bacterial EF-Tu, or endogenous Pep peptides (Chinchilla et al., 2006; Sun et al., 2013b; Yamaguchi et al., 2006; Zipfel et al., 2006), while PRRs containing other domains are involved in the recognition of carbohydrate-containing molecules, such as fungal chitin, bacterial peptidoglycans, extracellular ATP, or plant-cell-wall-derived oligogalacturonides (Brutus et al., 2010; Choi et al., 2014; Kaku et al., 2006; Miya et al., 2007; Willmann et al., 2011). Plant genomes encode a plethora of RLKs and RLPs that may function as PRRs, and several PRRs have actually been shown genetically to participate in innate immunity (for comprehensive recent reviews on plant PRRs, see Boller and Felix, 2009; Wu and Zhou, 2013). However, the exact identity of matching ligands is still unknown for most PRRs. Nevertheless, recent biochemical, structural, and genetic studies have started to shed light on the molecular mechanisms underlying PAMP binding to plant PRRs (Figure 1). Homodimerization: the Example of Chitin Perception in Arabidopsis The LysM-RLK CERK1/RLK1/LYK1 from the model plant Arabidopsis thaliana (hereafter Arabidopsis) is required for chitin perception (Miya et al., 2007; Wan et al., 2008). CERK1 contains three extracellular LysM domains that bind oligomers of fungal chitin (Liu et al., 2012; Miya et al., 2007; Petutschnig et al., 2010). Long chitin oligomers (seven to eight GlcNAc residues) act as bivalent ligands, leading to the homodimerization of CERK1 and generating an active receptor complex that directly initiates chitin-induced immune signaling (Liu et al., 2012); Figure 1A). Interestingly, CERK1 can also bind shorter chitin oligomers (4-5 GlcNac residues), but their perception does not induce receptor homodimerization and does not trigger immune responses (Liu et al., 2012). Thus, chitin-induced homodimerization of CERK1 is essential for signaling initiation, most likely by bringing together CERK1 cytoplasmic domains, which contain an active kinase (Petutschnig et al., 2010), enabling intermolecular transphosphorylation. Chitin-triggered CERK1 homodimerization is reminiscent of the activation mechanism of animal receptor tyrosine kinases, where ligand-induced dimerization triggers receptor activation and initiation of downstream signaling (Lemmon and Schlessinger, 2010). Interestingly, while CERK1 is the major chitin-binding protein in Arabidopsis and is strictly required for chitin-triggered immune responses in this species (Miya et al., 2007; Petutschnig et al., 2010; Wan et al., 2008), the related LysM-RLK LYK4 can also bind chitin (Petutschnig et al., 2010) and is involved in chitin perception (Wan et al., 2012). Whether LYK4 is also activated through chitin-induced homodimerization remains to be determined. Heterodimerization: The Example of Flagellin Perception in Arabidopsis One of the best-studied plant PRRs is the Arabidopsis LRR-RLK FLS2, which recognizes bacterial flagellin via perception of the conserved 22-aminoacid epitope flg22 (Gómez-Gómez and Boller, 2000). The FLS2 ectodomain contains 28 LRRs and directly binds flg22 (Chinchilla et al., 2006). FLS2 can form homodimers even in the absence of elicitation, but such homodimerization is not required for flg22 binding, and its relevance is unclear (Sun et al., 2012, 2013b). FLS2 is conserved in most plant species (Boller and Felix, 2009), but differences in recognition specificities exist. For example, the shorter peptide flg15 is an agonist in tomato, while it acts as an antagonist in Arabidopsis (Bauer et al., 2001; Felix et al., 1999; Mueller et al., 2012; Robatzek et al., 2007). Comparative analysis coupled with mutagenesis studies on both the FLS2 LRRs and flg peptides have defined potential LRRs involved in flg22 recognition (Dunning et al., 2007; Helft et al., 2011; Mueller et al., 2012). Furthermore, these studies have revealed that flg22 perception engages an address message -type of recognition, with the N-terminal part of flg22 being required for receptor binding and the C-terminal part being required for activation of immune responses (Meindl et al., 2000; Sun et al., 2013b). Interestingly, FLS2 forms a complex with the regulatory LRR- RLK BAK1/SERK3 quasi-instantaneously upon flg22 perception (Chinchilla et al., 2007; Schulze et al., 2010); Figure 1B), suggesting that FLS2 and BAK1 already exist in close proximity in the plasma membrane. A recent structural study based on a cocrystal of the ectodomains of FLS2 and BAK1 in complex with flg22 revealed the mechanisms underlying flg22 perception by FLS2 264 Molecular Cell 54, April 24, 2014 ª2014 Elsevier Inc.

3 (Sun et al., 2013b). Flg22 binds to the concave surface of the FLS2 superhelical ectodomain spanning 14 LRRs (LRR3-16). The flg22-bound FLS2 ectodomain directly interacts with the BAK1 ectodomain, with the C-terminal region of the FLS2-bound flg22 stabilizing the FLS2-BAK1 dimerization by acting as molecular glue between the two ectodomains. Thus, the FLS2-BAK1 heterodimerization is both ligand and receptor mediated. Hence, while BAK1 is not required for overall flg22 binding to FLS2 (Chinchilla et al., 2007), it acts as a coreceptor for flg22 that is essential for signaling activation (Sun et al., 2013b; see below for discussion). Notably, a similar activation mechanism has been recently reported for the Arabidopsis LRR-RLK BRI1 (the receptor for the plant hormone brassinosteroid, which regulates growth and development) and BAK1 or the BAK1-related protein SERK1 (Santiago et al., 2013; Sun et al., 2013a). This suggests that many (if not all) LRR-containing RLKs (and RLPs) may engage in similar heterodimeric complexes with BAK1 or related SERK proteins. The growing number of LRR-RLKs and LRR-RLPs acting as PRRs shown to associate with BAK1 and related SERK proteins, and depending on them for full activity (Liebrand et al., 2013b), supports this hypothesis. It is of note that some mechanistic differences may exist between plant species, as a rice (Oryza sativa) ortholog of BAK1, OsSERK2, forms a constitutive complex with the LRR-RLK XA21, which confers resistance to the bacterium Xanthomonas oryzae pv. oryzae (Chen et al., 2014). However, the absence of confirmed ligand for XA21 (Bahar et al., 2014) currently precludes testing if the XA21-OsSERK2 interaction would be enhanced by PAMP perception. In addition, while FLS2-BAK1 dimerization occurs independently of kinase activity and of the presence of the intracellular domains (Schwessinger et al., 2011; Sun et al., 2013b), the association of OsSERK2 with the intracellular domains of ligand-binding receptors seems to be kinase activity dependent (Chen et al., 2014). Also, the tomato ortholog of BAK1 interacts with the tomato LRR-RLP Eix1, and allows the negative regulation it exerts on the signaling mediated by the related LRR-RLP Eix2 that perceives fungal xylanase (Bar et al., 2010). The molecular mechanisms underlying this phenomenon are still unknown. Heteromultimerization: The Example of Chitin Perception in Rice In rice, the major chitin-binding protein is the LysM-RLP CEBiP (Kaku et al., 2006; Kouzai et al., 2014). CEBiP is a predicted GPI-anchored protein with three extracellular LysM domains and a C-terminal tail (Hayafune et al., 2014; Kaku et al., 2006). It has been recently shown that OsCEBiP homodimerizes to bind long chitin oligomers (Hayafune et al., 2014), which resembles the chitin-binding mechanism shown for the Arabidopsis chitin receptor AtCERK1 (Liu et al., 2012; Figure 1A). However, as with other RLPs, the absence of obvious known signaling motifs in the C-terminal region of CEBiP suggested that it must function in cooperation with additional proteins to initiate signaling (Kaku et al., 2006). Indeed, CEBiP forms a hetero-oligomeric receptor complex with OsCERK1, the rice ortholog of AtCERK1, in the presence of biologically active chitin (Shimizu et al., 2010; Figure 1C). Unlike its Arabidopsis ortholog, OsCERK1 has a single extracellular LysM domain and does not bind chitin, but is required for chitin-mediated signaling (Shimizu et al., 2010). These findings show that the chitin perception system in rice is significantly different to the one in Arabidopsis, and requires a hetero-oligomeric receptor complex formed by dimers of an elicitor-binding LysM-RLP (CEBiP) and a nonligand-binding signaling-active LysM-RLK (OsCERK1) (Kaku et al., 2006; Shinya et al., 2012), which form a sandwich-type receptor for chitin oligomers (Hayafune et al., 2014; Figure 1C). In addition to CEBiP, the LysM-RLPs OsLYP4 and OsLYP6 also bind chitin and are involved in chitin responsiveness (Liu et al., 2012). Interestingly, CEBiP orthologs in Arabidopsis do not seem required for classical chitin immune responses, such as ROS burst or immune gene expression (Shinya et al., 2012; Wan et al., 2012). Nevertheless, the closest ortholog of CEBiP in Arabidopsis, AtLYM2, is able to bind chitin (Petutschnig et al., 2010; Shinya et al., 2012). Indeed, AtLYM2 localizes to the plasmodesmata (symplastic connections between neighboring cells) and is involved in CERK1-independent chitin-induced plasmodesmata closure and resistance to fungal pathogens (Faulkner et al., 2013; Narusaka et al., 2013). Therefore, it is possible that some localized cellular responses triggered by chitin in Arabidopsis also involve hetero-oligomerization between a chitin-binding LysM-RLP (AtLYM2) and a yet-unknown LysM-RLK potentially related to CERK1. In addition to chitin, the CEBiP paralogs OsLYP4 and OsLYP6 also bind peptidoglycan (PGN), a major component of bacterial cell walls (Liu et al., 2012). In Arabidopsis, two other orthologs of CEBiP, AtLYM1 and AtLYM3, specifically bind PGN, but not chitin (Willmann et al., 2011). PGN-induced responses in Arabidopsis also require CERK1, which does not bind PGN itself (Willmann et al., 2011), showing that CERK1 is a multifaceted RLK, able to function as a ligand-binding PRR for chitin, and as a positive regulator of PGN responses. While a complex between AtLYM1, AtLYM3, and AtCERK1 has not yet been confirmed biochemically, these observations suggest that the PGN perception system in Arabidopsis resembles that of the rice chitin receptor, involving a combination of ligand-binding RLPs and a signaling RLK in a hetero-oligomeric complex. Roles of PRR-Associated RLKs in the Activation of PRR Complexes As summarized above, most PRRs either contain a kinase domain or associate with RLKs. The paradigm of signaling activation by receptor kinases would imply that ligand binding by the extracellular domain causes the activation of the intracellular kinase domain and the phosphorylation of substrates that contribute to intracellular signal transduction. However, as mentioned before, the LRR-RLKs identified to date still require dimerization with BAK1 (or a related SERK protein) to transduce the signal, even though they have an intracellular kinase domain with the potential for signaling (Figure 2). Most plant RLK PRRs (e.g., FLS2, EFR, XA21) are non-rd kinases, having another amino acid in place of an otherwise conserved arginine residue in the catalytic loop of the kinase domain, whereas BAK1 is an RD kinase (Dardick et al., 2012). Puzzlingly, non-rd kinases seem associated with immune functions across kingdoms, and the association between non-rd Molecular Cell 54, April 24, 2014 ª2014 Elsevier Inc. 265

4 Figure 2. Examples of Arabidopsis and Rice PRR Complexes (A) In Arabidopsis, flg22 perception triggers the phosphorylation of the cytoplasmic domains of FLS2 and BAK1, as well as the RLCK BIK1. Activated BIK1 gets released from the receptor complex, leading to phosphorylation and activation of the NADPH oxidase AtRBOHD. Flg22-triggered ROS burst additionally requires the RLCK BSK1 and the endocytosis regulator SCD1. Likewise, BIK1 is required for the ROS burst triggered by the chitin-induced activation of AtCERK1. In both cases, it is unclear how PRR activation leads to the activation of MAPKs and other downstream substrates. (B) In rice, XA21 requires the E3 ligase XB3 and the PANK protein XB25 for stability. Additionally, it forms a constitutive complex with OsSERK2, although the mechanisms of signal transduction upon perception of Xanthomonas oryzae pv. oryzae (Xoo) remain unknown. The multimeric chitin receptor complex formed by OsCEBiP and OsCERK1 associates with several cytoplasmic proteins that are required for signal transduction. After chitin perception, OsCERK1 phosphorylates OsRLCK185, which in turn gets released from the receptor complex and contributes to the activation of MAPKs. Additionally, OsCERK1 activates the Os- RacGEF1/OsRac1 module, which mediates the activation of chitin-induced ROS burst. and RD kinases for the initiation of PTI has also been observed in Drosophila and humans (for a review, see Dardick et al., 2012). Since the kinase activity detected in FLS2 or EFR kinase domains is considerably weaker than that of other RD kinases (e.g., BAK1 or BRI1) (Schwessinger et al., 2011), it has been hypothesized that non-rd ligand-binding RLK PRRs could require the association with a strong RD kinase (such as BAK1) to boost phosphorylation in the receptor complex and initiate signaling (Dardick et al., 2012). Consistent with this hypothesis, mutations that impair complex formation between FLS2 and BAK1 abolish the phosphorylation of both proteins and the initiation of downstream signaling (Sun et al., 2013b). Additionally, the kinase activities of both FLS2 and EFR are required for flg22- and elf18-triggered responses (Cao et al., 2013; Schwessinger et al., 2011). In contrast, CERK1 contains an RD kinase domain and does not require BAK1 to initiate chitin-triggered signaling in Arabidopsis (Gimenez-Ibanez et al., 2009; Heese et al., 2007). However, signaling mediated by RD RLKs, such as BRI1 or the PRRs PEPR1/PEPR2, still relies on BAK1 (and other SERKs) (Gou et al., 2012; Krol et al., 2010; Li et al., 2002a; Nam and Li, 2002; Roux et al., 2011), suggesting that SERK proteins are critical for the function of both RD and non-rd ligand-binding RLKs. In addition to their role as kinase activity enhancers, SERK proteins could generate specific phosphorylation events in cis and trans within PRR complexes regulating interaction with specific substrates and activation of specific signaling branches leading to different downstream responses, similar to the observed generation of docking sites for downstream substrates of animal receptor kinases driven by phosphorylation on specific residues (Lemmon and Schlessinger, 2010). Consistent with this hypothesis, while BAK1 forms a complex with both BRI1 (regulating growth) and FLS2 (regulating immunity), BAK1 contributes to signaling specificity in a phosphorylation-dependent manner (Schwessinger et al., 2011). Further work remains necessary to understand the sequence and nature of the phosphorylation events triggered after ligand perception and how they contribute to signaling initiation and specificity. RLPs, which lack intracellular signaling domains, depend on the association with kinases for signaling. As such, LRR-type and LysM-type RLPs acting as PRRs have been shown to associate with BAK1 (or other SERKs) and CERK1, respectively, which can fulfill here the role of signaling kinase domains activated upon ligand perception. Interestingly, in addition to SERK proteins, the tomato LRR-RLK SOBIR1 was recently found to associate with several LRR-RLP PRRs, such as Eix2, Ve1, and Cf4 (Liebrand et al., 2013a). SOBIR1 is required for the accumulation of Cf4 and Ve1, and, therefore, silencing of SOBIR1 expression compromises Cf4- and Ve1-mediated responses (Liebrand et al., 2013a). Because SOBIR1 localizes to mobile cytoplasmic vesicles, it has been proposed that SOBIR1 may be necessary for adequate trafficking and, consequently, stability of LRR-RLP-containing complexes (Liebrand et al., 2013b). Interestingly, Arabidopsis SOBIR1 is also required for the function of several RLPs involved in innate immunity (Jehle et al., 2013; Zhang et al., 2013), suggesting that SOBIR1 is a common regulator of LRR-RLP PRRs in different plant species. While it is still unclear how SOBIR1 regulates LRR-RLP accumulation, it is worth noting that the rice PRR XA21 (an LRR-RLK) seems to be also subjected to a phosphorylation-dependent mechanism controlling protein stability, requiring autophosphorylation of several Ser and Thr amino acids (Xu et al., 2006). In 266 Molecular Cell 54, April 24, 2014 ª2014 Elsevier Inc.

5 addition, several nonkinase proteins that associate with XA21 are required for its accumulation. The ubiquitin ligase XB3 and the plant-specific ankyrin-repeat (PANK) protein XB25 interact with XA21 in planta and are transphosphorylated by XA21 kinase domain in vitro (Jiang et al., 2013; Wang et al., 2006; Figure 2B). Reduced expression of XB3 or XB25 results in reduced accumulation of XA21 and, consequently, compromised XA21-mediated immunity (Jiang et al., 2013; Wang et al., 2006). Additional RLKs can be found as part of PRR complexes. For example, the LRR-RLK BIR2 was recently found to interact with BAK1 in the absence of PAMP perception (Halter et al., 2014). BIR2 is a pseudokinase that negatively regulates BAK1 interaction with FLS2. Flg22 perception leads to the dissociation of BIR1 from BAK1, allowing FLS2-BAK1 dimerization and signaling initiation (Halter et al., 2014; Figure 3A). Lectin receptor kinases (LecRKs) are also emerging as potential components and regulators of PRR complexes (Singh and Zimmerli, 2013). LecRK-VI.2 was found to be differentially required for the activation of distinct signaling components downstream of the FLS2 complex after flg22 perception (Singh et al., 2012), and LecRK-I.9 is required for flg22-induced callose deposition (Bouwmeester et al., 2011). These results suggest that these LecRKs may exist in complex with FLS2 or associated proteins. However, the genetic contribution of these LecRKs to FLS2-mediated responses may also be indirect. Indeed, LecRK-I.9, for example, was recently identified as a PRR for extracellular ATP (Choi et al., 2014), which may be considered a DAMP released upon cell rupture. DAMP perception may be part of a PTI amplification loop, as recently demonstrated for the perception of AtPep peptides by PEPR1/2 in Arabidopsis (Liu et al., 2013; Ma et al., 2012; Tintor et al., 2013; Zipfel, 2013). Link with Downstream Events: RLCKs as Direct Substrates of PRR Complexes PRRs and associated transmembrane proteins require cytoplasmic partners to link PRR activation with downstream intracellular signaling. In recent years, receptor-like cytoplasmic kinases (RLCKs) have emerged as direct substrates of PRR complexes and key positive regulators of PTI signaling (Figure 2). The Arabidopsis RLCK BIK1 associates with FLS2 and BAK1 in the absence of flg22 (Lu et al., 2010; Zhang et al., 2010). After flg22 perception, BAK1 phosphorylates BIK1, which then phosphorylates both FLS2 and BAK1, and dissociates from the FLS2- BAK1 complex (Lu et al., 2010; Zhang et al., 2010; Figure 2A). BIK1 is also phosphorylated after elf18 perception and interacts with EFR and CERK1 (Lu et al., 2010; Zhang et al., 2010). Accordingly, bik1 mutants are compromised in specific immune responses triggered by flg22, elf18, and chitin, such as the rapid ROS burst and PAMP-induced defense gene expression, and are more susceptible to infection by the pathogenic fungus Botrytis cinerea and nonadapted bacterial pathogens (Laluk et al., 2011; Lu et al., 2010; Veronese et al., 2006; Zhang et al., 2010). Furthermore, BIK1 also interacts with PEPR1 and participates in an amplification mechanism of PTI involving the gaseous hormone ethylene and some responses triggered by ethylene itself (Laluk et al., 2011; Liu et al., 2013; Tintor et al., 2013; Zipfel, 2013). These results highlight the important role of BIK1 for the activation of PRR complexes that mediate Arabidopsis innate immunity. BIK1 is part of the large multigenic RLCK-VII subfamily that contains 46 members (Zhang et al., 2010), and at least PBL1, PBL2, and PBL5 from this family can also regulate flg22-induced ROS burst (Liu et al., 2013; Zhang et al., 2010). Interestingly, BIK1 and PBL1 are not required for flg22-induced activation of MAPKs (Feng et al., 2012). However, flg22-triggered MAPK activation was reduced upon expression of the bacterial uridine 5 0 -monophosphate transferase AvrAC, which inhibits phosphorylation in conserved residues located in the activation loop of BIK1 and related kinases (Feng et al., 2012). This suggests that additional BIK1-related proteins, potentially PBL2 and PBL5, may be required for this response. In rice, OsRLCK185 is a substrate of OsCERK1, and regulates chitin- and PGN-induced immune responses (Yamaguchi et al., 2013). OsCERK1 phosphorylates OsRLCK185, which partially dissociates from the OsCERK1 complex after chitin perception (Yamaguchi et al., 2013; Figure 2B). The RLCK BSK1 was initially identified as a substrate of the brassinosteroid receptor BRI1, acting as a positive regulator of brassinosteroid responses (Tang et al., 2008). Recently, BSK1 was found to also associate with FLS2, partially dissociating after flg22 perception, and to be required for a subset of flg22- triggered responses, but not MAPK activation (Shi et al., 2013; Figure 2A). Interestingly, while BSK1 seems a common positive regulator of both BRI1- and PRR-mediated signaling, BIK1 also interacts with BRI1, but in this case acts as a negative regulator of brassinosteroid-triggered responses (Lin et al., 2013). Upon brassinosteroid perception, BRI1 phosphorylates BIK1 independently of BAK1, triggering the release of BIK1 from the BRI1 receptor complex (Lin et al., 2013). Importantly, although an antagonism exists between the BRI1 and FLS2 pathways (Albrecht et al., 2012; Belkhadir et al., 2012), this does not appear to be caused by a competition between these common regulators at the plasma membrane (Albrecht et al., 2012; Gro Malinovsky et al., 2014; Lozano-Duran et al., 2013), but rather is driven by an indirect crosstalk mediated by the key transcriptional regulator BZR1 (Gro Malinovsky et al., 2014; Lozano-Duran et al., 2013). Interestingly, different PRRs seem to recruit distinct RLCKs. Indeed, FLS2-mediated ROS burst depends on BIK1, PBL1, PBL2, and PBL5, while only BIK1 and PBL1 are involved in the ROS burst triggered by PEPR1/2 activation (Liu et al., 2013; Zhang et al., 2010). Similarly, BSK1 is involved in flg22- but not elf18-triggered ROS burst (Shi et al., 2013). Also, the requirement of different RLCKs for specific PTI responses suggests that the choice of specific RLCKs as PRR substrates constitutes another layer in the regulation of signaling branching from PRR complexes (Figure 2). The PAMP-induced ROS burst and MAPK activation are appealing responses to study signaling branching from PRR complexes, since both responses are detectable rapidly after PAMP treatment (<5 min) and constitute independent signaling events (Ranf et al., 2011; Segonzac et al., 2011; Xu et al., 2013). The prominent role of RLCKs in the initiation and specificity of PTI signaling has also prompted the search for RLCK substrates. Interestingly, we have recently identified the NADPH oxidase Molecular Cell 54, April 24, 2014 ª2014 Elsevier Inc. 267

6 Figure 3. Negative Regulation of PRR Complexes (A) In Arabidopsis, BIR2 interacts with BAK1 in the absence of elicitor, thus inhibiting FLS2-BAK1 heterodimerization. Moreover, phosphatases and other potential negative regulators target kinases within the PRR complex. Flg22 binding triggers the release of BIR2 and the dissociation or inactivation of phosphatases, allowing FLS2-BAK1 heterodimerization and complex activation. (B) In rice, the ATPase XB24 keeps XA21 in an inactive state by promoting phosphorylation of specific XA21 residues. After ligand binding, XB24 is released and the XA21 complex gets activated. Subsequently, the phosphatase XB15 interacts specifically with activated XA21, dephosphorylating it and attenuating XA21-mediated immune responses. (C) In Arabidopsis, the formation of the receptor complex triggers the recruitment of the E3 ligases PUB12/13 alongside BAK1. Activated BAK1 phosphorylates PUB12/13, and those in turn promote ubiquitination (Ub) of FLS2 and target it for degradation. Additionally, FLS2 is endocytosed and targeted for degradation, potentially with the contribution of SCD1. AtRBOHD as a direct target of BIK1 (Kadota et al., 2014). AtRBOHD is the main enzyme responsible for the rapid production of apoplastic ROS upon PAMP perception (Nühse et al., 2007; Zhang et al., 2007). Upon flg22 treatment, BIK1 gets activated and directly phosphorylates AtRBOHD residues that are essential for AtROBHD activation, ROS burst, and subsequent immunity (Kadota et al., 2014; Figure 2A). The identification of additional RLCK substrates will be essential to understand how activation of PRR complexes is linked to the execution of PTI responses. In addition to RLCKs, other substrates of PRR complexes have been found to play a role in PTI signaling. In rice, chitin treatment leads to OsCERK1 phosphorylation of the Rac GDP/GTP exchange factor 1 (OsRacGEF1), which in turn activates OsRac1 (Akamatsu et al., 2013; Figure 2B). Coupled to the activation of the OsCEBiP/OsCERK1 complex activation, the OsRacGEF1/OsRac1 module is required for chitin-triggered responses and resistance to fungal pathogens in rice (Akamatsu et al., 2013; Ono et al., 2001; Suharsono et al., 2002; Figure 2B). Power Is Nothing without Control: Negative Regulation of PRR Complexes PRR complexes are powerful molecular machines capable of initiating responses that readjust the cellular dynamics, diverting resources toward immunity. This compels a scrupulous control of immune signaling activation and a prompt switch-off when pathogen threat is over. Regulation mechanisms should also ensure that PRR complexes return to a steady state, ready to get activated in case of further pathogen attack. Given the key role of phosphorylation for the activation of PRR complexes, negative regulation of PRR complexes can be mediated by protein phosphatases (Figure 3). Several phosphatases have been shown to associate with PRRs and/or associated kinases to keep the complexes inactive through dephosphorylation in the absence of ligand binding. Early work identified the protein phosphatase 2C (PP2C) KAPP as an FLS2-interacting protein that negatively regulates flg22-triggered responses (Gómez-Gómez et al., 2001; Figure 3A). KAPP actually associates with numerous plant RLKs (Ding et al., 2007), but its role is still unclear. Notably, additional phosphatases start to emerge as part of PRR complexes. In rice, the PP2C XB15 associates with activated XA21 after pathogen perception, leading to its dephosphorylation and inactivation (Park et al., 2008; Figure 3B). XB15 interaction with XA21 is phosphorylation dependent, suggesting that the affinity of XB15 may rely on the presence of potential phosphorylation-mediated docking sites in the activated XA21 (Park et al., 2008). Further work will be required to determine if PRRassociated kinases are also targeted by phosphatases. The rice ATPase XB24 provides another mean to regulate the phosphorylation status of a PRR. XB24 interacts with XA21 and uses ATP to promote the phosphorylation of specific Ser and Thr sites on XA21, keeping it in an inactive and steady state (Chen et al., 2010; Figure 3B). XB24 dissociates from XA21 after pathogen recognition, allowing XA21 activation and immune responses (Chen et al., 2010). 268 Molecular Cell 54, April 24, 2014 ª2014 Elsevier Inc.

7 An important regulatory aspect of plasma-membrane ligandbinding RLKs is their degradation after ligand binding and consequent activation to enable the de novo replenishment of ligand-free receptors at the plasma membrane. For example, FLS2 is subject to endocytosis and degraded after flg22 perception (Lu et al., 2011; Robatzek et al., 2006; Smith et al., 2014; Figure 3C). FLS2 degradation seems to be controlled by the E3-ubiquitin ligases PUB12 and PUB13, which exist in a constitutive complex with BAK1 and are therefore recruited into the FLS2 complex upon flg22 binding (Lu et al., 2011; Figure 3C). PUB12 and PUB13 are phosphorylated by BAK1 and in turn polyubiquitinate FLS2, leading to FLS2 degradation (Lu et al., 2011). Whether PUB12 and PUB13 play a role in FLS2 endocytosis itself is currently unknown. It has been hypothesized that the ligand-induced endocytosis and degradation of FLS2 may serve to prevent continuous signaling from activated receptors at the plasma membrane (Smith et al., 2014). Accordingly, newly synthesized FLS2 is incorporated into the plasma membrane at later times, restoring the sensitivity of the cell to additional or subsequent pathogen attacks (Smith et al., 2014). Appropriate PRR trafficking after ligand perception may also be required for specific signaling responses, in a manner regulated by specific components of the PRR complex. For example, the DENN domain protein SCD1 was found recently to function in clathrin-mediated endocytosis during cytokinesis and cell expansion (McMichael et al., 2013). SCD1 also interacts with FLS2 and is required for a subset of flg22-triggered responses, including ROS burst (Korasick et al., 2010). Together with the observation that chemical inhibition of vesicular trafficking impairs flg22-triggered ROS burst (Smith et al., 2014), these reports suggest a potential link between endocytosis and the activation of early flg22- triggered responses. Moreover, after flg22 perception, FLS2 colocalizes with subunits of the endosomal sorting complex required for transport (ESCRT)-I, which is required for flg22- triggered stomatal responses (Spallek et al., 2013), suggesting that an adequate FLS2 endosomal sorting is important for FLS2-mediated stomatal immunity. It is also formally possible that chemical or genetic alterations of the overall endocytic pathways in plant cells affect some PTI responses in a direct or indirect manner. Concluding Remarks Plant PRR immune complexes are emerging as extremely complex signaling molecular machines. A simplistic scenario for the perception of danger signals would involve a transmembrane receptor that binds a ligand and initiates signaling autonomously. However, the most likely scenario involves, at least, coreceptors, regulatory proteins, substrates that link PRR activation to the induction of early signaling components, and negative regulators, suggesting that we are just scratching on the surface of extremely complex regulatory systems. Phosphorylation has been shown to drive the initiation of signaling from PRRs, but the activation mechanisms of PRR kinases and the specific phosphorylation events that mediate signaling initiation, interaction with downstream substrates, and subsequent signaling remain unknown. It will be important to decipher whether phosphorylation at specific sites is key for the recruitment or dissociation of specific signaling components to the receptor complex and/or to determine the fate of the activated PRRs, as previously shown in animal receptor kinases (Lemmon and Schlessinger, 2010). In such a scenario, specific phosphorylated sites would therefore regulate directly or indirectly the branching of signaling from the PRR complex. Biochemical evidences show dynamic association of several proteins within PRR complexes. In that sense, PRR complexes resemble supramolecular structures comprised of several interacting proteins, although there is currently no biochemical proof that all these proteins are part of a single dynamic megacomplex or belong to multiple smaller complexes within a single cell. Moreover, some interactions may be extremely dynamic, and thus different components of PRR complexes may not physically interact simultaneously. In addition, the expression patterns of the different proteins comprising PRR complexes at the cell, tissue, or organ level are not necessarily known. In conclusion, understanding the basis of PRR complex formation, organization, activation, and the subsequent connection to downstream signaling networks leading to actual immunity will be a key challenge for the future. ACKNOWLEDGMENTS We apologize to colleagues whose work could not be cited because of space limitations. Work on immune signaling in the Zipfel laboratory is funded by the Gatsby Charitable Foundation, the European Research Council, and the United Kingdom Biotechnology and Biological Sciences Reseach Council. A.P.M. was funded by a postdoctoral fellowship from the Federation of European Biochemical Societies (FEBS). We thank Rosa Lozano-Durán for critical reading of the manuscript and all members of the Zipfel laboratory for stimulating discussions and helpful comments. 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