Molecular Cell

Review

Plant PRRs and the Activation

of Innate Immune Signaling
Alberto P. Macho1 and Cyril Zipfel1,*
1The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, UK

*Correspondence: cyril.zipfel@tsl.ac.uk
http://dx.doi.org/10.1016/j.molcel.2014.03.028

Despite being sessile organisms constantly exposed to potential pathogens and pests, plants are surpris-
ingly 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 acti-
vation 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 percep-
tion system (Dodds and Rathjen, 2010). The first layer is medi-
ated by surface-localized pattern recognition receptors (PRRs)
leading to PRR-triggered immunity (PTI). The second layer in-
volves intracellular immune receptors, most often of the NOD-
like 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 pat-
terns, 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 pro-
gression. Early cellular PTI events include the rapid generation of
reactive oxygen species (ROS), the activation of mitogen-acti-
vated protein kinases (MAPKs), and the expression of immune-
related 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 patho-
gens 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 adapt-
ed 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 organ-
isms, 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 sur-
vival; (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 mem-
brane, 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 sur-
face-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

ligand binding, a single-pass transmembrane domain, and an
intracellular kinase domain. RLPs have a similar structural orga-
nization but lack the intracellular kinase domain. PRR ectodo-
mains 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 mole-
cules, such as fungal chitin, bacterial peptidoglycans, extracel-
lular 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 biochem-
ical, 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 Arabi-
dopsis 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 olig-
omers (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 homodimeriza-
tion 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 intermolec-
ular transphosphorylation. Chitin-triggered CERK1 homodimeri-
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Molecular Cell
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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 re-
ceptor 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.
zation is reminiscent of the activation mechanism of animal
receptor tyrosine kinases, where ligand-induced dimerization
triggers receptor activation and initiation of downstream sig-
naling (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 acti-
vated 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 Bol-
ler, 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 specific-
ities 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-termi-
nal 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), suggest-
ing that FLS2 and BAK1 already exist in close proximity in the
plasma membrane. A recent structural study based on a cocrys-
tal of the ectodomains of FLS2 and BAK1 in complex with flg22
revealed the mechanisms underlying flg22 perception by FLS2
Molecular Cell
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(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 re-
ported 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 be-
tween 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 pre-
cludes 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 phe-
nomenon 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 resem-
bles 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 sig-
naling (Kaku et al., 2006). Indeed, CEBiP forms a hetero-oligo-
meric 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 percep-
tion system in rice is significantly different to the one in Arabidop-
sis, and requires a hetero-oligomeric receptor complex formed
by dimers of an elicitor-binding LysM-RLP (CEBiP) and a non-
ligand-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 Ara-
bidopsis, AtLYM2, is able to bind chitin (Petutschnig et al., 2010;
Shinya et al., 2012). Indeed, AtLYM2 localizes to the plasmodes-
mata (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 Arabi-
dopsis also require CERK1, which does not bind PGN itself (Will-
mann et al., 2011), showing that CERK1 is a multifaceted RLK,
able to function as a ligand-binding PRR for chitin, and as a pos-
itive regulator of PGN responses. While a complex between
AtLYM1, AtLYM3, and AtCERK1 has not yet been confirmed bio-
chemically, 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 acti-
vation 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 func-
tions across kingdoms, and the association between non-RD
Molecular Cell 54, April 24, 2014 ª2014 Elsevier Inc. 265
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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 do-
mains is considerably weaker than that of other RD kinases
(e.g., BAK1 or BRI1) (Schwessinger et al., 2011), it has been hy-
pothesized 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 initi-
ation 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; Schwes-
singer 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 crit-
ical for the function of both RD and non-RD ligand-binding RLKs.
In addition to their role as kinase activity enhancers, SERK pro-
teins 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 gen-
eration of docking sites for downstream substrates of animal re-
ceptor kinases driven by phosphorylation on specific residues
(Lemmon and Schlessinger, 2010). Consistent with this hypoth-
esis, 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 asso-
ciate with BAK1 (or other SERKs) and CERK1, respectively,
which can fulfill here the role of signaling kinase domains acti-
vated 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 re-
sponses (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 accu-
mulation, 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 autophosphor-
ylation of several Ser and Thr amino acids (Xu et al., 2006). In

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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 accumu-
lation 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 interac-
tion with FLS2. Flg22 perception leads to the dissociation of
BIR1 from BAK1, allowing FLS2-BAK1 dimerization and sig-
naling initiation (Halter et al., 2014; Figure 3A).
Lectin receptor kinases (LecRKs) are also emerging as poten-
tial 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 associ-
ated 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 percep-
tion may be part of a PTI amplification loop, as recently demon-
strated 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 cyto-
plasmic partners to link PRR activation with downstream intra-
cellular 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 phos-
phorylates 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). Accord-
ingly, bik1 mutants are compromised in specific immune re-
sponses 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 partic-
ipates 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 Arabi-
dopsis 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
50 -monophosphate transferase AvrAC, which inhibits phosphor-
ylation 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 indepen-
dently 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 regula-
tors at the plasma membrane (Albrecht et al., 2012; Gro Malinov-
sky et al., 2014; Lozano-Duran et al., 2013), but rather is driven
by an indirect crosstalk mediated by the key transcriptional regu-
lator 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 com-
plexes (Figure 2). The PAMP-induced ROS burst and MAPK acti-
vation are appealing responses to study signaling branching
from PRR complexes, since both responses are detectable
rapidly after PAMP treatment (<5 min) and constitute indepen-
dent 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

AtRBOHD as a direct target of BIK1 (Kadota et al., 2014).
AtRBOHD is the main enzyme responsible for the rapid produc-
tion of apoplastic ROS upon PAMP perception (Nühse et al.,
2007; Zhang et al., 2007). Upon flg22 treatment, BIK1 gets acti-
vated 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 acti-
vates 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 (Aka-
matsu 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
268 Molecular Cell 54, April 24, 2014 ª2014 Elsevier Inc.
Molecular Cell
Review
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 hetero-
dimerization 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, dephosphorylat-
ing 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 pro-
mote ubiquitination (Ub) of FLS2 and target it for
degradation. Additionally, FLS2 is endocytosed
and targeted for degradation, potentially with the
contribution of SCD1.
resources toward immunity. This com-
pels 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 pro-
tein 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 phos-
phatase 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 com-
plexes. 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 af-
finity of XB15 may rely on the presence of potential phosphory-
lation-mediated docking sites in the activated XA21 (Park
et al., 2008). Further work will be required to determine if PRR-
associated 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 path-
ogen recognition, allowing XA21 activation and immune re-
sponses (Chen et al., 2010).
Molecular Cell
Review
An important regulatory aspect of plasma-membrane ligand-
binding 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 percep-
tion (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 consti-
tutive 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 endocy-
tosis 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 com-
plex signaling molecular machines. A simplistic scenario for the
perception of danger signals would involve a transmembrane re-
ceptor 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 regula-
tors, 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, interac-
tion with downstream substrates, and subsequent signaling
remain unknown. It will be important to decipher whether phos-
phorylation at specific sites is key for the recruitment or dissoci-
ation 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 inter-
acting proteins, although there is currently no biochemical proof
that all these proteins are part of a single dynamic ‘‘megacom-
plex’’ 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 Euro-
pean Biochemical Societies (FEBS). We thank Rosa Lozano-Durán for critical
reading of the manuscript and all members of the Zipfel laboratory for stimu-
lating discussions and helpful comments.
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