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Structure, biochemical function, and signaling
mechanism of plant NLRs
Jizong Wang
1,5,
*, Wen Song
3,4,
*and Jijie Chai
2,3,4,
*
1
State Key Laboratory of Protein and Plant Gene Research, School of Advanced Agricultural Sciences, Peking University, Beijing 100871, China
2
Tsinghua-Peking Joint Center for Life Sciences, Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China
3
Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany
4
Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany
5
Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences at Weifang, Weifang, Shandong 261000,
China
*Correspondence: Jizong Wang (wangjizong@pku.edu.cn), Wen Song (wesong@mpipz.mpg.de), Jijie Chai (chai@mapipz.mpg.de)
https://doi.org/10.1016/j.molp.2022.11.011
ABSTRACT
To counter pathogen invasion, plants have evolved a large number of immune receptors, including
membrane-resident pattern recognition receptors (PRRs) and intracellular nucleotide-binding and
leucine-rich repeat receptors (NLRs). Our knowledge about PRR and NLR signaling mechanisms has
expanded significantly over the past few years. Plant NLRs form multi-protein complexes called resisto-
somes in response to pathogen effectors, and the signaling mediated by NLR resistosomes converges
on Ca
2+
-permeable channels. Ca
2+
-permeable channels important for PRR signaling have also been iden-
tified. These findings highlight a crucial role of Ca
2+
in triggering plant immune signaling. In this review, we
first discuss the structural and biochemical mechanisms of non-canonical NLR Ca
2+
channels and then
summarize our knowledge about immune-related Ca
2+
-permeable channels and their roles in PRR and
NLR signaling. We also discuss the potential role of Ca
2+
in the intricate interaction between PRR and
NLR signaling.
Key words: plant immunity, PRR, NLR, resistosome, Ca
2+
-permeable channels, Ca
2+
signaling, second
messenger
Wang J., Song W., and Chai J. (2023). Structure, biochemical function, and signaling mechanism of plant NLRs.
Mol. Plant. 16, 1–21.
INTRODUCTION
The plant immune system mainly relies on two types of receptors
to mediate immune responses. One type is cell-surface-located
pattern recognition receptors (PRRs) sensing the conserved
signatures of invading pathogens, called pathogen-associated
molecular patterns (PAMPs) or host-derived damage-associated
molecular patterns (DAMPs), to initiate pattern-triggered immu-
nity (PTI) (Yu et al., 2017;DeFalco and Zipfel, 2021). The other
type is intracellular nucleotide-binding (NB), leucine-rich repeat
(LRR) receptors (NLRs). Plant NLRs mediate direct or indirect
recognition of race-specific pathogen effectors delivered into
plant cells, initiating effector-triggered immunity (ETI) (Cui et al.,
2015;Jones et al., 2016;Zhou and Zhang, 2020). Despite
their different structures and subcellular localizations, PRRs
and NLRs share a suite of downstream defense responses,
including Ca
2+
influx; bursts of reactive oxygen species (ROS);
activation of mitogen-activated protein kinase (MAPK) cascades;
production of phytocytokines and defense hormones, including
salicylic acid (SA) and ethylene; and massive transcriptional re-
programming (Ngou et al., 2022;Figure 1). Probably because of
their similarity, PTI and ETI are tightly connected and mutually
potentiate (Ngou et al., 2021;Yuan et al., 2021). However, PTI
and ETI differ in timing, amplitude, and duration of defense.
Compared with PTI, ETI involves prolonged and more robust
immune responses and is frequently accompanied by a
hypersensitive response (HR), a form of localized programmed
cell death associated with pathogen restriction or killing. The
pathogen resistance and cell death activity of the plant HR can
be physiologically, genetically and temporally uncoupled
(K€
unstler et al., 2016).
Overview of PRRs and PTI signaling
Plant PRRs are primarily receptor-like kinases (RLKs) or receptor-
like proteins (RLPs). RLKs and RLPs have a tripartite domain
organization, containing an extracellular domain (ECD), a
Published by the Molecular Plant Shanghai Editorial Office in association with
Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1
Molecular Plant
Review Article
llll
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
transmembrane domain, and a cytoplasmic serine/threonine ki-
nase domain in RLKs or a short cytoplasmic tail in RLPs (Boutrot
and Zipfel, 2017;Wang and Chai, 2020b;Lee et al., 2021;Song
et al., 2021). For example, FLAGELLIN-SENSITIVE 2 (FLS2) is an
LRR-RLK because its ECD encodes LRRs that recognize the bac-
terial PAMP flg22 (Felix et al., 1999;Gomez-Gomez and Boller,
2000), whereas the LRR-RLKs PEP RECEPTOR 1,2 (PEPR1,2)
are receptors of the phytocytokine Pep1and homologous peptides
in Arabidopsis thaliana (hereafter called Arabidopsis)(Huffaker
et al., 2006;Yamaguchi et al., 2006,2010;Krol et al., 2010). The
ECDs of PRRs are necessary and sufficient for PAMP/DAMP
recognition (Liu et al., 2012,2016a;Sun et al., 2013,2022;Tang
et al., 2015;Xiao et al., 2019;Xu et al., 2022b). Ligand binding
induces trans-phosphorylation of cytoplasmic kinase domains in
the PRR complexes (Figure 1). BRI1-ASSOCIATED KINASE 1
(BAK1; also called SOMATIC EMBRYOGENESIS RECEPTOR KI-
NASE 3 [SERK3]) belongs to the SERK family of LRR-RLKs and
acts as a co-receptor of various LRR-RLKs, including FLS2 and
PEPR1,2 (Chinchilla et al., 2007;Schulze et al., 2010;Sun et al.,
2013;Tang et al., 2015). Because of the lack of the C-terminal
kinase domain for signal transduction, LRR-RLPs constitutively
interact with the LRR-RLK SUPPRESSOR OF BIR-1 (SOBIR1),
forming binary RLKs (Gust and Felix, 2014;Albert et al., 2015;Bi
et al., 2016) to recruit BAK1.
Receptor-like cytoplasmic kinases (RLCKs) are direct downstream
components of PRRs (Figure 1). Lycopersicon esculentum
(tomato) AVR9/CF-9 INDUCED KINASE 1 (ACIK1) is the first
RLCK identified to participate in PRR-mediated immunity
(Rowland et al., 2005), and its Arabidopsis ortholog BOTRYTIS-
INDUCED KINASE 1 (BIK1) has a crucial role in PTI signaling
(Zhang et al., 2010a;Lu et al., 2010). BIK1 and its homologous
protein PBS1-LIKE PROTEIN 1 (PBL1), which belong to the PBL
family, constitute a key signaling node downstream of multiple
PRR complexes. Activated RLCKs phosphorylate multiple down-
stream immune signaling components to transduce immune sig-
nals (Liang and Zhou, 2018). For example, BIK1 phosphorylation
of different residues of RESPIRATORY BURST OXIDASE
HOMOLOGUE D (RBOHD) is critical for PAMP-induced ROS burst
and antibacterial immunity (Kadota et al., 2014;Li et al., 2014). BIK1
can also phosphorylate and activate Ca
2+
channels, including
CYCLIC NUCLEOTIDE-GATED CHANNEL (CNGC) 2-CNGC4
(Tian et al., 2019) and REDUCED HYPEROSMOLALITY
INDUCED CA
2+
INCREASE 1.3 (OSCA1.3) (Thor et al., 2020), to
trigger Ca
2+
influx and stomatal closure, respectively. Ca
2+
as a
secondary messenger activates downstream components, such
as Ca
2+
-dependent protein kinases (CPKs), including CPK4/5/6/
11, which, in turn,phosphorylate and activate RBOHD for ROS pro-
duction (Boudsocq et al., 2010;Dubiella et al., 2013). Two MAPK
cascade signaling pathways, MAPK Kinase Kinase (MAPKKK
or MEKK) 1-MAPK Kinase (MAPKK or MKK) 1/2-MPK4 and
MAPKKK3/5-MKK4/5-MPK3/6, participate in PTI responses
(Meng and Zhang, 2013). A recent study has shown that the
RLCK VII-4 subfamily member PBL19 directly phosphorylates
MEKK1 andMAPKKK5 to activateMPK4 and MPK3/6, respectively
(Yamada et al., 2016;Wang et al., 2017;Bi et al., 2018).
Overview of NLRs and ETI signaling
NLRs are conserved in plants and animals. Plant and animal
NLRs belong to the signal transduction adenosine triphospha-
tases (ATPases) with numerous domains (STAND) family and
share a similar domain organization (Wang and Chai, 2020a;
Duxbury et al., 2021). NLRs are characterized by a variable
N-terminal domain, a central conserved nucleotide binding and
oligomerization domain (NOD), and a C-terminal LRR domain
(Figure 1). The NOD module consists of three subdomains:
nucleotide-binding domain (NBD), helical domain 1 (HD1), and
winged-helix domain (WHD). A major biochemical function of
the NOD is to act as a nucleotide switch regulating NLR
oligomerization (Yang et al., 2019;Wang and Chai, 2020a;
Duxbury et al., 2021;Forderer et al., 2022). Based on the
domain structures of their N termini, plant NLRs are mainly
classified into three groups: coiled-coil (CC) NLRs (CNLs), Toll/
interleukin-1 receptor (TIR) NLRs (TNLs), and CC
R
-NLRs
(RNLs). RNLs possess a CC domain similar to the membrane-
anchored resistance protein RESISTANCE TO POWDERY
MILDEW 8 (RPW8), hence known as CC
R
(Collier et al., 2011).
Overexpression of the CC or TIR domain is sufficient to
recapitulate pathogen-induced immune responses of full-length
NLRs (Swiderski et al., 2009;Krasileva et al., 2010;Bernoux
et al., 2011;Collier et al., 2011;Maekawa et al., 2011;Cesari
et al., 2016;Schreiber et al., 2016;Baudin et al., 2017). Atypical
NLRs, such as C-terminally truncated NLRs, also exist in
plants. For instance, Arabidopsis RESPONSE TO THE
BACTERIAL TYPE III EFFECTOR PROTEIN HOPBA1 (RBA1) is
a TIR-only protein (Nishimura et al., 2017), and Arabidopsis TN2
(TIR-NB 2) lacks the LRR domain (Zhao et al., 2015).
Functionally, plant NLRs can be divided into sensor NLRs and
helper NLRs, which are responsible for recognition of pathogen
effectors and immune signal outputs, respectively (Jubic et al.,
2019;Feehan et al., 2020). Examples of helper NLRs include
ACTIVATED DISEASE RESISTANCE 1 (ADR1), N
REQUIREMENT GENE 1 (NRG1) (Peart et al., 2005;Bonardi
et al., 2011;Collier et al., 2011;Qi et al., 2018;Castel et al.,
2019;Lapin et al., 2019;Wu et al., 2019), and NB-LRR
PROTEIN REQUIRED FOR HR-ASSOCIATED CELL DEATH
(NRC) (Gabriels et al., 2007;Wu et al., 2016,2017). There is
also a class of singleton NLRs in plants that can recognize
effectors and execute downstream signaling. Representative
members of singleton NLRs include the Arabidopsis CNLs
RESISTANCE TO P. SYRINGAE PV MACULICOLA 1 (RPM1)
(Grant et al., 1995) and HOPZ-ACTIVATED RESISTANCE 1
(ZAR1) (Lewis et al., 2010;Wang et al., 2015) and the Triticum
monococcum (wheat) CNL STEM RUST RESISTANCE 35 (Sr35)
(Salcedo et al., 2017). In some cases, effector recognition
requires two genetically linked NLRs (called paired NLRs) with
one functioning as the sensor and the other as the executor.
Well-characterized paired NLRs include the CNL pairs R-GENE
ANALOG (RGA) 5/RGA4 (Cesari et al., 2013)inOryza
sativa (rice) and the Arabidopsis TNL pair RESISTANCE TO
RALSTONIA SOLANACEARUM 1 (RRS1)/RESISTANT TO P.
SYRINGAE 4 (RPS4) (Le Roux et al., 2015;Sarris et al., 2015).
Recognition of pathogen effectors frequently involves the C-ter-
minal LRR domain, which results in oligomerization of plant
NLRs and formation of large NLR-containing complexes called
resistosomes (Wang et al., 2019a;Ma et al., 2020;Martin et al.,
2020;F€
orderer et al., 2022;Zhao et al., 2022)(Figure 1). CNL
resistosomes, such as ZAR1 in Arabidopsis, function as Ca
2+
-
permeable influx channels (Bi et al., 2021). Signaling mediated
by CNLs typically requires NON RACE-SPECIFIC DISEASE
2Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
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RESISTANCE 1 (NDR1) (Century et al., 1997;Day et al., 2006;
Knepper et al., 2011). However, the mechanism of how NDR1
contributes to CNL signaling remains unclear. NRC helpers are
required for many CNLs in solanaceous plants to mediate
immune responses (Wu et al., 2016,2017). NRCs have
been proposed to form resistosomes upon activation (Adachi
et al., 2019;Duggan et al., 2021;Ahn et al., 2022;Contreras
et al., 2022). In contrast with the CNL resistosomes, TNL
resistosomes have nicotinamide adenine dinucleotide (NAD)
nucleosidase (NADase) activity encoded in the N-terminal TIR
domains (Horsefield et al., 2019;Wan et al., 2019;Ma et al.,
2020;Martin et al., 2020). All tested TNLs require the RNLs
ADR1 and NRG1 (Lapin et al., 2022) and the lipase-like protein
ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and its two pa-
ralogs, PHYTOALEXIN DEFICIENT 4 (PAD4) and SENESCENCE-
ASSOCIATED GENE 101 (SAG101), in Arabidopsis (Bhandari
et al., 2019;Gantner et al., 2019;Lapin et al., 2019;Dongus
and Parker, 2021). Like the ZAR1 resistosome, self-activating
mutants of ADR1 and NRG1 act as Ca
2+
-permeable channels
(Jacob et al., 2021).
RESISTOSOMES: STRUCTURE AND
BIOCHEMICAL FUNCTION
Assembly of resistosomes is required for plant NLR
signaling
Oligomerization is a common theme of signaling mediated by
NLRs (Wang and Chai, 2020a;Duxbury et al., 2021). This notion
is well exemplified by assemblies of apoptosomes (Qi et al.,
2010;Zhou et al., 2015) and NLR inflammasomes (Hu
et al., 2015;Zhang et al., 2015), which mediate apoptosis
and pyroptosis in animals, respectively. Cytochrome c
binding induces oligomerization of APOPTOTIC PEPTIDASE
ACTIVATING FACTOR 1 (Apaf-1) in the presence of ATP or
Figure 1. Plant immune system and signaling network.
Recognition of pathogen PAMPs or host DAMPs by cell surface immune receptor PRRs leads to pattern-triggered immunity (PTI). Activation of PRRs
triggers phosphorylation of intracellular kinase RLCKs and MAPKs. The activated RLCKs phosphorylate and activate membrane-residing calcium
channels (CNGCs, GLRs, and OSCA1.3) and the NADPH oxidase RBOHD to induce Ca
2+
influx and ROS burst, respectively. MAPK phosphorylation
cascades induce massive transcriptional reprogramming, leading to expression of defense-related genes. Pathogens deliver race-specific effector
proteins to suppress host PTI in various modes to facilitate infection. Under these circumstances, intracellular NLR immune receptors, including CNLs
and TNLs, sense these pathogen effectors and lead to another layer of plant immunity known as ETI. Recognition of effectors by singleton CNLs leads to
formation of oligomeric CNL resistosomes, which can function as a calc ium-permeable channel to mediate ETI signaling. The membrane-localized NDR1
has been demonstrated to participate in CNL-induced cell death, but the detailed mechanism remains elusive. TNL resistosomes activated by perception
of pathogen effectors typically function as NADase holoenzymes to produce the secondary signaling messengers pRib-AMP/ADP and ADPr-ADP/di-
ADPR, which are, respectively, recognized by EDS1-PAD4 and EDS1-SAG101 in Arabidopsis. Activated EDS1-PAD4 and EDS1-SAG101 interact with
the helper RNLs ADR1 and NRG1, respectively. EDS1-activated ADR1 and NRG1 form resistosomes and function as Ca
2+
-permeable channels to induce
HR cell death. EDS1-activated ADR1 plays roles in regulation of resistance via an unknown mechanism. PTI also activates EDS1-PAD4 complex-
mediated ETI. TIR-only proteins can also function as 2030-cNMP synthetases with dsRNA as the substrate to promote EDS1 signaling through an
unknown mechanism.
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 3
Biochemical function and signaling mechanism of NLRs Molecular Plant
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deoxyadenosine triphosphate (dATP), forming a large protein
complex called the Apaf-1 apoptosome. The Apaf-1 apoptosome,
mainly heptameric, recruits the downstream cysteine protease
caspase-9, leading to formation of the Apaf-1/caspase-9 holoen-
zyme to mediate activation of the cysteine protease (Li et al.,
2017). A highly conserved paradigm has been demonstrated in
NLR signaling in animals (Hu and Chai, 2016;Wang et al.,
2021b). A well-studied example of this is NLR FAMILY CARD
DOMAIN CONTAINING 4 (NLRC4), which recognizes bacterial
flagellin and the type III secretion system component PrgJ through
NEURONAL APOPTOSIS INHIBITOR PROTEIN 5/6 (NAIP5/6) and
NAIP2, respectively (Kofoed and Vance, 2011;Zhao et al., 2011;
Tenthorey et al., 2017;Yang et al., 2018). Flagellin or PrgJ
binding induces NAIP interaction with NLRC4, resulting in assem-
bly of substoichiometric NAIP-NLRC4 complexes, called
NLRC4 inflammasomes, with a stoichiometry of 1:9 or 1:10 be-
tween NAIP and NLRC4 (Hu et al., 2015;Zhang et al., 2015).
The inflammasomes directly interact with caspase-1 or through
the adaptor ASC (APOPTOSIS-ASSOCIATED SPECK-LIKE
PROTEIN CONTAINING A CARD) to proteolytically mature the
protease. The Apaf-1 apoptosome and NLRC4 inflammasomes
act as platforms for protease activation (Figure 2).
NLRs have been known to function as a nucleotide switch for
many years, but the underlying mechanism was not demon-
strated until recently. ZAR1 recognizes the Xanthomonas
campestris pv. campestris uridylylase effector protein AvrAC to
mediate ETI (Wang et al., 2015). In unchallenged cells, ZAR1
forms a constitutive complex with the RLCK RESISTANCE-
RELATED KINASE 1 (RKS1). Upon X. campestris pv. campestris
infection, AvrAC uridylylates another RLCK member, PBL2.
The modified PBL2 (PBL2
UMP
) associates with RKS1 and conse-
quently activates ZAR1-mediated immune signaling. The ZAR1-
RKS1-PBL2
UMP
complex reconstituted using purified proteins
is momoneric (Wang et al., 2019b). Structural comparison
between ZAR1-RKS1 and ZAR1-RKS1-PBL2
UMP
reveals a strik-
ing conformational change in the ZAR1 NBD, which is predicted
to impair adenosine diphosphate (ADP) binding as confirmed
by biochemical data. This would facilitate ZAR1 ATP binding
because of its much higher concentrations in cells. PBL2
UMP
,
however, makes no direct contact with the ZAR1 NBD, suggest-
ing an allosteric mechanism of PBL2
UMP
-induced impairment
of the ADP binding activity of ZAR1 (Wang et al., 2019b). As
observed in other inactive NLRs (Hu et al., 2013;Maekawa
et al., 2016;Sharif et al., 2019;Steele et al., 2019;Hochheiser
Figure 2. Structures and functions of the apoptosome, inflammasome, and resistosome.
Domain organization, N-terminal signaling domain structures, oligomeric structures, biochemical functions, and downstream signaling components of
the NLR proteins Apaf-1, NLRC4, NLRP3, ZAR1, and RPP1. The heptameric Apaf-1 apoptosome contains cytochrome c(salmon) and Apaf-1 (domains
are colored differently). The Apaf-1 apoptosome functions as a platform to recruit caspase-9 through CARD-CARD interaction. The NLRC4 in-
flammasomes are decamers or undecamers (NAIP:NLRC4 1:9 or 1:10) and function as a platform to recruit caspase-1/11. The CNL ZAR1 resistosome,
containing the pathogen effector AvrAC-uridylylated PBL2 (green), RKS1 (light yellow) , and ZAR1 (domain-based colors), is pentameric and functions as a
PM Ca
2+
-permeable channel to trigger ETI signaling. The tetrameric TNL RPP1 resistosome contains the pathogen effector ATR1 (green) and RPP1
(domain-based colors) and functions as an NADase holoenzyme to produce immune molecules recognized by EDS1 family proteins to activate helper
NLRs (RNLs).
4Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
et al., 2022) and the NLR-like Apaf-1 (Riedl et al., 2005;Reubold
et al., 2011), ADP is deeply bound in inactive ZAR1 in the
cryoelectron microscopy structure of ZAR1-RKS1, making it diffi-
cult, if not impossible, for ADP to be directly replaced by ATP or
dATP for activation. Ligand-induced allosteric substitution of
ADP by ATP can also avoid accidental activation of NLRs under
resting conditions.
dATP/ATP induces oligomerization of the reconstituted monomeric
ZAR1-RKS1-PBL2
UMP
complex (Wang et al., 2019a), similar to
dATP-induced assembly of the Apaf-1 apoptosome (Zhou et al.,
2015). The oligomeric ZAR1 complex, called the resistosome,
forms a wheel-like pentamer entirely mediated by ZAR1. A remark-
ably conserved structure is also found for the Sr35 resistosome
containing the wheat CNL Sr35, its cognate effector AvrSr35,
and ATP molecules (F€
orderer et al., 2022;Zhao et al., 2022),
suggesting that the pentameric assembly may be conserved
among plant CNL channels (Figure 2). Some sensor CNLs, such
as Rpi-amr1/3 (Rpi, R genes against Phytophthora infestans;amr,
AVRamr from P. infestans) and RESISTANCE TO POTATO VIRUS
X (Rx) appear not to require oligomerization for activation of
the NRC helper (Ahn et al., 2022;Contreras et al., 2022), but the
precise active forms of these CNLs remain unclear. Mutations
disrupting the ZAR1 or Sr35 resistosomes substantially suppress
the HR cell death and resistance activities of the two CNLs (Wang
et al., 2019a;F€
orderer et al., 2022;Zhao et al., 2022). Formation
of the ZAR1 resistosome has been demonstrated in plant cells
(Hu et al., 2020;Bi et al., 2021). These data support the biological
relevance of the CNL resistosomes.
The TNLs RECOGNITION OF PERONOSPORA PARASITICA 1
(RPP1) from Arabidopsis (Ma et al., 2020) and RECOGNITION
OF XOPQ 1 (ROQ1) from Nicotiana benthamiana (Martin et al.,
2020) have been shown to form resistosomes. Mutations
disrupting the RPP1 and ROQ resistosomes abolish the cell
death activity, supporting the biological significance of these
two TNL resistosomes. RPP1 and ROQ1 recognize the
oomycete effector ATR1 and bacterial pathogen effector XopQ,
respectively. The RPP1-ATR1 (RPP1 resistosome) and ROQ1-
XopQ (ROQ1 resistosome) complexes were purified from insect
cells and Nicotiana benthamiana plants, respectively. Despite
the difference, these two TNL resistosomes form highly
conserved tetramers (Figure 2). A conserved C-terminal domain
called C-terminal jelly-roll and IG-like domain is directly involved
in RPP1 and ROQ1 recognition of ATR1 and XopQ, respectively
(Ma et al., 2020;Martin et al., 2020). In contrast with the
ATP-bound ROQ1, ZAR1, and Sr35resistosomes, the RPP1 re-
sistosome binds ADP in the P-loop region of RPP1. The
unexpected nucleotide binding of active RPP1 results from
acquisition of an additional b2-a2 loop, which promotes an in-
ter-protomer NBD-WHD interaction and compensates for the
loss of interactions mediated by the g-phosphate group of ATP
(Ma et al., 2020).
NLR resistosomes function as Ca
2+
-permeable
channels or NADase holoenzymes
CNL and TNL resistosomes form wheel-like structures similar to
those of the Apaf-1 apoptosome and the NLR inflammasomes.
However, striking differences exist in their N-terminal signaling
domains among these large protein complexes (Figure 2). In
contrast with the flexible N-terminal caspase recruitment
domains (CARDs) in the Apaf-1 apoptosome (Zhou et al., 2015;
Li et al., 2017) and the NLRC4 inflammasomes (Hu et al., 2015;
Zhang et al., 2015), the CC domains of the CNL resistosomes
(Wang et al., 2019a;F€
orderer et al., 2022;Zhao et al., 2022)or
the TIR domains of the TNL resistosomes (Ma et al., 2020;
Martin et al., 2020) are well defined, suggesting that the CNL
and TNL resistosomes may use different mechanisms for signal
transduction.
In the ZAR1 resistosome, the N-terminal helix a1 forms a funnel-
shaped structure, which is the only exposed portion of the CC
domain (Figure 2). These structural observations suggest that the
funnel-shaped structure is important for ZAR1 resistosome func-
tion. Simultaneous mutations of two negatively charged residues
(Glu11 and Glu18) at the inner surface of the funnel-shaped struc-
ture abolish ZAR1-mediated immune responses, suggesting that
the ZAR1 resistosome may have pore- or channel-related activity
(Wang et al., 2019a). Protein fractionation analysis shows AvrAC-
induced ZAR1 association with the plasma membrane (PM).
PM localization has been demonstrated for the CNLs RESISTANT
TO P. SYRINGAE 2 (RPS2) (Elmore et al., 2012) and RPM1
(Gao et al., 2011;El Kasmi et al., 2017). The ZAR1 resistosome is
formed in Arabidopsis protoplasts and displays calcium-
permeable cation-selective channel activity in lipid bilayers (Bi
et al., 2021). ZAR1 activation in the plant cell triggers Ca
2+
influx,
perturbation of subcellular structures, and immune responses (Bi
et al., 2021). These data support PM channel activity of the ZAR1
resistosome (Figure 2).
The available data suggest that the Ca
2+
-permeable channel ac-
tivity may be evolutionarily conserved in CNLs of different plant
species. The Sr35 resistosome from wheat not only has a struc-
ture strikingly similar to the ZAR1 resistosome (Figure 2) but
also displays Ca
2+
-permeable channel activity when expressed
in Xenopus oocytes (F€
orderer et al., 2022;Zhao et al., 2022). In
contrast with that of the ZAR1 resistosome, however, the
channel activity of the Sr35 resistosome appears to be
independent of acidic residues predicted to line the inner
surface of the channel formed by the a1 helix in the Sr35
resistosome. It may be that the N termini of the Sr35
resistosome are structurally and functionally distinct from those
of the ZAR1 resistosome. Auto-active RNL NRG1 and ADR1
also act as Ca
2+
-permeable channels. Like those from the a1 he-
lix of ZAR1, the negatively charged residues at the a1 helix of
NRG1 and ADR1 are required for Ca
2+
influx and cell death
(Jacob et al., 2021). Bioinformatics data show that the ZAR1 a1
helix is conserved among CNLs of distantly related plant
species, with 20% of them including the helper NLRs NRCs
sharing the consensus ‘‘MADA’’ motif (Adachi et al., 2019). An
N-terminal 29-residue peptide of NRC4 with yellow fluorescent
protein (YFP) fused to the C terminus is sufficient to induce HR
cell death when expressed in Nicotiana benthamiana, providing
direct evidence of the biological function of a1 in a CNL. However,
many Solanaceae CNLs have a large domain called the
Solanaceae domain prior to their CC domain. Some of them
cooperate with NRCs to mediate immune responses (Wu et al.,
2016,2017).
The CC domain of many CNL/RNLs is sufficient to induce HR-like
cell death when expressed in plant cells (Collier et al., 2011;
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 5
Biochemical function and signaling mechanism of NLRs Molecular Plant
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
Maekawa et al., 2011;Cesari et al., 2016;Baudin et al., 2017). In
the structures of the ZAR1 and Sr35 resistosomes, the inter-CC
domain interaction results in formation of a similar helical barrel
structure, which is stabilized by the conserved NOD (Wang
et al., 2019a;F€
orderer et al., 2022;Zhao et al., 2022). High
concentrations of the domain protein might bypass the
requirement of the NOD for oligomerization, but direct evidence
of whether the CC domain alone can form a channel structure
like that from the ZAR1 or Sr35 resistosomes is lacking.
The TIR domain is a conserved immune module in animals,
plants, and bacteria (Lapin et al., 2022). TIR domains were
first found as intracellular domains of animal transmembrane
immune receptor interleukin-1 receptors (IL-1Rs) and Toll-like
receptors (TLRs) and function as scaffolds (Gay et al., 2014).
Recognition of immune signals by the ECDs of TLRs or IL-IRs in-
duces receptor dimerization, thereby forming intracellular TIR
domain homo-dimers and recruiting downstream cytoplasmic
TIR adaptor proteins (Yin et al., 2015). The biochemical function
of plant TIR domains was not identified until recent biochemical
and structural studies (Horsefield et al., 2019;Wan et al., 2019;
Ma et al., 2020;Martin et al., 2020). The discovery of NADase
activity of plant TIR domains was inspired by the animal
STERILE ALPHA AND TIR MOTIF CONTAINING 1 (SARM1) TIR
domain (Gerdts et al., 2015;Essuman et al., 2017). SARM1 is
the only animal TIR adaptor protein coupled with enzymatic
activity. Plant TNL- and TIR-only proteins shared the conserved
NADase catalytic residue glutamate (Horsefield et al., 2019;
Wan et al., 2019). Biochemical studies have demonstrated the
NADase activity of plant TIR domain proteins (Horsefield et al.,
2019;Wan et al., 2019) and TNL resistosomes (Ma et al., 2020;
Figure 2). Mutations of the catalytic glutamate residue abrogate
TNL- and TIR-mediated immunity and cell death (Horsefield
et al., 2019;Wan et al., 2019;Ma et al., 2020;Martin et al.,
2020), indicating that the NADase activity is essential for TNL
and TIR domain signaling in plants. Assembly of the TNL
resistosomes promotes the TIR-encoded NADase activity (Ma
et al., 2020;Martin et al., 2020). Tetrameric assembly of the TIR
domain in the TNL resistosomes results in formation of two
composite catalytic sites, each of which is formed by an
asymmetric TIR dimer (Ma et al., 2020;Martin et al., 2020). A
similar mechanism is also involved in activation of the NADase
activity of SARM1 (Shi et al., 2022), the bacterial TIR domain
proteins Acinetobacter baumannii TIR (Manik et al., 2022)
and TIR-SAVED (SMODS-associated and fused to various
effector domain) (Hogrel et al., 2022), and the endonuclease
activity of the bacterial NLRs Avs (antiviral STAND) and Avs4
(Gao et al., 2022).
UNIFIED MECHANISMS IN PLANT NLR
SIGNALING
Ca
2+
-permeable channel activity is a unified mechanism
of plant NLR signaling
Despite their variations in signaling input, composition, and struc-
ture, NLR inflammasomes converge on inflammatory caspases
for activation of a conserved suite of immune responses (Cao
et al., 2022;Ohto, 2022). The underlying mechanism has been
well documented. Assembly of NLR inflammasomes, including
NLR FAMILY PYRIN DOMAIN CONTAINING 3 (NLRP3) and
NLRC4, induces proximity of their N-terminal pyrin domains or
CARDs for recruitment and activation of caspase-1 or another in-
flammatory caspase directly or via the adaptor ASC through ho-
motypic interactions (Swanson et al., 2019). Activated caspase-1
proteolytically matures the cytokines IL-1band IL-18 and cleaves
the gasdermin D (GSDMD) substrate to release its N-terminal
pore-forming domain (He et al., 2015;Kayagaki et al., 2015;Shi
et al., 2015). The GSDMD pores formed in the PM facilitate secre-
tion of mature inflammatory cytokines and other DAMPs (Liu
et al., 2016b;Ding et al., 2016;Ruan et al., 2018;Xia et al.,
2021) and trigger initiation of lytic cell death, called pyroptosis
(Figure 3).
Like that of animal NLRs, signaling mediated by CNL/RNLs and
TNLs also shares similar immune outputs (Figure 3). This
observation is rationalized by TNL-activated ADR1 and NRG1
of the RNL class (Peart et al., 2005;Bonardi et al., 2011;Collier
et al., 2011;Qi et al., 2018;Castel et al., 2019;Lapin et al.,
2019;Wu et al., 2019,2021;Sun et al., 2021;Huang et al.,
2022;Jia et al., 2022). Ca
2+
-permeable channel activity appears
to be evolutionarily conserved among CNL/RNLs and NRCs
(Figure 3;Bi et al., 2021;Jacob et al., 2021;Ahn et al., 2022;
Contreras et al., 2022;Feehan et al., 2022;F€
orderer et al.,
2022), suggesting that Ca
2+
-permeable channel activity is likely
to be a unified mechanism for plant NLR signaling. However,
the mechanisms of how NLR-induced Ca
2+
influx is decoded
remain poorly understood. Presumably, calcium-binding pro-
teins, including calmodulins (CaMs), CAM-like proteins (CMLs),
calcineurin B-like proteins (CBLs), and CPKs, translate the
elevated cytosolic Ca
2+
levels into a downstream immune
response (Luan and Wang, 2021).
The notion that CNL resistosomes act as Ca
2+
channels is of sub-
stantial significance for understanding NLR signaling because it
suggests that extracellular Ca
2+
influx into plant cells is a major
NLR-activated signal to initiate host defense and cell death
(Figure 3). Multiple lines of evidence support NLR-activated
Ca
2+
influx as a trigger for immune signaling. The ZAR1 resisto-
some forms hours before loss of PM integrity (an indicator of
HR cell death) (Bi et al., 2021), suggesting that the resistosome
is a trigger but not a direct executor of the HR cell death. Gain
of function of the CNGC20 mutant with increased Ca
2+
influx ac-
tivity constitutively activates EDS1- and SA-dependent Arabi-
dopsis immunity (Zhao et al., 2021), indicating that elevations in
cytosolic Ca
2+
accumulation can be sufficient to induce immune
signaling. Like Ca
2+
influx, ROS production is also one of the
earliest events of plant immune responses (Thordal-Christensen
et al., 1997) and is largely dependent on the activity of the
PM-localized nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase RBOHD (Torres et al., 2002). ROS act as
second messengers to amplify immune signaling.
Pharmacological and genetic studies suggested that Ca
2+
functions upstream of ROS during ETI (Grant et al., 2000). The
NADPH oxidase inhibitor diphenylene iodonium chloride has little
effect on ETI signaling (Grant et al., 2000). In contrast, treatment
with lanthanum chloride (LaCl
3
), an extracellular calcium
antagonist, markedly suppresses ETI immune responses,
including increases in cytosolic Ca
2+
concentration, H
2
O
2
accu-
mulation, and HR cell death (Gao et al., 2013). The pathogen
effector AvrRps4 strongly induces ion leakage activity of Arabi-
dopsis leaves but not ROS and HR cell death (Ngou et al.,
6Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
2020). It remains unknown whether CNL resistosomes also have
conductivity to deliver other signaling molecules. The pore size of
the funnel-shaped structure in the ZAR1 resistosome is less than
10 A
˚in diameter (Wang et al., 2019a), which does not seem to be
large enough to pass large signaling molecules. The possibility
cannot be excluded that conformational changes in the pore
during ETI allow passage of non-ion signaling molecules.
The increase in cytosolic Ca
2+
during plant immunity can also
be caused by release from internal Ca
2+
pools (Spalding and
Harper, 2011;Edel et al., 2017;Resentini et al., 2021;Koster
et al., 2022). Supporting a role of Ca
2+
released from these
pools in plant immunity, inhibition of Ca
2+
release from intracel-
lular compartments by ruthenium red (RR) blocks HR cell death
induced by the pathogen effectors AvrRpt2 or AvrRpm1 (Gao
et al., 2013). However, RR inhibition appears to be less
efficient than LaCl
3
(Gao et al., 2013), supporting the idea that
Ca
2+
influx is a trigger for ETI responses. NLR-activated Ca
2+
influx as a signaling trigger agrees with the cell death activity
of CNL resistosomes in animal cells. Co-expression of Sr35
and AvrSr35, but not either alone, triggers cell death in insect
cells (F€
orderer et al., 2022). Similar cell death activity has also
been demonstrated for auto-active ADR1 and NRG1 mutants
in human HeLa cells (Jacob et al., 2021). These results
suggest that unregulated channel activity is sufficient to
recapitulate plant CNL/RNL-mediated cell death in eukaryotic
cells. Although the mechanisms underlying the cell death medi-
ated by plant CNL/RNLs in animal and insect cells remain un-
known, these findings support the idea that the Ca
2+
ion can
act as a cell death trigger to kill animal cells (Orrenius et al.,
2003). Ca
2+
as a trigger of cell death was first suggested to
be involved in the cardiac pathology that occurs after ischemia
Figure 3. NLR signaling mechanisms in animals and plants.
Cartoon representations of NLR signaling in animals (left) and plants (right). Left: recognition of PAMPs or DAMPs induces assembly of NLRs, such as
NLRC4 and NLRP3 inflammasomes. In the case of NLRP3, transcriptional upregulation of inflammasome-related genes by Toll-like receptors (TLRs) is
needed to activate the inflammasome. The NLR inflammasomes interact with the ASC adaptor protein, which, in turn, recruits and activates caspase-1.
When activated, caspase-1 proteolytically processes gasdermin D (GSDMD ) to release its N-terminal pore-forming domain, which forms pores on the PM
to execute pyroptosis. Caspase-1 also cleaves pro-IL-1band pro-IL-18 into IL-1band IL-18, which are released through the GSDMD pores. The NLRC4
inflammasomes can also directly recruit caspase-1 for activation. Right: plant CNL recognition of pathogen effectors induces pentameric resistosomes
that act as PM Ca
2+
-permeable channels to induce ETI. Recognition of pathogen effectors by plant TNLs results in formation of tetrameric resistosomes
that function as NADase holoenzymes to catalyze production of the secondary messengers pRib-AMP/ADP and ADPr-ATP/di-ADPR, which bind to and
activate EDS1-PAD4 and EDS1-SAG101, respectively. The second messenger-activated EDS1-PAD4 and EDS1-SAG101 allosterically induce the PM
Ca
2+
-permeable channel activity of helper NLRs (RNLs), including ADR1 and NRG1, triggering TNL-mediated ETI signaling. Activation of PRRs by PAMPs
facilitates NLR signaling by upregulation of ETI-related genes.
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 7
Biochemical function and signaling mechanism of NLRs Molecular Plant
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doi.org/10.1016/j.molp.2022.11.011
(Fleckenstein et al., 1974). Now it is well established that the
Ca
2+
ion has a central role in activating distinct parts of
apoptosis through itself or in conjunction with other apoptotic
programs to kill the cell (Orrenius et al., 2003).
ETI involves massive transcriptional reprograming. How can
this be reconciled with the PM channel activity of CNL/RNLs?
One might expect that Ca
2+
itself enters the nucleus and activates
Ca
2+
-dependent proteins in the nucleus. Consistent with
this possibility, many Ca
2+
sensors, including CaMs, CPKs, and
even transcriptional factors such as CALMODULIN-BINDING
PROTEIN 60g (CBP60g) (Li et al., 2021) and CaM-binding tran-
scription activator (CAMTA) 3 (Jacob et al., 2018;Sun et al.,
2020), are nucleus localized. Increases in nuclear free Ca
2+
concentrations have been reported in response to various
stresses (van Der Luit et al., 1999;Pauly et al., 2001;Xiong
et al., 2004). However, this model is not supported by the
observation that dynamic cyto-nuclear trafficking is required for
the disease resistance activity of many NLRs, including CNLs
(Gu et al., 2016,2017;Ludke et al., 2022). An alternative model
for the transcriptional activity of CNLs/RNLs may be that they
adopt different structural forms from those of the resistosomes
to mediate immunity in the nucleus. The CC domain of ZAR1
adopts different fold architectures in its inactive and active
structures (Wang et al., 2019a), a phenomenon called protein
metamorphosis (Bryan and Orban, 2010), that can confer them
different functions. Several CNLs have been shown to interact
with transcriptional factors (Wang et al., 2021a), suggesting that
CNLs may directly regulate transcriptional programming. Given
that overexpression of the CC domain is sufficient to induce
pathogen resistance (Wroblewski et al., 2018), it is reasonable
to assume that this structural domain is involved in interaction
with CNL-interacting partners for regulation of transcriptional
programming. The CC domain of the Hordeum vulgare (barley)
CNL MILDEW-A (MLA) 10 interacts with the transcriptional fac-
tors WRKY DNA-BINDING PROTEIN (WRKY) 1/2 (Shen et al.,
2007). A recent study shows that NRG1 is PM and nucleus
localized upon activation, but only the PM-resident NRG1 forms
oligomers (Feehan et al., 2022), suggesting oligomerization-
independent NRG1 functions.
TIR-catalyzed small molecules function as second
messengers to link TNL signaling to RNLs
EDS1, PAD4, and SAG101 constitute a plant-specific family
sharing similar domain structures, including an N-terminal lipase-
like domain and a highly conserved C-terminal EDS1-PAD4 (EP)
domain (Wagner et al., 2013). EDS1 forms exclusive dimers with
PAD4 and SAG101. Genetic and biochemical data support
functional cooperation of EDS1-PAD4 with ADR1 and EDS1-
SAG101 with NRG1 (Peart et al., 2005;Bonardi et al., 2011;
Collier et al., 2011;Qi et al., 2018;Castel et al., 2019;Lapin et al.,
2019;Wu et al., 2019,2021;Sun et al., 2021). However, the
mechanism of how the EDS1 dimers integrate signals from TNLs
to activate ADR1 and NRG1 of the RNL class have remained
elusive until recently. The TIR NADase activity produces a variant
cyclic adenosine diphosphate ribose (v-cADPR) that is a potential
immune signaling molecule (Wan et al., 2019). Variant cyclic
ADPR formed by 100-20glycosidic linkage between the two
riboses of NAD
+
(20cADPR) is generated by plant TIR domain
proteins (Leavitt et al., 2022;Manik et al., 2022). Our recent
findings demonstrate RPP1 resistosome-induced specific EDS1-
PAD4 interaction with ADR1 and EDS1-SAG101 interaction with
NRG1 in insect cells (Huang et al., 2022;Jia et al., 2022;
Figure 4). Similar activity also exists in the TIR domain of the
Arabidopsis TNL RPS4 (RPS4-TIR). The RPP1 resistosome- or
EDS1-PAD4
EDS1-SAG101
EDS1-PAD4-pRib-ADP
EDS1-SAG101-ADPr-ATP
pRib-ADP
ADPr-ATP
EDS1 PAD 4
EDS1 SAG101
EDS1 PAD4
EDS1 SAG101
ADR1
NRG1
EDS1-PAD4-ADR1
EDS1-SAG101-NRG1
Resistance
Cell death
pRib-ADP
ADPr-ATP
A B
Figure 4. EDS1 signaling activated by TIR-catalyzed small molecules.
(A) The plant TIR-catalyzed small molecules pRib-ADP and ADPr-ATP bind specifically to EDS1-PAD4 and EDS1-SAG101 dimers, respectively, trig-
gering conformational changes in PAD4 and SAG101 EP domains. EDS1, PAD4, and SAG101 are shown in blue, yellow, and pink, respectively. pRib-ADP
and ADPr-ATP are shown in green. The small molecule binding grooves of the EDS1 complexes are highlighted.
(B) Small molecule binding allosterically induces EDS1-PAD4 and EDS1-SAG101 complex interaction with ADR1 and NRG1, respectively.
8Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
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RPS4-TIR-catalyzed small molecules bind to and stimulate EDS1
heterodimer interactions with ADR1 or NRG1. With high-resolution
mass spectrometry, crystallography, and cryoelectron micro-
scopy, the small molecules have been identified as structurally
related 2-(500-phosphoribosyl)-500-adenosine monophosphate
(pRib-AMP), pRib-ADP, ADP-ribosylated ATP (ADPr-ATP), and
ADP-ribosylated ADPR (di-ADPR). Chemically synthesized pRib-
AMP/ADP induce strong EDS1-PAD4 interaction with ADR1 but
weak EDS1-SAG101 interaction with NRG1. In contrast, ADPr-
ATP/di-ADPR are highly specific for promoting formation of the
EDS1-SAG101-NRG1 complex.The overlapping but distinct activ-
ity of the TIR-catalyzed products is consistent with the shared and
specific immune outputs mediated by EDS1-PAD4-ADR1 and
EDS1-SAG101-NRG1 (Dongus and Parker, 2021). Thus, specific
immune functions of the two EDS1 heterodimers could be
conferred by their recognized TIR-catalyzed small molecules.
TIR-induced assemblies of the EDS1-PAD4-ADR1 and EDS1-
SAG101-NRG1 signaling complexes explain the functional incom-
patibility between EDS1-SAG101 and NRG1 in different plant
species (Gantner et al., 2019;Lapin et al., 2019).
Structural studies reveal the mechanisms of selective EDS1-
SAG101 activation by ADRr-ATP/di-ADPR and preferential EDS1-
PAD4 activation by pRib-ADP/AMP. pRib-ADP and ADPr-ATP
interact with a similar groove formed by the C-terminal EP domains
of EDS1-PAD4 and EDS1-SAG101, respectively (Huang et al.,
2022;Jia et al., 2022;Figure 4). The pRib-ADP-contacting
residues of PAD4 are largely conserved in SAG101. In contrast,
SAG101 residues for interacting with the ADPR moiety of ADPr-
ATP are variable in PAD4. These variations result in a much
smaller ligand binding groove in EDS1-PAD4, explaining why the
complex is unable to recognize the comparatively bulkier ADPr-
ATP/di-ADPR. Mutations of amino acids at the ligand binding
sites reduce EDS1-PAD4 and EDS1-SAG101 association with
ADR1 and SAG101, respectively, and compromise immunity func-
tions in vivo(Lapin et al., 2019;Sun et al.,2021;Dongus et al., 2022).
These results support the biological significance of the EDS1-
PAD4-ADR1 and EDS1-SAG101-NRG1 complexes. pRib-ADP
and ADPr-ATP become nearly completely buried after binding to
EDS1 heterodimers. Their binding induces similar conformational
changesin the C-terminal EP domains of PAD4 andSAG101. These
results suggest that these two small molecules allosterically
induce EDS1 heterodimer interactions with downstream ADR1
and NRG1. However, whether this NRG1/ADR1 activation
mechanism is conserved among different plant species remains
unknown. One recent study indicates that nuclear accumulation
of EDS1 complexes is sufficient for ROQ1-induced cell death in
N. benthamiana (Zonnchen et al., 2022), suggesting different
activation mechanisms of EDS1.
The available data support the four TIR-catalyzed small mole-
cules as second messengers (Figure 3). A second messenger is
produced by an enzyme whose formation is regulated by a
first messenger, induces specific effectors to exert specific
biological effects, and is removed by a signal-terminating system
(Seifert, 2015). pRib-AMP and pRib-ADP are rapidly degraded in
N. benthamiana extracts (Huang et al., 2022), suggesting the
existence of mechanisms involved in eliminating TIR-catalyzed
products to terminate their signaling. The enzymes responsible
for this remain unknown, but Nudix hydrolases, which hydrolyze
a wide range of organic pyrophosphates with varying degrees
of substrate specificity (McLennan, 2006), can be potential
candidates. TIR-catalyzed small molecules are likely conserved
in different plant species. Brachypodium distachyon TIR
domain-containing protein (BdTIR), triggers cell death when
expressed in N. benthamiana (Wan et al., 2019) and catalyzes
small molecules that can activate Arabidopsis EDS1-SAG101
interaction with NRG1 in vitro (Jia et al., 2022).
In addition to being NADases, plant TIR domain proteins
also have ADPR-transferase activity (Jia et al., 2022).
In contrast to the canonical poly-ADP-ribosylation (PAR)
polymerases (Alemasova and Lavrik, 2019), however, TIRs
catalyze transfer of ADPR from NAD
+
to small molecules. When
NAD
+
is the only substrate, the NAD
+
-derived ADPR is transferred
to the NAD hydrolyzed product ADPR, resulting in formation of di-
ADPR. ADPr-ATP can be formed when NAD
+
and ATP are used
as the substrates (Figure 5). It will be of interest to investigate
whether TIRs can catalyze transfer of ADPR to other small
molecules. The configuration of C’’ (arrangement of its
covalently linking atoms) from ADPr of ADPr-ATP or di-ADPR re-
mains the same as that of NAD
+
. This contrasts with ADPR chain
extension catalyzed by PAR polymerases (Alemasova and Lavrik,
2019). Thus, di-ADPR, when generated by hydrolysis of PARs, is
an enantiomer of that catalyzed by TIRs.
TIR-catalyzed production of di-ADPR and ADPr-ATP strongly
supports the ribosyl-transferase activity of plant TNLs and
TIR domain-containing proteins (Jia et al., 2022). In vitro
biochemical data suggest that hydrolysis of these two
compounds leads to production of pRib-AMP or pRib-ADP (Jia
et al., 2022). This could be a mechanism by which plants
balance ADR1 and NRG1 signaling. Regardless of the
biosynthetic pathways of these nucleotide derivatives, plant TIR
domain proteins are multi-functional enzymes. It will be of interest
to investigate whether these TIR-catalyzed products can be
metabolized to generate novel signaling molecules.
TIR domain proteins are 20,30-cyclic AMP (cAMP)/cyclic
guanosine monophosphate (cGMP) synthetases
In addition to the enzyme activities discussed in the last section,
plant TIR domains, including the Arabidopsis TIR-only protein
RBA1 and the TIR domain of the flax TNL L7, also display
20,30-cAMP/cGMP synthetase activity with double-stranded
RNA (dsRNA) or double-stranded DNA (dsDNA) as substrates
(Yu et al., 2022)(Figure 1). In vitro, the two TIR domain proteins
strongly prefer dsRNA as a substrate for production of these
two non-canonical cyclic nucleotide monophosphates (cNMPs),
although in vivo substrates remain to be identified. In contrast,
ATP and guanosine triphosphate are not in vitro substrates of
TIRs to generate 20,30-cAMP/cGMP, suggesting that nuclease
activity is required for the synthetase activity. The nuclease activ-
ity, however, is not sufficient for TIR-mediated cell death in
N. benthamiana because a mutation of RBA1 Cys83 has little
effect on nuclease activity but substantially suppresses the cell
death activity in N. benthamiana (Yu et al., 2022). 20,30-cNMPs
were initially identified as intermediates of RNA cleavage by
RNases, which also generate 20,30-cyclophosphate-terminated
RNA oligonucleotides (Jackson, 2017). In contrast with these
RNases, no similar products are produced by plant TIR
domains when dsRNA is used as the substrate.
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 9
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RBA1 Cys83 is highly conserved among plant TIR domain proteins,
and mutations of this RBA1 residue or its equivalents in other TIRs
significantly impair the cell death activity in N. benthamiana
(Bernoux et al., 2011;Williams et al., 2016). RBA1 C83A is greatly
reduced in 20,30-cAMP/cGMP synthetase activity but still retains
wild-type-like NADase activity, supporting a critical role of
20,30-cAMP/cGMP in RBA1-mediated cell death (Yu et al., 2022).
Accumulation of these two non-canonical 20,30-cNMPs in
N. benthamiana plants expressing the wild typebut not the catalytic
mutant E86A of RBA1 is significantly enhanced. Evidence of the
significance of 20,30-cAMP/cGMP in the cell death activity also
comes from the nucleotide hydrolase NUDIX HYDROLASE
HOMOLOG 7 (NUDT7), a negative regulator of EDS1 signaling in
Arabidopsis (Bartsch et al., 2006;Ge et al., 2007). NUDT7 hydro-
lyzes 20,30-cAMP/cGMP but not 30,50-cAMP/cGMP, and the hydro-
lysis activity is completely abolished by the catalytic mutation
E154Q (Yu et al., 2022). Co-expression with wild-type NUDT7 but
not the E154Q mutant greatly suppresses RBA1-mediated cell
death activity in N. benthamiana. These results suggest that TIR-
catalyzed 20,30-cAMP/cGMP are upstream of EDS1. However,
expression of RBA1 in eds1 mutants of N. benthamiana induces
no pronounced accumulation of 20,30-cAMP/cGMP. To reconcile
these data, a model is proposed wherepositive feedback is formed
between 20,30-cAMP/cGMP and EDS1 in EDS1 signaling (Yu
et al., 2022).
Interestingly, asymmetric TIR dimers similar to those in the
RPP1 and ROQ1 resistosomes required for NADase activity
are not present in the filament structure of L7-TIR bound by dsDNA
(Figure 6).The TIR tetramer in theTNL resistosomes is incompatible
with the oligomeric TIR in the filament structure, suggesting that
TNL resistosomes might not have synthetase activity. RPP1
alone, but not the RPP1-ATR1 resistosome, exhibits 20,30-cAMP/
cGMP synthetase activity in vitro (D.Y and J.C., perosnal commu-
nication, D.Y and J.C). A more recent study shows that a mutation
of Cys90 in the Arabidopsis TNL SUPPRESSOR OF NPR1-1,
CONSTITUTIVE 1 (SNC1), predicted to be important for
20,30-cAMP/cGMP synthetase activity, has no effect on immune
response, but the enzymatic activity of the TNL remains untested
(Tian et al., 2022). It currently remains unclear whether and how
the 20,30-cAMP/cGMP synthetase activity contributes to TNL
function. It is possible that TIR-only genes are induced during
TNL activation and that 20,30-cAMP/cGMP synthesized by these
TIR-only proteins function as signal amplifiers in TNL signaling.
Alternatively spliced TIR domain proteins are required for full func-
tion of some TNLs,suggesting that coordinated expressionof alter-
native and regular transcripts of truncated TNLs and full-length
TNLs is required for their full immune activity (Jordan et al., 2002).
RBA1 mutant C83A is compromised in 20,30-cAMP/cGMP syn-
thetase activity but retains wild-type NADase activity (Yu et al.,
2022). However, the RBA1 mutant displays activity in inducing
EDS1-SAG101 interaction with NRG1 (Jia et al., 2022). This is in
concert with the observations that NADase activity is not
sufficient for TIR signaling (Horsefield et al., 2019;Wan et al.,
2019;Duxbury et al., 2020) and that inducible expression of
TIR
TIR
T
T
T
T
T
I
I
I
I
I
I
I
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
TIR
Plant TIR+NAD+Plant TIR+NAD++ATP
PT
A
-rPDARPDA-rPDA
PDA-biRpPMA-biRp
TIR
T
T
T
T
T
T
T
T
T
I
I
I
I
R
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R
R
R
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R
TIR
T
T
T
T
T
T
I
I
I
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R
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R
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R
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R
R
R
RR
R
R
R
R
R
R
R
R
R
pRib-AMP
2' cADPR
(V-cADPR)
3' cADPR
(V2-cADPR)
Bacteria and Plant TIR+NAD+
?
NADase + ADP-ribosylation
NADase
Animal TIR+NAD+
TIR
T
T
T
T
T
I
I
I
I
I
R
R
R
R
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R
R
R
R
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R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
cADPR
ADPR
Figure 5. Production of immune signal molecules by TIR-mediated NAD
+
degradation in different species.
The SARM1 TIR domain hydrolyzes NAD
+
to produce ADPR and cADPR, whereas bacterial and plant TIRs hydrolyze NAD
+
to produce ADPR, 20cADPR,
and 30cADPR. On the other hand, plant TIRs can use NAD
+
or NAD
+
/ATP as substrates to produce pRib-AMP/pRib-ADP and di-ADPR/ADPr-ATP,
respectively via their NADase and ribosyl-transferase activities. Production of pRib-ADP/AMP likely occurs through hydrolysis of ADPr-ATP/di-ADPR.
pRib-AMP might also be produced by hydrolysis of 20cADPR. Plant TIR domains can also catalyze dsRNA hydrolysis to generate 20,30-cAMP/cGMP.
The TIR domains are shown in filled blue circles. The conserved catalytic residue glutamic residue is indicated.
10 Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
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doi.org/10.1016/j.molp.2022.11.011
AvrRps4 induces no cell death and MAPK activation (Ngou et al.,
2020). How can these in vitro and in vivo data be reconciled? It
should be borne in mind that all components have to be in
place for ligands to activate receptor signaling in vivo. This is
well exemplified by activation of the animal NLR NLRP3, which
requires a priming signal for transcriptional up-regulation of
NLRP3 and pro-IL-1bin addition to NLRP3-specific stimuli
(Swanson et al., 2019). 20,30-cAMP induces many stress-related
genes in Arabidopsis (Kosmacz et al., 2018;Chodasiewicz
et al., 2022), raising the possibility that this non-canonical
cNMP and probably 20,30-cGMP as well might upregulate genes
required for activation of EDS1 signaling.
TIR domains are multi-functional enzymes
As discussed above, plant TIR domains encode multiple enzy-
matic activities catalyzing metabolism of NAD
+
, NAD
+
+ATP,
and nucleic acids, resulting in production of structurally diversi-
fied nucleotide metabolites, including pRib-ADP/AMP, di-
ADPR/ADPr-ATP, and 20,30-cAMP/cGMP. These nucleotide
compounds function to induce EDS1 signaling. Besides these
TIR-catalyzed products, plant TIR domain proteins, such as B.
distachyon TIR domain-containing protein, can also produce
20cADPR and 30cADPR (Bayless et al., 2022;Manik et al.,
2022). However, the biological function of these two nucleotide
derivatives in plant immune signaling remains unclear.
Hydrolysis of 20cADPR has been proposed to produce pRib-
AMP (Manik et al., 2022;Figure 5). However, this model cannot
explain how TIR-catalyzed pRib-ADP is produced. Alternatively,
it is formally possible that 20cADPR results from circularization
of pRib-AMP via phosphate-phosphate linkages. Bacterial TIR
domains, such as A. baumannii TIR and Thoeris B from the
bacterial antiphage defense system, also have the function of
catalyzing production of 20cADPR and 30cADPR (Ofir et al.,
2021;Manik et al., 2022;Figure 5). 30cADPR and, to a lesser
extent, 20cADPR activate the NADase activity of Thoeris A,
depleting cellular NAD
+
and causing death of bacteria. The
phytobacterial TIR domain-containing effector HopAM1 can
generate 30cADPR when hydrolyzing NAD
+
(Eastman et al.,
2022;Manik et al., 2022). Production of this compound is
associated with HopAM1 suppression of plant immunity, but it
cannot be excluded that HopAM1 also produces other small mol-
ecules for virulence. The mechanism of how HopAM1 inhibits
plant immunity remains elusive, but NAD
+
depletion could have
a role in this process. The mammalian TIR domain-containing
protein SARM1 can hydrolyze NAD
+
into nicotinamide, ADPR,
or cADPR (Shi et al., 2022). The NADase activity of SARM1 is
essential to induce axon degeneration. The underlying mecha-
nism may be direct NAD
+
depletion or production of signaling
molecules to kill cells indirectly.
CALCIUM SIGNALING IN PLANT
IMMUNITY
Ca
2+
is a shared trigger for PTI and ETI signaling
It is becoming increasingly evident that PRRs and NLRs share
many signaling components, including calcium channels,
Hydrolysis of NAD+
Hydrolysis of DNA/RNA
RPP1 resistosome
L7TIR-dsDNA filament
A
B
pRib-ADP
pRib-AMP
ADPr-ATP
di-ADPR
2’ cADPR
3’ cADPR
2’3’cAMP
2’3’cGMP
Figure 6. Plant TIR domains form different oligomers to hydrolyze NAD
+
and dsDNA/RNA.
(A) TIR domains from the RPP1 resistosome (left) form two asymmetric TIR dimers, each of which forms a composite active site. The two active sites are
important for NAD
+
hydrolysis to produce the small molecules pRib-ADP/AMP, ADPr-ATP/di-ADPR, 20cADPR, and 30cADPR.
(B) Left: the L7 TIR domains bound by dsDNA (left). Right: a tetrameric L7 TIR in the complex. The symmetric TIR dimer required for formation of the
composite active sites in the RPP1 resistosome is not present in the L7 TIR complex. Formation of the L7 TIR-dsDNA or, presumably, L7 TIR-dsRNA is
important for production of 20,30cAMP/cGMP.
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 11
Biochemical function and signaling mechanism of NLRs Molecular Plant
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doi.org/10.1016/j.molp.2022.11.011
NADPH oxidases, and MAPKs (Cui et al., 2015;Yu et al., 2017;
Zhou and Zhang, 2020;DeFalco and Zipfel, 2021). This is
supported by transcriptional profiling of activated NLRs,
including the barley CNL MLA1 (Jacob et al., 2018), the
Arabidopsis TNL RPS4 (Sohn et al., 2014), and various PRRs
(Bjornson et al., 2021), which show significant overlap in early
response genes. For example, two pathogen-induced transcrip-
tion factors, SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1
(SARD1) and CBP60g, are a convergent point in PTI and ETI
signaling (Huang et al., 2021b;Li et al., 2021). These results
suggest that PTI and ETI signaling may converge somewhere to
mediate activation of shared downstream components.
Many PAMPs and DAMPs have been shown to be sufficient to
induce rapid Ca
2+
signals in plant cell culture (Atkinson et al.,
1996;Levine et al., 1996;Gelli et al., 1997;Zimmermann et al.,
1997) and Arabidopsis plants (Ranf et al., 2008,2011;
Vadassery et al., 2009;Krol et al., 2010). Treatment with LaCl
3
completely abrogates the PAMP/DAMP-induced Ca
2+
signals,
suggesting that PM-localized, calcium-permeable channels are
required for the Ca
2+
signals. Supporting this conclusion, inhibi-
tion of Ca
2+
release from intracellular compartments by RR barely
affects Pep-13-triggered immune responses in Petroselinum
crispum (parsley) cells (Blume et al., 2000). rbohd mutants only
show a slight defect in elicitor-triggered Ca
2+
signals in Arabidop-
sis seedlings (Ranf et al., 2011). In contrast, elicitor-induced ROS
production is severely attenuated by treatment with Ca
2+
channel
blockers (Ranf et al., 2011). These results suggest that Ca
2+
sig-
nals act upstream of ROS production to trigger PTI signaling.
Pharmacological data also support involvement of Ca
2+
signaling
upstream of MAPK pathways in plant immunity (Lebrun-Garcia
et al., 1998;Romeis et al., 1999;Link et al., 2002). Several
classes of PM-localized channels, including CNGCs (Tian et al.,
2019), OSCAs (Thor et al., 2020), and glutamate-like receptors
(GLRs) (Kwaaitaal et al., 2011;Bjornson et al., 2021), implicated
in conducting Ca
2+
during plant immunity, have been identified.
Because Ca
2+
is a trigger for the PTI and ETI signaling pathways
(Figure 7), and many components are also shared in these two
processes, how can they lead to different biological outcomes?
The different origins and amplitudes of calcium signals could be
important in determining the different physiological responses of
PTI and ETI signaling. Although PAMPs/DAMPs induce a transient
rise in cytosolic Ca
2+
(Tian et al., 2019), a sustained increase in
Figure 7. Calcium signaling in plant immunity.
In PTI, PAMP-activated PRRs trigger phosphorylation of RLCKs and MAPK cascades. Activation of RLCKs leads to phosphorylation and activation of
PM-localized Ca
2+
channels, including CNGCs, GLRs, and OSCAs, for Ca
2+
influx. In ETI, pathogen effectors are perceived by the intracellular NLRs.
Pathogen effector-activated CNLs or TNL-activated RNLs form resistosomes. CNL/RNL resistosomes act as PM Ca
2+
-permeable channels to allow
extracellular Ca
2+
influx. Calcium transporters such as ACAs mediate calcium efflux to counterbalance Ca
2+
signaling. Ca
2+
signaling is involved in many
amplification loops, including Ca
2+
-ROS, Ca
2+
-SA, Ca
2+
-Ca
2+
, and PTI-ETI. The CPK, CaM, RBOHD, CAMTA3, and CBP60g transcription factors;
metacaspase; and TIR proteins play critical roles in these amplification loops. Compared with PTI, ETI triggers strong and long-lasting cytosolic Ca
2+
influx, leading to HR cell death.
12 Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
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doi.org/10.1016/j.molp.2022.11.011
cytosolic Ca
2+
is observed in NLR-mediated ETI (Bi et al., 2021;
Jacob et al., 2021). These data appear to support the Ca
2+
signa-
ture hypothesis, where spatial and temporal characteristics of
stimulus-specific Ca
2+
signals contribute to the specificity of
the biological outcome (Kim et al., 2022;Koster et al., 2022).
However, strong evidence of specific information encoded
by Ca
2+
signatures leading to a specific outcome is scarce
(Hetherington and Brownlee, 2004). Different PAMPs/DAMPs
induce distinct cytosolic Ca
2+
signatures (Ranf et al.,
2011) despite conserved downstream components of PTI
signaling. There is evidence of an alternative model to explain the
specificity of Ca
2+
signals (Scrase-Field and Knight, 2003). This
model hypothesizes that Ca
2+
simply acts as a switch to activate
Ca
2+
-dependent components and that the signaling specificity is
dictated by components other than Ca
2+
. The switch model can
also explain the differences in PTI and ETI responses. For example,
it may be that PTI- and ETI-activating channels associate with
different Ca
2+
-sensing partners to decode the different origins of
Ca
2+
signals, leading to different physiological responses. The
signatureand switch mechanismsmight operate in PTI andETI, de-
pending on the circumstances in plant cells.
In addition to Ca
2+
influx (Spalding and Harper, 2011;Edel et al.,
2017;Resentini et al., 2021), Ca
2+
efflux mediated by active trans-
porters is involved in shaping Ca
2+
signals (Figure 7). Ca
2+
in the
cytoplasm can also be pumped to the intracellular Ca
2+
stores,
including the vacuole and endoplasmic reticulum (ER), through
Ca
2+
pumps and Ca
2+
/H
+
exchangers (CAXs). The coordinate ac-
tion of Ca
2+
channels and transporters generates stimulus-specific
signals. In addition to CAXs, active Ca
2+
transporters include auto-
inhibited Ca
2+
-ATPases (ACAs) and ER Ca
2+
-ATPases (Geisler
et al., 2000;Shigaki and Hirschi, 2000;Garcia Bossi et al., 2020).
Double knockout of the PM-localized ACA8 and its homolog
ACA10 compromises flg22-induced Ca
2+
signals and resistance
to Pseudomonas syringae infection (Frei dit Frey et al., 2012)and
stomatal closure upon PAMP perception (Yang et al., 2017).
Besides PM-localized ACA8/10, ACA4/11 on tonoplasts and
ACA1/2/7 on the ER are also involved in PTI-triggered Ca
2+
signaling, although the mechanism remains unclear (Hilleary
et al., 2020;Rahmati Ishka et al., 2021). CNGCs and ACAs
are subject to regulation by CaM and phosphorylation; for
example, to control Ca
2+
transport across the PM. Binding of
CaM can activate and inactivate CNGCs (Hua et al., 2003).
CaM7-gated CNGC2-CNGC4 channel activity has been demon-
strated recently in Arabidopsis.OverexpressionofCaM7substan-
tially compromises flg22-induced PTI signaling (Tian et al., 2019).
The biological significance of CaM7 inhibition of CNGC2-CNGC4
remains unclear, but it might provide negative feedback restricting
Ca
2+
flux into plant cells. CaM stimulates theactivity of ACA pumps
by preventing their auto-inhibition. The activity of ACAs can also be
inhibited by phosphorylation in the N-terminal domain (Geisler
et al., 2000). However, more studies are needed to connect ACAs
and CAXs to the immunity signaling pathways.
Ca
2+
signals and amplification of plant immune
responses
Formation of high-order assembly is a common strategy adopted
by immune receptors in animals for amplification of immune
signaling (Cai et al., 2017). For example, filament formation of
ASC seeded by NLR inflammasomes can markedly improve the
efficiency of caspase activation by recruiting a large number of
inactive caspase molecules (Lu et al., 2014). Although higher-
order oligomers (Yu et al., 2022) and phase separation (Huang
et al., 2021a) have been shown to be involved in plant immune
signaling, the plant immune system appears to rely more on
signal amplification loops to mount quick and robust immune
responses. These amplification loops typically involve synergistic
interactions between Ca
2+
, ROS, and SA. In this section, we
discuss plant immune signaling amplification involving Ca
2+
.
ROS and intracellular calcium signaling interact with and amplify
each other, forming the ROS-Ca
2+
self-amplifying loop (Figure 7).
The major ROS-producing enzymeRBOHD, during plant immunity,
can be activated by directly binding cytosolic Ca
2+
with its N-termi-
nal EF-hand motif or phosphorylation by Ca
2+
-binding proteins
(Ogasawara et al., 2008). CPK5 has been shown to activate ROS
signaling by phosphorylating RBOHD (Boudsocq et al., 2010;
Dubiella et al., 2013). CBL1/CBL9-interacting protein kinase 26
phosphorylates RBOHF to produce ROS (Drerup et al., 2013;
Kimura et al., 2013). Abrogation of ROS accumulation in the
rbohD mutant or through inhibitor application leads to loss of a
second Ca
2+
peak during ETI, demonstrating a feedback effect of
ROS on Ca
2+
signaling (Gao et al., 2013). In addition, ROS can be
sensed by the PM-anchored RLK HYDROGEN-PEROXIDE-
INDUCED CA
2+
INCREASES 1 (HPCA1), leading to downstream
signaling, including activation of Ca
2+
influx (Tian et al., 2019;
Wu et al., 2020). A more recent study depicts a role of HPCA1 in
this ROS-Ca
2+
self-amplifying loop by showing that HPCA1
triggers an increase in cytosolic Ca
2+
levels via the calcium-
permeable channel MECHANOSENSITIVE ION CHANNEL-LIKE
3 (MSL3). HPCA1 is required for systemic cell-to-cell ROS and
calcium signaling in response to local bacterial infection and a
broad range of abiotic stresses (Fichman et al., 2022).
Ca
2+
influx and SA can form self-amplification loops to enhance
immune response. For instance, Ca
2+
influx modulates the activ-
ity of the calcium-binding transcription factor CAMTA3 and
CBP60g, which results in further upregulation of expression of
SA-biosynthesis genes, including ISOCHORISMATE SYNTHASE
1(ICS1), ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5), and
AVRPPHB-SUSCEPTIBLE 3 (PBS3)(Zhang et al., 2010b;Sun
et al., 2015;Huang et al., 2020;Figure 7). SA perception by
NONEXPRESSER OF PR GENES (NPR) 1 or NPR3/NPR4 induces
a plethora of defense-related genes, including a number of
PRRs, NLRs, and their signaling components (Ding et al., 2018).
In SA biosynthesis- or perception-defective mutants, PRR-
and NLR-mediated immunity is compromised (Zhang and Li,
2019). These data indicate that calcium influx of PRR or NLR
immune signaling promotes accumulation of SA, which, in turn,
enhances calcium signaling and facilitates PTI and ETI
responses.
Elevations in the cytosolic concentrations of Ca
2+
can activate
Ca
2+
-dependent metacaspases (Figure 7), which mature and
release an endogenous elicitor, Pep1, in Arabidopsis (Hander
et al., 2019;Shen et al., 2019). The DAMP initiates a feedback
mechanism to amplify the original PTI signaling, including
Ca
2+
influx. Ca
2+
signals can also be amplified by positive
regulation of Ca
2+
transporters and channels, including ACAs,
CNGCs, and GLRs modulated by CaMs or CPKs (Geisler et al.,
2000;Zhou et al., 2014;Pan et al., 2019). Amplification of initial
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 13
Biochemical function and signaling mechanism of NLRs Molecular Plant
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
Ca
2+
signals by secondary Ca
2+
signals generated by endomem-
brane Ca
2+
channels has been reported (Swarbreck et al., 2013),
although the identities of the secondary channels remain
unknown.
Activation of ETI requires participation of PRRs and their co-
receptors, and ETI can further amplify PTI, indicating that PRRs
and NLRs cooperatively potentiate each other to enhance plant
immune responses (Ngou et al., 2021;Pruitt et al., 2021;Tian
et al., 2021;Yuan et al., 2021). Given that Ca
2+
is a shared trigger
for plant immunity, this raises the question of whether Ca
2+
has a
role in mutual potentiation of PTI and ETI. ETI-bolstered PTI is
proposed to occur through elevation in intracellular Ca
2+
concen-
trations (Bjornson and Zipfel, 2021). This model is consistent with
Ca
2+
-dependent transcriptional regulation of immune genes dur-
ing ETI (Gao et al., 2013). Ca
2+
influx promotes expression of TIR
domain protein-encoding genes during PTI, and activation of
these TIRs is important for PTI signaling in Arabidopsis (Pruitt
et al., 2021;Tian et al., 2021). Activation of TIR signaling
presumably results in Ca
2+
-permeable channel activity of ADR1
and NRG1 (Jacob et al., 2021). These results indicate a critical
role of self-amplification of Ca
2+
signals in PTI. However, it cannot
be excluded that activation of TIRs leads to Ca
2+
-unrelated activ-
ity to promote PTI signaling. It currently remains unknown
whether Ca
2+
has a role in potentiating ETI by PTI, but there is ev-
idence indicating that the ETI-potentiating signals seem not to be
PTI specific. The intensity of cell death of the RPS4-expressing
N. benthamiana plants is strictly correlated with expression levels
of the TNL protein (Zhang et al., 2004). This result suggests that
elevation in protein concentrations can have a role similar to
PTI signaling in promoting RPS4-mediated cell death.
PERSPECTIVES
Ca
2+
is a universalsecond messenger involved in diverse biological
processes, including development and immunity (Tian et al., 2020).
Many findings in the past few years highlight a critical role of Ca
2+
-
permeable channels in triggering initiation of PTI and ETI signaling
(Xu et al., 2022a;Kim et al., 2022;Koster et al., 2022). These
discoveries started to decipher the mechanisms underlying
pathogen-induced Ca
2+
influx, and some PM-localized channels
involved in conducting Ca
2+
during plant immunity were identified.
Particularly, recent findings regarding plant resistosomes revealed
that CNLs and TNLs can converge on Ca
2+
signals to mediate ETI
responses. However, CNLs resistosomes are non-canonical Ca
2+
-
permeablechannels, and little is knownabout their channel proper-
ties. It remains completely unknown whether they are subjected to
regulation like CNGCs and other canonical calcium channels.
Biophysical and electrophysiologicalcharacterizationof CNL resis-
tosomes and Ca
2+
channels involved in PTI signaling will facilitate
our understanding of their gatingproperties. Such studiesmay pro-
vide insight into the quick and transient Ca
2+
currents during PTI
and sustained Ca
2+
currents during ETI. Future studies directed to-
ward deciphering how Ca
2+
influx is translated into downstream
transcriptional signals will advance our understanding of immune
signaling.Many Ca
2+
sensors,particularly CaMs and CMLs,are en-
coded in plant genomes. Therefore,it remains a challenge to define
the biological role of a specific Ca
2+
sensor in immune signaling.
Valuable information to dissect specific functions of CaM/CMLs
could be gained through analyses of gene expression, subcellular
localization of proteins, and identification of their targets. It would
be also challenging to disentangle the interplay among different
Ca
2+
channels and the relationship between Ca
2+
channels and
Ca
2+
transporters. Such studies may provide clues about how
Ca
2+
signaling specificity is achieved. PTI and ETI inter-potentiate
in plant immunity to achieve resistance against pathogens, but
the underlying molecular mechanisms remain largely unknown.
FUNDING
This work was supported by the Young Elite Scientists Sponsor-
ship Program by CAST (grant YESS20210018 to J.W.), the Na-
tional Natural Science Foundation of China (grant 32 271 253 to
J.W.), the Alexander von Humboldt Foundation (professorship
to J.C.), the Max-Planck-Gesellschaft (a Max Planck fellowship
to J.C.), Deutsche Forschungsgemeinschaft (grant SFB-1403-
414 786 233 to J.C.), Germany’s Excellence Strategy CEPLAS
(EXC-2048/1, project 390 686 111 to J.C.).
ACKNOWLEDGMENTS
No conflict of interest is declared.
Received: September 15, 2022
Revised: November 7, 2022
Accepted: November 21, 2022
Published: November 21, 2022
REFERENCES
Adachi, H., Contreras, M., Harant, A., Wu, C.H., Derevnina, L., Sakai,
T., Duggan, C., Moratto, E., Bozkurt, T.O., Maqbool, A., et al.
(2019). An N-terminal motif in NLR immune receptors is functionally
conserved across distantly related plant species. Elife 8:e49956.
Ahn, H.-K., Lin, X., Olave-Achury, A.C., Derevnina, L., Contreras, M.P.,
Kourelis, J., Kamoun, S., and Jones, J.D.G. (2022). Effector-
dependent Activation and Oligomerization of NRC Helper NLRs by
Rpi-Amr3 and Rpi-Amr1. Preprint at bioRxiv. https://doi.org/10.1101/
2022.04.25.489359.
Albert, I., Bohm, H., Albert, M., Feiler, C.E., Imkampe, J., Wallmeroth,
N., Brancato, C., Raaymakers, T.M., Oome, S., Zhang, H., et al.
(2015). An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered
immunity. Native Plants 1:15140.
Alemasova, E.E., and Lavrik, O.I. (2019). Poly(ADP-ribosyl)ation by
PARP1: reaction mechanism and regulatory proteins. Nucleic Acids
Res. 47:3811–3827.
Atkinson, M.M., Midland, S.L., Sims, J.J., and Keen, N.T. (1996).
Syringolide 1 triggers Ca2+ influx, K+ efflux, and extracellular
alkalization in soybean cells carrying the disease-resistance gene
Rpg4. Plant Physiol. 112:297–302.
Bartsch, M., Gobbato, E., Bednarek, P., Debey, S., Schultze, J.L.,
Bautor, J., and Parker, J.E. (2006). Salicylic acid-independent
ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis
immunity and cell death is regulated by the monooxygenase FMO1
and the Nudix hydrolase NUDT7. Plant Cell 18:1038–1051.
Baudin, M., Hassan, J.A., Schreiber, K.J., and Lewis, J.D. (2017).
Analysis of the ZAR1 immune complex reveals determinants for
immunity and molecular interactions. Plant Physiol. 174:2038–2053.
Bayless, A.M., Chen, S., Ogden, S.C., Xu, X., Sidda, J.D., Manik, M.K.,
Li, S., Kobe, B., Ve, T., Song, L., et al. (2022). Plant and prokaryotic
TIR domains generate distinct cyclic ADPR NADase products.
Preprint at bioRxiv. https://doi.org/10.1101/2022.09.19.508568.
Bernoux, M., Ve, T., Williams, S., Warren, C., Hatters, D., Valkov, E.,
Zhang, X., Ellis, J.G., Kobe, B., and Dodds, P.N. (2011). Structural
and functional analysis of a plant resistance protein TIR domain
14 Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
reveals interfaces for self-association, signaling, and autoregulation.
Cell Host Microbe 9:200–211.
Bhandari, D.D., Lapin, D., Kracher, B., von Born, P., Bautor, J.,
Niefind, K., and Parker, J.E. (2019). An EDS1 heterodimer signalling
surface enforces timely reprogramming of immunity genes in
Arabidopsis. Nat. Commun. 10:772.
Bi, G., Liebrand, T.W., Bye, R.R., Postma, J., van der Burgh, A.M.,
Robatzek, S., Xu, X., and Joosten, M.H. (2016). SOBIR1 requires
the GxxxG dimerization motif in its transmembrane domain to form
constitutive complexes with receptor-like proteins. Mol. Plant Pathol.
17:96–107.
Bi, G., Su, M., Li, N., Liang, Y., Dang, S., Xu, J., Hu, M., Wang, J., Zou,
M., Deng, Y., et al. (2021). The ZAR1 resistosome is a calcium-
permeable channel triggering plant immune signaling. Cell 184:3528–
3541.e3512.
Bi, G., Zhou, Z., Wang, W., Li, L., Rao, S., Wu, Y., Zhang, X., Menke,
F.L.H., Chen, S., and Zhou, J.M. (2018). Receptor-like cytoplasmic
kinases directly link diverse pattern recognition receptors to the
activation of mitogen-activated protein kinase cascades in
Arabidopsis. Plant Cell 30:1543–1561.
Bjornson, M., Pimprikar, P., Nurnberger, T., and Zipfel, C. (2021). The
transcriptional landscape of Arabidopsis thaliana pattern-triggered
immunity. Native Plants 7:579–586.
Bjornson, M., and Zipfel, C. (2021). Plant immunity: crosstalk between
plant immune receptors. Curr. Biol. 31:R796–R798.
Blume, B., Nurnberger, T., Nass, N., and Scheel, D. (2000). Receptor-
mediated increase in cytoplasmic free calcium required for activation
of pathogen defense in parsley. Plant Cell 12:1425–1440.
Bonardi, V., Tang, S., Stallmann, A., Roberts, M., Cherkis, K., and
Dangl, J.L. (2011). Expanded functions for a family of plant
intracellular immune receptors beyond specific recognition of
pathogen effectors. Proc. Natl. Acad. Sci. USA 108:16463–16468.
Boudsocq,M., Willmann,M.R., McCormack,M., Lee, H., Shan, L.,He, P.,
Bush, J., Cheng,S.H., and Sheen, J. (2010). Differential innate immune
signalling via Ca(2+) sensor protein kinases. Nature 464:418–422.
Boutrot, F., and Zipfel, C. (2017). Function, discovery, and Exploitation of
plant pattern recognition receptors for broad-spectrum disease
resistance. Annu. Rev. Phytopathol. 55:257–286.
Bryan, P.N., and Orban, J. (2010). Proteins that switch folds. Curr. Opin.
Struct. Biol. 20:482–488.
Cai, X., Xu, H., and Chen, Z.J. (2017). Prion-like polymerization in
immunity and inflammation. Cold Spring Harbor Perspect. Biol.
9:a023580.
Cao, J., Nash, G., and Zhang, L. (2022). Structural mechanisms of
inflammasome regulation revealed by cryo-EM studies. Curr. Opin.
Struct. Biol. 75:102390.
Castel, B., Ngou, P.M., Cevik, V., Redkar, A., Kim, D.S., Yang, Y., Ding,
P., and Jones, J.D.G. (2019). Diverse NLR immune receptors activate
defence via the RPW8-NLR NRG1. New Phytol. 222:966–980.
Century, K.S., Shapiro, A.D., Repetti, P.P., Dahlbeck, D., Holub, E.,
and Staskawicz, B.J. (1997). NDR1, a pathogen-induced
component required for Arabidopsis disease resistance. Science
278:1963–1965.
Cesari, S., Moore, J., Chen, C., Webb, D., Periyannan, S., Mago, R.,
Bernoux, M., Lagudah, E.S., and Dodds, P.N. (2016). Cytosolic
activation of cell death and stem rust resistance by cereal MLA-
family CC-NLR proteins. Proc. Natl. Acad. Sci. USA 113:10204–10209.
Cesari, S., Thilliez, G., Ribot, C., Chalvon, V., Michel, C., Jauneau, A.,
Rivas, S., Alaux, L., Kanzaki, H., Okuyama, Y., et al. (2013). The rice
resistance protein pair RGA4/RGA5 recognizes the Magnaporthe
oryzae effectors AVR-Pia and AVR1-CO39 by direct binding. Plant
Cell 25:1463–1481.
Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger,
T., Jones, J.D., Felix, G., and Boller, T. (2007). A flagellin-induced
complex of the receptor FLS2 and BAK1 initiates plant defence.
Nature 448:497–500.
Chodasiewicz, M., Kerber, O., Gorka, M., Moreno, J.C., Maruri-Lopez,
I., Minen, R.I., Sampathkumar, A., Nelson, A.D.L., and Skirycz, A.
(2022). 2’,3’-cAMP treatment mimics the stress molecular response
in Arabidopsis thaliana. Plant Physiol. 188:1966–1978.
Collier, S.M., Hamel, L.P., and Moffett, P. (2011). Cell death mediated by
the N-terminal domains of a unique and highly conserved class of NB-
LRR protein. Mol. Plant Microbe Interact. 24:918–931.
Contreras, M.P., Pai, H., Tumtas, Y., Duggan, C., Him Yuen, E.L.,
Cruces, A.V., Kourelis, J., Ahn, H.-K., Wu, C.-H., Bozkurt, T.O.,
et al. (2022). Sensor NLR Immune Proteins Activate Oligomerization
of Their NRC Helper. Preprint at bioRxiv. https://doi.org/10.1101/
2022.04.25.489342.
Cui, H., Tsuda, K., and Parker, J.E. (2015). Effector-triggered immunity:
from pathogen perception to robust defense. Annu. Rev. Plant Biol.
66:487–511.
Day, B., Dahlbeck, D., and Staskawicz, B.J. (2006). NDR1 interaction
with RIN4 mediates the differential activation of multiple disease
resistance pathways in Arabidopsis. Plant Cell 18:2782–2791.
DeFalco, T.A., and Zipfel, C. (2021). Molecular mechanisms of early plant
pattern-triggered immune signaling. Mol. Cell 81:3449–3467.
Ding, J., Wang, K., Liu, W., She, Y., Sun, Q., Shi, J., Sun, H., Wang, D.C.,
and Shao, F. (2016). Pore-forming activity and structural autoinhibition
of the gasdermin family. Nature 535:111–116.
Ding, Y., Sun, T., Ao, K., Peng, Y., Zhang, Y., Li, X., and Zhang, Y.
(2018). Opposite roles of salicylic acid receptors NPR1 and NPR3/
NPR4 in transcriptional regulation of plant immunity. Cell 173:1454–
1467.e1415.
Dongus, J.A., Bhandari, D.D., Penner, E., Lapin, D., Stolze, S.C.,
Harzen, A., Patel, M., Archer, L., Dijkgraaf, L., Shah, J., et al.
(2022). Cavity surface residues of PAD4 and SAG101 contribute to
EDS1 dimer signaling specificity in plant immunity. Plant J.
110:1415–1432.
Dongus, J.A., and Parker, J.E. (2021). EDS1 signalling: at the nexus of
intracellular and surface receptor immunity. Curr. Opin. Plant Biol.
62:102039.
Drerup, M.M., Schlucking, K., Hashimoto, K., Manishankar, P.,
Steinhorst, L., Kuchitsu, K., and Kudla, J. (2013). The Calcineurin
B-like calcium sensors CBL1 and CBL9 together with their
interacting protein kinase CIPK26 regulate the Arabidopsis NADPH
oxidase RBOHF. Mol. Plant 6:559–569.
Dubiella, U., Seybold, H., Durian, G., Komander, E., Lassig, R., Witte,
C.P., Schulze, W.X., and Romeis, T. (2013). Calcium-dependent
protein kinase/NADPH oxidase activation circuit is required for rapid
defense signal propagation. Proc. Natl. Acad. Sci. USA 110:8744–8749.
Duggan, C., Moratto, E., Savage, Z., Hamilton, E., Adachi, H., Wu,
C.H., Leary, A.Y., Tumtas, Y., Rothery, S.M., Maqbool, A., et al.
(2021). Dynamic localization of a helper NLR at the plant-pathogen
interface underpins pathogen recognition. Proc. Natl. Acad. Sci.
USA 118.
Duxbury, Z., Wang, S., MacKenzie, C.I., Tenthorey, J.L., Zhang, X.,
Huh, S.U., Hu, L., Hill, L., Ngou, P.M., Ding, P., et al. (2020).
Induced proximity of a TIR signaling domain on a plant-mammalian
NLR chimera activates defense in plants. Proc. Natl. Acad. Sci. USA
117:18832–18839.
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 15
Biochemical function and signaling mechanism of NLRs Molecular Plant
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
Duxbury, Z., Wu, C.H., and Ding, P. (2021). A comparative overview of
the intracellular guardians of plants and animals: NLRs in innate
immunity and beyond. Annu. Rev. Plant Biol. 72:155–184.
Eastman, S., Smith, T., Zaydman, M.A., Kim, P., Martinez, S.,
Damaraju, N., DiAntonio, A., Milbrandt, J., Clemente, T.E., Alfano,
J.R., et al. (2022). A phytobacterial TIR domain effector manipulates
NAD(+) to promote virulence. New Phytol. 233:890–904.
Edel, K.H., Marchadier, E., Brownlee, C., Kudla, J., and Hetherington,
A.M. (2017). The evolution of calcium-based signalling in plants. Curr.
Biol. 27:R667–R679.
El Kasmi, F., Chung, E.H., Anderson, R.G., Li, J., Wan, L., Eitas, T.K.,
Gao, Z., and Dangl, J.L. (2017). Signaling from the plasma-
membrane localized plant immune receptor RPM1 requires self-
association of the full-length protein. Proc. Natl. Acad. Sci. USA
114:E7385–E7394.
Elmore, J.M., Liu, J., Smith, B., Phinney, B., and Coaker, G. (2012).
Quantitative proteomics reveals dynamic changes in the plasma
membrane during Arabidopsis immune signaling. Mol. Cell.
Proteomics 11. M111 014555.
Essuman, K., Summers, D.W., Sasaki, Y., Mao, X., DiAntonio, A., and
Milbrandt, J. (2017). The SARM1 toll/interleukin-1 receptor domain
possesses intrinsic NAD(+) cleavage activity that promotes
pathological axonal degeneration. Neuron 93:1334–1343.e1335.
Feehan, J.M., Castel, B., Bentham, A.R., and Jones, J.D. (2020). Plant
NLRs get by with a little help from their friends. Curr. Opin. Plant Biol.
56:99–108.
Feehan, J.M., Wang, J., Sun, X., Choi, J., Ahn, H.-K., Ngou, B.P.M.,
Parker, J.E., and Jones, J.D.G. (2022). Oligomerisation of a plant
helper NLR requires cell-surface and intracellular immune receptor
activation. Preprint at bioRxiv. https://doi.org/10.1101/2022.2006.
2016.496440.
Felix, G., Duran, J.D., Volko, S., and Boller, T. (1999). Plants have a
sensitive perception system for the most conserved domain of
bacterial flagellin. Plant J. 18:265–276.
Fichman, Y., Zandalinas, S.I., Peck, S., Luan, S., and Mittler, R. (2022).
HPCA1 is required for systemic reactive oxygen species and calcium
cell-to-cell signaling and plant acclimation to stress. Plant Cell
34:4453–4471.
Fleckenstein, A., Janke, J., Doring, H.J., and Leder, O. (1974).
Myocardial fiber necrosis due to intracellular Ca overload-a new
principle in cardiac pathophysiology. Recent Adv. Stud. Card. Struct.
Metabol. 4:563–580.
F€
orderer, A., Li, E., Lawson, A., Deng, Y.-n., Sun, Y., Logemann, E.,
Zhang, X., Wen, J., Han, Z., Chang, J., et al. (2022). A wheat
resistosome defines common principles of immune receptor
channels. Nature 610:532–539.
Forderer, A., Yu, D., Li, E., and Chai, J. (2022). Resistosomes at the
interface of pathogens and plants. Curr. Opin. Plant Biol. 67:102212.
Frei dit Frey, N., Mbengue, M., Kwaaitaal, M., Nitsch, L., Altenbach, D.,
Haweker, H., Lozano-Duran, R., Njo, M.F., Beeckman, T., Huettel,
B., et al. (2012). Plasma membrane calcium ATPases are important
components of receptor-mediated signaling in plant immune
responses and development. Plant Physiol. 159:798–809.
Gabriels, S.H., Vossen, J.H., Ekengren, S.K., van Ooijen, G., Abd-El-
Haliem, A.M., van den Berg, G.C., Rainey, D.Y., Martin, G.B.,
Takken, F.L., de Wit, P.J., et al. (2007). An NB-LRR protein required
for HR signalling mediated by both extra- and intracellular resistance
proteins. Plant J. 50:14–28.
Gantner, J., Ordon, J., Kretschmer, C., Guerois, R., and Stuttmann, J.
(2019). An EDS1-SAG101 complex is essential for TNL-mediated
immunity in Nicotiana benthamiana. Plant Cell 31:2456–2474.
Gao, L.A., Wilkinson, M.E., Strecker, J., Makarova, K.S., Macrae, R.K.,
Koonin, E.V., and Zhang, F. (2022). Prokaryotic innate immunity
through pattern recognition of conserved viral proteins. Science
377:eabm4096.
Gao, X., Chen, X., Lin, W., Chen, S., Lu, D., Niu, Y., Li, L., Cheng, C.,
McCormack, M., Sheen, J., et al. (2013). Bifurcation of Arabidopsis
NLR immune signaling via Ca(2)(+)-dependent protein kinases. PLoS
Pathog. 9:e1003127.
Gao, Z., Chung, E.H., Eitas, T.K., and Dangl, J.L. (2011). Plant
intracellular innate immune receptor Resistance to Pseudomonas
syringae pv. maculicola 1 (RPM1) is activated at, and functions on,
the plasma membrane. Proc. Natl. Acad. Sci. USA 108:7619–7624.
Garcia Bossi, J., Kumar, K., Barberini, M.L., Dominguez, G.D.,
Rondon Guerrero, Y.D.C., Marino-Buslje, C., Obertello, M.,
Muschietti, J.P., and Estevez, J.M. (2020). The role of P-type IIA
and P-type IIB Ca2+-ATPases in plant development and growth. J.
Exp. Bot. 71:1239–1248.
Gay, N.J., Symmons, M.F., Gangloff, M., and Bryant, C.E. (2014).
Assembly and localization of Toll-like receptor signalling complexes.
Nat. Rev. Immunol. 14:546–558.
Ge, X., Li, G.J., Wang, S.B., Zhu, H., Zhu, T., Wang, X., and Xia, Y.
(2007). AtNUDT7, a negative regulator of basal immunity in
Arabidopsis, modulates two distinct defense response pathways and
is involved in maintaining redox homeostasis. Plant Physiol.
145:204–215.
Geisler, M., Axelsen, K.B., Harper, J.F., and Palmgren, M.G. (2000).
Molecular aspects of higher plant P-type Ca(2+)-ATPases. Biochim.
Biophys. Acta 1465:52–78.
Gelli, A., Higgins, V.J., and Blumwald, E. (1997). Activation of plant
plasma membrane Ca2+-permeable channels by race-specific fungal
elicitors. Plant Physiol. 113:269–279.
Gerdts, J., Brace, E.J., Sasaki, Y., DiAntonio, A., and Milbrandt, J.
(2015). SARM1 activation triggers axon degeneration locally via
NAD(+) destruction. Science 348:453–457.
Gomez-Gomez, L., and Boller, T. (2000). FLS2: an LRR receptor-like
kinase involved in the perception of the bacterial elicitor flagellin in
Arabidopsis. Mol. Cell 5:1003–1011.
Grant, M., Brown, I., Adams, S., Knight, M., Ainslie, A., and Mansfield,
J. (2000). The RPM1 plant disease resistance gene facilitates a rapid
and sustained increase in cytosolic calcium that is necessary for the
oxidative burst and hypersensitive cell death. Plant J. 23:441–450.
Grant, M.R., Godiard, L., Straube, E., Ashfield, T., Lewald, J., Sattler,
A., Innes, R.W., and Dangl, J.L. (1995). Structure of the Arabidopsis
RPM1 gene enabling dual specificity disease resistance. Science
269:843–846.
Gu, Y., Zavaliev, R., and Dong, X. (2017). Membrane trafficking in plant
immunity. Mol. Plant 10:1026–1034.
Gu, Y., Zebell, S.G., Liang, Z., Wang, S., Kang, B.H., and Dong, X.
(2016). Nuclear pore permeabilization is a convergent signaling event
in effector-triggered immunity. Cell 166:1526–1538.e11.
Gust, A.A., and Felix, G. (2014). Receptor like proteins associate with
SOBIR1-type of adaptors to form bimolecular receptor kinases. Curr.
Opin. Plant Biol. 21:104–111.
Hander, T., Fernandez-Fernandez, A.D., Kumpf, R.P., Willems, P.,
Schatowitz, H., Rombaut, D., Staes, A., Nolf, J., Pottie, R., Yao,
P., et al. (2019). Damage on plants activates Ca(2+)-dependent
metacaspases for release of immunomodulatory peptides. Science
363:eaar7486.
He, W.T., Wan, H., Hu, L., Chen, P., Wang, X., Huang, Z., Yang, Z.H.,
Zhong, C.Q., and Han, J. (2015). Gasdermin D is an executor of
pyroptosis and required for interleukin-1beta secretion. Cell Res.
25:1285–1298.
16 Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
Hetherington, A.M., and Brownlee, C. (2004). The generation of Ca(2+)
signals in plants. Annu. Rev. Plant Biol. 55:401–427.
Hilleary, R., Paez-Valencia, J., Vens, C., Toyota, M., Palmgren, M., and
Gilroy, S. (2020). Tonoplast-localized Ca(2+) pumps regulate Ca(2+)
signals during pattern-triggered immunity in Arabidopsis thaliana.
Proc. Natl. Acad. Sci. USA 117:18849–18857.
Hochheiser, I.V., Pilsl, M., Hagelueken, G., Moecking, J., Marleaux,
M., Brinkschulte, R., Latz, E., Engel, C., and Geyer, M. (2022).
Structure of the NLRP3 decamer bound to the cytokine release
inhibitor CRID3. Nature 604:184–189.
Hogrel, G., Guild, A., Graham, S., Rickman, H., Gruschow, S.,
Bertrand, Q., Spagnolo, L., and White, M.F. (2022). Cyclic
nucleotide-induced helical structure activates a TIR immune effector.
Nature 608:808–812.
Horsefield, S., Burdett, H., Zhang, X., Manik, M.K., Shi, Y., Chen, J., Qi,
T., Gilley, J., Lai, J.S., Rank, M.X., et al. (2019). NAD(+) cleavage
activity by animal and plant TIR domains in cell death pathways.
Science 365:793–799.
Hu, M., Qi, J., Bi, G., and Zhou, J.M. (2020). Bacterial effectors induce
oligomerization of immune receptor ZAR1 in vivo. Mol. Plant
13:793–801.
Hu, Z., and Chai, J. (2016). Structural mechanisms in NLR inflammasome
assembly and signaling. Curr. Top. Microbiol. Immunol. 397:23–42.
Hu, Z., Yan, C., Liu, P., Huang, Z., Ma, R., Zhang, C., Wang, R., Zhang,
Y., Martinon, F., Miao, D., et al. (2013). Crystal structure of NLRC4
reveals its autoinhibition mechanism. Science 341:172–175.
Hu, Z., Zhou, Q., Zhang, C., Fan, S., Cheng, W., Zhao, Y., Shao, F.,
Wang, H.W., Sui, S.F., and Chai, J. (2015). Structural and
biochemical basis for induced self-propagation of NLRC4. Science
350:399–404.
Hua, B.-G., Mercier, R.W., Zielinski, R.E., and Berkowitz, G.A. (2003).
Functional interaction of calmodulin with a plant cyclic nucleotide
gated cation channel. Plant Physiol. Biochem. 41:945–954.
Huang, S., Jia, A., Song, W., Hessler, G., Meng, Y., Sun, Y., Xu, L.,
Laessle, H., Jirschitzka, J., Ma, S., et al. (2022). Identification and
receptor mechanism of TIR-catalyzed small molecules in plant
immunity. Science 377:eabq3297.
Huang, S., Zhu, S., Kumar, P., and MacMicking, J.D. (2021a). A phase-
separated nuclear GBPL circuit controls immunity in plants. Nature
594:424–429.
Huang, W., Wang, Y., Li, X., and Zhang, Y. (2020). Biosynthesis and
regulation of salicylic acid and N-hydroxypipecolic acid in plant
immunity. Mol. Plant 13:31–41.
Huang, W., Wu, Z., Tian, H., Li, X., and Zhang, Y. (2021b). Arabidopsis
CALMODULIN-BINDING PROTEIN 60b plays dual roles in plant
immunity. Plant Commun. 2:100213.
Huffaker, A., Pearce, G., and Ryan, C.A. (2006). An endogenous peptide
signal in Arabidopsis activates components of the innate immune
response. Proc. Natl. Acad. Sci. USA 103:10098–10103.
Jackson, E.K. (2017). Discovery and roles of 2’,3’-cAMP in biological
systems. Handb. Exp. Pharmacol. 238:229–252.
Jacob, F., Kracher, B., Mine, A., Seyfferth, C., Blanvillain-Baufume, S.,
Parker, J.E., Tsuda, K., Schulze-Lefert, P., and Maekawa, T. (2018).
A dominant-interfering camta3 mutation compromises primary
transcriptional outputs mediated by both cell surface and intracellular
immune receptors in Arabidopsis thaliana. New Phytol. 217:1667–16 80.
Jacob, P., Kim, N.H., Wu, F., El-Kasmi, F., Chi, Y., Walton, W.G.,
Furzer, O.J., Lietzan, A.D., Sunil, S., Kempthorn, K., et al. (2021).
Plant "helper" immune receptors are Ca(2+)-permeable nonselective
cation channels. Science 373:420–425.
Jia, A., Huang, S., Song, W., Wang, J., Meng, Y., Sun, Y., Xu, L.,
Laessle, H., Jirschitzka, J., Hou, J., et al. (2022). TIR-catalyzed
ADP-ribosylation reactions produce signaling molecules for plant
immunity. Science 377:eabq8180.
Jones, J.D., Vance, R.E., and Dangl, J.L. (2016). Intracellular innate
immune surveillance devices in plants and animals. Science
354:aaf6395.
Jordan, T., Schornack, S., and Lahaye, T. (2002). Alternative splicing of
transcripts encoding Toll-like plant resistance proteins - what’s the
functional relevance to innate immunity? Trends Plant Sci. 7:392–398.
Jubic, L.M., Saile, S., Furzer, O.J., El Kasmi, F., and Dangl, J.L. (2019).
Help wanted: helper NLRs and plant immune responses. Curr. Opin.
Plant Biol. 50:82–94.
Kadota, Y., Sklenar, J., Derbyshire, P., Stransfeld, L., Asai, S.,
Ntoukakis, V., Jones, J.D., Shirasu, K., Menke, F., Jones, A., et al.
(2014). Direct regulation of the NADPH oxidase RBOHD by the PRR-
associated kinase BIK1 during plant immunity. Mol. Cell 54:43–55.
Kayagaki, N., Stowe, I.B., Lee, B.L., O’Rourke, K., Anderson, K.,
Warming, S., Cuellar, T., Haley, B., Roose-Girma, M., Phung,
Q.T., et al. (2015). Caspase-11 cleaves gasdermin D for non-
canonical inflammasome signalling. Nature 526:666–671.
Kim, N.H., Jacob, P., and Dangl, J.L. (2022). Con-Ca(2+) -tenating plant
immune responses via calcium-permeable cation channels. New
Phytol. 234:813–818.
Kimura, S., Kawarazaki, T., Nibori, H., Michikawa, M., Imai, A., Kaya,
H., and Kuchitsu, K. (2013). The CBL-interacting protein kinase
CIPK26 is a novel interactor of Arabidopsis NADPH oxidase AtRbohF
that negatively modulates its ROS-producing activity in a
heterologous expression system. J. Biochem. 153:191–195.
Knepper, C., Savory, E.A., and Day, B. (2011). The role of NDR1 in
pathogen perception and plant defense signaling. Plant Signal.
Behav. 6:1114–1116.
Kofoed, E.M., and Vance, R.E. (2011). Innate immune recognition of
bacterial ligands by NAIPs determines inflammasome specificity.
Nature 477:592–595.
Kosmacz, M., Luzarowski, M., Kerber, O., Leniak, E., Gutierrez-
Beltran, E., Moreno, J.C., Gorka, M., Szlachetko, J., Veyel, D.,
Graf, A., et al. (2018). Interaction of 2’,3’-cAMP with Rbp47b plays a
role in stress granule formation. Plant Physiol. 177:411–421.
Koster, P., DeFalco, T.A., and Zipfel, C. (2022). Ca(2+) signals in plant
immunity. EMBO J. 41:e110741.
Krasileva, K.V., Dahlbeck, D., and Staskawicz, B.J. (2010). Activation of
an Arabidopsis resistance protein is specified by the in planta
association of its leucine-rich repeat domain with the cognate
oomycete effector. Plant Cell 22:2444–2458.
Krol, E., Mentzel, T., Chinchilla, D., Boller, T., Felix, G., Kemmerling,
B., Postel, S., Arents, M., Jeworutzki, E., Al-Rasheid, K.A., et al.
(2010). Perception of the Arabidopsis danger signal peptide 1
involves the pattern recognition receptor AtPEPR1 and its close
homologue AtPEPR2. J. Biol. Chem. 285:13471–13479.
K€
unstler, A., Bacso
´, R., Gullner, G., Hafez, Y.M., and Kira
´ly, L. (2016).
Staying alive – is cell death dispensable for plant disease resistance
during the hypersensitive response? Physiol. Mol. Plant Pathol.
93:75–84.
Kwaaitaal, M., Huisman, R., Maintz, J., Reinstadler, A., and
Panstruga, R. (2011). Ionotropic glutamate receptor (iGluR)-like
channels mediate MAMP-induced calcium influx in Arabidopsis
thaliana. Biochem. J. 440:355–365.
Lapin, D., Johanndrees, O., Wu, Z., Li, X., and Parker, J.E. (2022).
Molecular innovations in plant TIR-based immunity signaling. Plant
Cell 34:1479–1496.
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 17
Biochemical function and signaling mechanism of NLRs Molecular Plant
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
Lapin, D., Kovacova, V., Sun, X., Dongus, J.A., Bhandari, D.D., von
Born, P., Bautor, J., Guarneri, N., Rzemieniewski, J., Stuttmann,
J., et al. (2019). A coevolved EDS1-SAG101-NRG1 module mediates
cell death signaling by TIR-domain immune receptors. Plant Cell
31:2430–2455.
Le Roux, C., Huet, G., Jauneau, A., Camborde, L., Tremousaygue, D.,
Kraut, A., Zhou, B., Levaillant, M., Adachi, H., Yoshioka, H., et al.
(2015). A receptor pair with an integrated decoy converts pathogen
disabling of transcription factors to immunity. Cell 161:1074–1088.
Leavitt, A., Yirmiya, E., Amitai, G., Lu, A., Garb, J., Herbst, E.,
Morehouse, B.R., Hobbs, S.J., Antine, S.P., Sun, Z.J., et al. (2022).
Viruses inhibit TIR gcADPR signalling to overcome bacterial defence.
Nature. https://doi.org/10.1038/s41586-022-05375-9.
Lebrun-Garcia, A., Ouaked, F., Chiltz, A., and Pugin, A. (1998).
Activation of MAPK homologues by elicitors in tobacco cells. Plant J.
15:773–781.
Lee, D.H., Lee, H.S., and Belkhadir, Y. (2021). Coding of plant immune
signals by surface receptors. Curr. Opin. Plant Biol. 62:102044.
Levine, A., Pennell, R.I., Alvarez, M.E., Palmer, R., and Lamb, C. (1996).
Calcium-mediated apoptosis in a plant hypersensitive disease
resistance response. Curr. Biol. 6:427–437.
Lewis, J.D., Wu, R., Guttman, D.S., and Desveaux, D. (2010). Allele-
specific virulence attenuation of the Pseudomonas syringae HopZ1a
type III effector via the Arabidopsis ZAR1 resistance protein. PLoS
Genet. 6:e1000894.
Li, L., Li, M., Yu, L., Zhou, Z., Liang, X., Liu, Z., Cai, G., Gao, L., Zhang,
X., Wang, Y., et al. (2014). The FLS2-associated kinase BIK1 directly
phosphorylates the NADPH oxidase RbohD to control plant
immunity. Cell Host Microbe 15:329–338.
Li, L.S., Ying, J., Li, E., Ma, T., Li, M., Gong, L.M., Wei, G., Zhang, Y.,
and Li, S. (2021). Arabidopsis CBP60b is a central transcriptional
activator of immunity. Plant Physiol. 186:1645–1659.
Li, Y., Zhou, M., Hu, Q., Bai, X.C., Huang, W., Scheres, S.H., and Shi, Y.
(2017). Mechanistic insights into caspase-9 activation by the structure
of the apoptosome holoenzyme. Proc. Natl. Acad. Sci. USA
114:1542–1547.
Liang, X., and Zhou, J.M. (2018). Receptor-like cytoplasmic kinases:
central players in plant receptor kinase-mediated signaling. Annu.
Rev. Plant Biol. 69:267–299.
Link, V.L., Hofmann, M.G., Sinha, A.K., Ehness, R., Strnad, M., and
Roitsch, T. (2002). Biochemical evidence for the activation of distinct
subsets of mitogen-activated protein kinases by voltage and
defense-related stimuli. Plant Physiol. 128:271–281.
Liu, S., Wang, J., Han, Z., Gong, X., Zhang, H., and Chai, J. (2016a).
Molecular mechanism for fungal cell wall recognition by rice chitin
receptor OsCEBiP. Structure 24:1192–1200.
Liu, T., Liu, Z., Song, C., Hu, Y., Han, Z., She, J., Fan, F., Wang, J., Jin,
C., Chang, J., et al. (2012). Chitin-induced dimerization activates a
plant immune receptor. Science 336:1160–1164.
Liu, X., Zhang, Z., Ruan, J., Pan, Y., Magupalli, V.G., Wu, H., and
Lieberman, J. (2016b). Inflammasome-activated gasdermin D
causes pyroptosis by forming membrane pores. Nature 535:153–158.
Lu, A., Magupalli, V.G., Ruan, J., Yin, Q., Atianand, M.K., Vos, M.R.,
Schroder, G.F., Fitzgerald, K.A., Wu, H., and Egelman, E.H.
(2014). Unified polymerization mechanism for the assembly of ASC-
dependent inflammasomes. Cell 156:1193–1206.
Lu, D., Wu, S., Gao, X., Zhang, Y., Shan, L., and He, P. (2010). A
receptor-like cytoplasmic kinase, BIK1, associates with a flagellin
receptor complex to initiate plant innate immunity. Proc. Natl. Acad.
Sci. USA 107:496–501.
Luan, S., and Wang, C. (2021). Calcium signaling mechanisms across
kingdoms. Annu. Rev. Cell Dev. Biol. 37:311–340.
Ludke, D., Yan, Q., Rohmann, P.F.W., and Wiermer, M. (2022). NLR we
there yet? Nucleocytoplasmic coordination of NLR-mediated
immunity. New Phytol. 236:24–42.
Ma, S., Lapin, D., Liu, L., Sun, Y., Song, W., Zhang, X., Logemann, E.,
Yu, D., Wang, J., Jirschitzka, J., et al. (2020). Direct pathogen-
induced assembly of an NLR immune receptor complex to form a
holoenzyme. Science 370:eabe3069.
Maekawa, S., Ohto, U., Shibata, T., Miyake, K., and Shimizu, T. (2016).
Crystal structure of NOD2 and its implications in human disease. Nat.
Commun. 7:11813.
Maekawa, T., Cheng, W., Spiridon, L.N., Toller, A., Lukasik, E., Saijo,
Y., Liu, P., Shen, Q.H., Micluta, M.A., Somssich, I.E., et al. (2011).
Coiled-coil domain-dependent homodimerization of intracellular
barley immune receptors defines a minimal functional module for
triggering cell death. Cell Host Microbe 9:187–199.
Manik, M.K., Shi, Y., Li, S., Zaydman, M.A., Damaraju, N., Eastman, S.,
Smith, T.G., Gu, W., Masic, V., Mosaiab, T., et al. (2022). Cyclic ADP
ribose isomers: production, chemical structures, and immune
signaling. Science, eadc8969.
Martin, R., Qi, T., Zhang, H., Liu, F., King, M., Toth, C., Nogales, E., and
Staskawicz, B.J. (2020). Structure of the activated ROQ1 resistosome
directly recognizing the pathogen effector XopQ. Science
370:eabd9993.
McLennan, A.G. (2006). The Nudix hydrolase superfamily. Cell. Mol. Life
Sci. 63:123–143.
Meng, X., and Zhang, S. (2013). MAPK cascades in plant disease
resistance signaling. Annu. Rev. Phytopathol. 51:245–266.
Ngou, B.P.M., Ahn, H.K., Ding, P., and Jones, J.D.G. (2021). Mutual
potentiation of plant immunity by cell-surface and intracellular
receptors. Nature 592:110–115.
Ngou, B.P.M., Ahn, H.K., Ding, P., Redkar, A., Brown, H., Ma, Y.,
Youles, M., Tomlinson, L., and Jones, J.D.G. (2020). Estradiol-
inducible AvrRps4 expression reveals distinct properties of TIR-NLR-
mediated effector-triggered immunity. J. Exp. Bot. 71:2186–2197.
Ngou, B.P.M., Ding, P., and Jones, J.D.G. (2022). Thirty years of
resistance: zig-zag through the plant immune system. Plant Cell
34:1447–1478.
Nishimura, M.T., Anderson, R.G., Cherkis, K.A., Law, T.F., Liu, Q.L.,
Machius, M., Nimchuk, Z.L., Yang, L., Chung, E.H., El Kasmi, F.,
et al. (2017). TIR-only protein RBA1 recognizes a pathogen effector
to regulate cell death in Arabidopsis. Proc. Natl. Acad. Sci. USA
114:E2053–E2062.
Ofir, G., Herbst, E., Baroz, M., Cohen, D., Millman, A., Doron, S., Tal,
N., Malheiro, D.B.A., Malitsky, S., Amitai, G., et al. (2021). Antiviral
activity of bacterial TIR domains via immune signalling molecules.
Nature 600:116–120.
Ogasawara, Y., Kaya, H., Hiraoka, G., Yumoto, F., Kimura, S., Kadota,
Y., Hishinuma, H., Senzaki, E., Yamagoe, S., Nagata, K., et al.
(2008). Synergistic activation of the Arabidopsis NADPH oxidase
AtrbohD by Ca2+ and phosphorylation. J. Biol. Chem. 283:8885–8892.
Ohto, U. (2022). Activation and regulation mechanisms of NOD-like
receptors based on structural biology. Front. Immunol. 13:953530.
Orrenius, S., Zhivotovsky, B., and Nicotera, P. (2003). Regulation of cell
death: the calcium-apoptosis link. Nat. Rev. Mol. Cell Biol. 4:552–565.
Pan, Y., Chai, X., Gao, Q., Zhou, L., Zhang, S., Li, L., and Luan, S.
(2019). Dynamic interactions of plant CNGC subunits and
calmodulins drive oscillatory Ca(2+) channel activities. Dev. Cell
48:710–725.e715.
18 Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
Pauly, N., Knight, M.R., Thuleau, P., Graziana, A., Muto, S., Ranjeva,
R., and Mazars, C. (2001). The nucleus together with the cytosol
generates patterns of specific cellular calcium signatures in tobacco
suspension culture cells. Cell Calcium 30:413–421.
Peart, J.R., Mestre, P., Lu, R., Malcuit, I., and Baulcombe, D.C. (2005).
NRG1, a CC-NB-LRR protein, together with N, a TIR-NB-LRR protein,
mediates resistance against tobacco mosaic virus. Curr. Biol.
15:968–973.
Pruitt, R.N., Locci, F., Wanke, F., Zhang, L., Saile, S.C., Joe, A.,
Karelina, D., Hua, C., Frohlich, K., Wan, W.L., et al. (2021). The
EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered
immunity. Nature 598:495–499.
Qi, S., Pang, Y., Hu, Q., Liu, Q., Li, H., Zhou, Y., He, T., Liang, Q., Liu, Y.,
Yuan, X., et al. (2010). Crystal structure of the Caenorhabditis elegans
apoptosome reveals an octameric assembly of CED-4. Cell
141:446–457.
Qi, T., Seong, K., Thomazella, D.P.T., Kim, J.R., Pham, J., Seo, E., Cho,
M.J., Schultink, A., and Staskawicz, B.J. (2018). NRG1 functions
downstream of EDS1 to regulate TIR-NLR-mediated plant immunity
in Nicotiana benthamiana. Proc. Natl. Acad. Sci. USA 115:E10979–
E10987.
Rahmati Ishka, M., Brown, E., Rosenberg, A., Romanowsky, S., Davis,
J.A., Choi, W.G., and Harper, J.F. (2021). Arabidopsis Ca2+-ATPases
1, 2, and 7 in the endoplasmic reticulum contribute to growth and
pollen fitness. Plant Physiol. 185:1966–1985.
Ranf, S., Eschen-Lippold, L., Pecher, P., Lee, J., and Scheel, D. (2011).
Interplay between calcium signalling and early signalling elements
during defence responses to microbe- or damage-associated
molecular patterns. Plant J. 68:100–113.
Ranf, S., Wunnenberg, P., Lee, J., Becker, D., Dunkel, M., Hedrich, R.,
Scheel, D., and Dietrich, P. (2008). Loss of the vacuolar cation
channel, AtTPC1, does not impair Ca2+ signals induced by abiotic
and biotic stresses. Plant J. 53:287–299.
Resentini, F., Ruberti, C., Grenzi, M., Bonza, M.C., and Costa, A.
(2021). The signatures of organellar calcium. Plant Physiol.
187:1985–2004.
Reubold, T.F., Wohlgemuth, S., and Eschenburg, S. (2011). Crystal
structure of full-length Apaf-1: how the death signal is relayed in the
mitochondrial pathway of apoptosis. Structure 19:1074–1083.
Riedl, S.J., Li, W., Chao, Y., Schwarzenbacher, R., and Shi, Y. (2005).
Structure of the apoptotic protease-activating factor 1 bound to
ADP. Nature 434:926–933.
Romeis, T., Piedras, P., Zhang, S., Klessig, D.F., Hirt, H., and Jones,
J.D. (1999). Rapid Avr9- and Cf-9 -dependent activation of MAP
kinases in tobacco cell cultures and leaves: convergence of
resistance gene, elicitor, wound, and salicylate responses. Plant Cell
11:273–287.
Rowland, O., Ludwig, A.A., Merrick, C.J., Baillieul, F., Tracy, F.E.,
Durrant, W.E., Fritz-Laylin, L., Nekrasov, V., Sjolander, K.,
Yoshioka, H., et al. (2005). Functional analysis of Avr9/Cf-9 rapidly
elicited genes identifies a protein kinase, ACIK1, that is essential for
full Cf-9-dependent disease resistance in tomato. Plant Cell
17:295–310.
Ruan, J., Xia, S., Liu, X., Lieberman, J., and Wu, H. (2018). Cryo-EM
structure of the gasdermin A3 membrane pore. Nature 557:62–67.
Salcedo, A., Rutter, W., Wang, S., Akhunova, A., Bolus, S., Chao, S.,
Anderson, N., De Soto, M.F., Rouse, M., Szabo, L., et al. (2017).
Variation in the AvrSr35 gene determines Sr35 resistance against
wheat stem rust race Ug99. Science 358:1604–1606.
Sarris, P.F., Duxbury, Z., Huh, S.U., Ma, Y., Segonzac, C., Sklenar, J.,
Derbyshire, P., Cevik, V., Rallapalli, G., Saucet, S.B., et al. (2015). A
plant immune receptor detects pathogen effectors that target WRKY
transcription factors. Cell 161:1089–1100.
Schreiber, K.J., Bentham, A., Williams, S.J., Kobe, B., and
Staskawicz, B.J. (2016). Multiple domain associations within the
Arabidopsis immune receptor RPP1 regulate the activation of
programmed cell death. PLoS Pathog. 12:e1005769.
Schulze, B., Mentzel, T., Jehle, A.K., Mueller, K., Beeler, S., Boller, T.,
Felix, G., and Chinchilla, D. (2010). Rapid heteromerization and
phosphorylation of ligand-activated plant transmembrane receptors
and their associated kinase BAK1. J. Biol. Chem. 285:9444–9451.
Scrase-Field, S.A., and Knight, M.R. (2003). Calcium: just a chemical
switch? Curr. Opin. Plant Biol. 6:500–506.
Seifert, R. (2015). cCMP and cUMP: emerging second messengers.
Trends Biochem. Sci. 40:8–15.
Sharif, H., Wang, L., Wang, W.L., Magupalli, V.G., Andreeva, L., Qiao,
Q., Hauenstein, A.V., Wu, Z., Nunez, G., Mao, Y., et al. (2019).
Structural mechanism for NEK7-licensed activation of NLRP3
inflammasome. Nature 570:338–343.
Shen, Q.H., Saijo, Y., Mauch, S., Biskup, C., Bieri, S., Keller, B., Seki,
H., Ulker, B., Somssich, I.E., and Schulze-Lefert, P. (2007).
Nuclear activity of MLA immune receptors links isolate-specific and
basal disease-resistance responses. Science 315:1098–1103.
Shen, W., Liu, J., and Li, J.F. (2019). Type-II metacaspases mediate the
processing of plant elicitor peptides in Arabidopsis. Mol. Plant
12:1524–1533.
Shi, J., Zhao, Y., Wang, K., Shi, X., Wang, Y., Huang, H., Zhuang, Y.,
Cai, T., Wang, F., and Shao, F. (2015). Cleavage of GSDMD by
inflammatory caspases determines pyroptotic cell death. Nature
526:660–665.
Shi, Y., Kerry, P.S., Nanson, J.D., Bosanac, T., Sasaki, Y., Krauss, R.,
Saikot, F.K., Adams, S.E., Mosaiab, T., Masic, V., et al. (2022).
Structural basis of SARM1 activation, substrate recognition, and
inhibition by small molecules. Mol. Cell 82:1643–1659.e1610.
Shigaki, T., and Hirschi, K. (2000). Characterization of CAX-like genes in
plants: implications for functional diversity. Gene 257:291–298.
Sohn, K.H., Segonzac, C., Rallapalli, G., Sarris, P.F., Woo, J.Y.,
Williams, S.J., Newman, T.E., Paek, K.H., Kobe, B., and Jones,
J.D. (2014). The nuclear immune receptor RPS4 is required for
RRS1SLH1-dependent constitutive defense activation in Arabidopsis
thaliana. PLoS Genet. 10:e1004655.
Song, W., Forderer, A., Yu, D., and Chai, J. (2021). Structural biology of
plant defence. New Phytol. 229:692–711.
Spalding, E.P., and Harper, J.F. (2011). The ins and outs of cellular
Ca(2+) transport. Curr. Opin. Plant Biol. 14:715–720.
Steele, J.F.C., Hughes, R.K., and Banfield, M.J. (2019). Structural and
biochemical studies of an NB-ARC domain from a plant NLR immune
receptor. PLoS One 14:e0221226.
Sun, T., Huang, J., Xu, Y., Verma, V., Jing, B., Sun, Y., Ruiz Orduna, A.,
Tian, H., Huang, X., Xia, S., et al. (2020). Redundant CAMTA
transcription factors negatively regulate the biosynthesis of salicylic
acid and N-hydroxypipecolic acid by modulating the expression of
SARD1 and CBP60g. Mol. Plant 13:144–156.
Sun, T., Zhang, Y., Li, Y., Zhang, Q., Ding, Y., and Zhang, Y. (2015).
ChIP-seq reveals broad roles of SARD1 and CBP60g in regulating
plant immunity. Nat. Commun. 6:10159.
Sun, X., Lapin, D., Feehan, J.M., Stolze, S.C., Kramer, K., Dongus, J.A.,
Rzemieniewski, J., Blanvillain-Baufume, S., Harzen, A., Bautor, J.,
et al. (2021). Pathogen effector recognition-dependent association of
NRG1 with EDS1 and SAG101 in TNL receptor immunity. Nat.
Commun. 12:3335.
Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author. 19
Biochemical function and signaling mechanism of NLRs Molecular Plant
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
Sun, Y., Li, L., Macho, A.P., Han, Z., Hu, Z., Zipfel, C., Zhou, J.M., and
Chai, J. (2013). Structural basis for flg22-induced activation of the
Arabidopsis FLS2-BAK1 immune complex. Science 342:624–628.
Sun, Y., Wang, Y., Zhang, X., Chen, Z., Xia, Y., Wang, L., Sun, Y.,
Zhang, M., Xiao, Y., Han, Z., et al. (2022). Plant receptor-like
protein activation by a microbial glycoside hydrolase. Nature
610:335–342.
Swanson, K.V., Deng, M., and Ting, J.P. (2019). The NLRP3
inflammasome: molecular activation and regulation to therapeutics.
Nat. Rev. Immunol. 19:477–489.
Swarbreck, S.M., Colaco, R., and Davies, J.M. (2013). Plant calcium-
permeable channels. Plant Physiol. 163:514–522.
Swiderski, M.R., Birker, D., and Jones, J.D. (2009). The TIR domain of
TIR-NB-LRR resistance proteins is a signaling domain involved in cell
death induction. Mol. Plant Microbe Interact. 22:157–165.
Tang, J., Han, Z., Sun, Y., Zhang, H., Gong, X., and Chai, J. (2015).
Structural basis for recognition of an endogenous peptide by the
plant receptor kinase PEPR1. Cell Res. 25:110–120.
Tenthorey, J.L., Haloupek, N., Lopez-Blanco, J.R., Grob, P.,
Adamson, E., Hartenian, E., Lind, N.A., Bourgeois, N.M., Chacon,
P., Nogales, E., et al. (2017). The structural basis of flagellin
detection by NAIP5: a strategy to limit pathogen immune evasion.
Science 358:888–893.
Thor, K., Jiang, S., Michard, E., George, J., Scherzer, S., Huang, S.,
Dindas, J., Derbyshire, P., Leitao, N., DeFalco, T.A., et al. (2020).
The calcium-permeable channel OSCA1.3 regulates plant stomatal
immunity. Nature 585:569–573.
Thordal-Christensen, H., Zhang, Z., Wei, Y., and Collinge, D.B. (1997).
Subcellular localization of H2O2 in plants. H2O2 accumulation in
papillae and hypersensitive response during the barley—powdery
mildew interaction. Plant J. 11:1187–1194.
Tian, H., Wu, Z., Chen, S., Ao, K., Huang, W., Yaghmaiean, H., Sun, T.,
Xu, F., Zhang, Y., Wang, S., et al. (2021). Activation of TIR signalling
boosts pattern-triggered immunity. Nature 598:500–503.
Tian, L., Lu, J., and Li, X. (2022). Differential requirement of TIR enzymatic
activities in TIR-type immune receptor SNC1-mediated immunity. Plant
Physiol. https://doi.org/10.1093/plphys/kiac452.
Tian, W., Hou, C., Ren, Z., Wang, C., Zhao, F., Dahlbeck, D., Hu, S.,
Zhang, L., Niu, Q., Li, L., et al. (2019). A calmodulin-gated calcium
channel links pathogen patterns to plant immunity. Nature
572:131–135.
Tian, W., Wang, C., Gao, Q., Li, L., and Luan, S. (2020). Calcium spikes,
waves and oscillations in plant development and biotic interactions.
Native Plants 6:750–759.
Torres, M.A., Dangl, J.L., and Jones, J.D. (2002). Arabidopsis gp91phox
homologues AtrbohD and AtrbohF are required for accumulation of
reactive oxygen intermediates in the plant defense response. Proc.
Natl. Acad. Sci. USA 99:517–522.
Vadassery, J., Ranf, S., Drzewiecki, C., Mithofer, A., Mazars, C.,
Scheel, D., Lee, J., and Oelmuller, R. (2009). A cell wall extract
from the endophytic fungus Piriformospora indica promotes growth
of Arabidopsis seedlings and induces intracellular calcium elevation
in roots. Plant J. 59:193–206.
van Der Luit, A.H., Olivari, C., Haley, A., Knight, M.R., and Trewavas,
A.J. (1999). Distinct calcium signaling pathways regulate calmodulin
gene expression in tobacco. Plant Physiol. 121:705–714.
Wagner, S., Stuttmann, J., Rietz, S., Guerois, R., Brunstein, E., Bautor,
J., Niefind, K., and Parker, J.E. (2013). Structural basis for signaling
by exclusive EDS1 heteromeric complexes with SAG101 or PAD4 in
plant innate immunity. Cell Host Microbe 14:619–630.
Wan, L., Essuman, K., Anderson, R.G., Sasaki, Y., Monteiro, F., Chung,
E.H., Osborne Nishimura, E.,DiAntonio, A., Milbrandt, J., Dangl, J.L.,
et al. (2019). TIR domains of plant immune receptors are NAD(+)-
cleaving enzymes that promote cell death. Science 365:799–803.
Wang, C., Wang, G., Zhang, C., Zhu, P., Dai, H., Yu, N., He, Z., Xu, L.,
and Wang, E. (2017). OsCERK1-Mediated chitin perception and
immune signaling requires receptor-like cytoplasmic kinase 185 to
activate an MAPK cascade in rice. Mol. Plant 10:619–633.
Wang, G., Roux, B., Feng, F., Guy, E., Li, L., Li, N., Zhang, X., Lautier,
M., Jardinaud, M.F., Chabannes, M., et al. (2015). The decoy
substrate of a pathogen effector and a pseudokinase specify
pathogen-induced modified-self recognition and immunity in plants.
Cell Host Microbe 18:285–295.
Wang, J., and Chai, J. (2020a). Molecular actions of NLR immune
receptors in plants and animals. Sci. China Life Sci. 63:1303–1316.
Wang, J., and Chai, J. (2020b). Structural insights into the plant immune
receptors PRRs and NLRs. Plant Physiol. 182:1566–1581.
Wang, J., Han, M., and Liu, Y. (2021a). Diversity, structure and function of
the coiled-coil domains of plant NLR immune receptors. J. Integr. Plant
Biol. 63:283–296.
Wang, J., Hu, M., Wang, J., Qi, J., Han, Z., Wang, G., Qi, Y., Wang, H.W.,
Zhou, J.M., and Chai, J. (2019a). Reconstitution and structure of a
plant NLR resistosome conferring immunity. Science 364:eaav5870.
Wang, J., Wang, J., Hu, M., Wu, S., Qi, J., Wang, G., Han, Z., Qi, Y., Gao,
N., Wang, H.W., et al. (2019b). Ligand-triggered allosteric ADP release
primes a plant NLR complex. Science 364:eaav5868.
Wang, L., Sharif, H., Vora, S.M., Zheng, Y., and Wu, H. (2021b).
Structures and functions of the inflammasome engine. J. Allergy Clin.
Immunol. 147:2021–2029.
Williams, S.J., Yin, L., Foley, G., Casey, L.W., Outram, M.A., Ericsson,
D.J., Lu, J., Boden, M., Dry, I.B., and Kobe, B. (2016). Structure and
function of the TIR domain from the grape NLR protein RPV1. Front.
Plant Sci. 7:1850.
Wroblewski, T., Spiridon, L., Martin, E.C., Petrescu, A.J., Cavanaugh,
K., Truco, M.J., Xu, H., Gozdowski, D., Pawlowski, K., Michelmore,
R.W., et al. (2018). Genome-wide functional analyses of plant coiled-
coil NLR-type pathogen receptors reveal essential roles of their
N-terminal domain in oligomerization, networking, and immunity.
PLoS Biol. 16:e2005821.
Wu, C.H., Abd-El-Haliem, A., Bozkurt, T.O., Belhaj, K., Terauchi, R.,
Vossen, J.H., and Kamoun, S. (2017). NLR network mediates
immunity to diverse plant pathogens. Proc. Natl. Acad. Sci. USA
114:8113–8118.
Wu, C.H., Belhaj, K., Bozkurt, T.O., Birk, M.S., and Kamoun, S. (2016).
Helper NLR proteins NRC2a/b and NRC3 but not NRC1 are required for
Pto-mediated cell death and resistance in Nicotiana benthamiana.
New Phytol. 209:1344–1352.
Wu, F., Chi, Y., Jiang, Z., Xu, Y., Xie, L., Huang, F., Wan, D., Ni, J., Yuan,
F., Wu, X., et al. (2020). Hydrogen peroxide sensor HPCA1 is an LRR
receptor kinase in Arabidopsis. Nature 578:577–581.
Wu, Z., Li, M., Dong, O.X., Xia, S., Liang, W., Bao, Y., Wasteneys, G.,
and Li, X. (2019). Differential regulation of TNL-mediated immune
signaling by redundant helper CNLs. New Phytol. 222:938–953.
Wu, Z., Tian, L., Liu, X., Zhang, Y., and Li, X. (2021). TIR signal promotes
interactions between lipase-like proteins and ADR1-L1 receptor and
ADR1-L1 oligomerization. Plant Physiol. 187:681–686.
Xia, S., Zhang, Z., Magupalli, V.G., Pablo, J.L., Dong, Y., Vora, S.M.,
Wang, L., Fu, T.M., Jacobson, M.P., Greka, A., et al. (2021).
Gasdermin D pore structure reveals preferential release of mature
interleukin-1. Nature 593:607–611.
20 Molecular Plant 16, 1–21, January 2 2023 ª2022 The Author.
Molecular Plant Biochemical function and signaling mechanism of NLRs
Please cite this article in press as: Wang et al., Structure, biochemical function, and signaling mechanism of plant NLRs, Molecular Plant (2022), https://
doi.org/10.1016/j.molp.2022.11.011
Xiao, Y., Stegmann, M., Han, Z., DeFalco, T.A., Parys, K., Xu, L.,
Belkhadir, Y., Zipfel, C., and Chai, J. (2019). Mechanisms of RALF
peptide perception by a heterotypic receptor complex. Nature
572:270–274.
Xiong, T.C., Jauneau, A., Ranjeva, R., and Mazars, C. (2004). Isolated
plant nuclei as mechanical and thermal sensors involved in calcium
signalling. Plant J. 40:12–21.
Xu, G., Moeder, W., Yoshioka, K., and Shan, L. (2022a). A tale of many
families: calcium channels in plant immunity. Plant Cell 34:1551–1567.
Xu, L., Wang, J., Xiao, Y., Han, Z., and Chai, J. (2022b). Structural insight
into chitin perception by chitin elicitor receptor kinase 1 of Oryza sativa.
J. Integr. Plant Biol. https://doi.org/10.1111/jipb.13279.
Yamada, K., Yamaguchi, K., Shirakawa, T., Nakagami, H., Mine, A.,
Ishikawa, K., Fujiwara, M., Narusaka, M., Narusaka, Y., Ichimura,
K., et al. (2016). The Arabidopsis CERK1-associated kinase PBL27
connects chitin perception to MAPK activation. EMBO J. 35:2468–2483.
Yamaguchi, Y., Huffaker, A., Bryan, A.C., Tax, F.E., and Ryan, C.A.
(2010). PEPR2 is a second receptor for the Pep1 and Pep2 peptides
and contributes to defense responses in Arabidopsis. Plant Cell
22:508–522.
Yamaguchi, Y., Pearce, G., and Ryan, C.A. (2006). The cell surface
leucine-rich repeat receptor for AtPep1, an endogenous peptide
elicitor in Arabidopsis, is functional in transgenic tobacco cells. Proc.
Natl. Acad. Sci. USA 103:10104–10109.
Yang, D.L., Shi, Z., Bao, Y., Yan, J., Yang, Z., Yu, H., Li, Y., Gou, M.,
Wang, S., Zou, B., et al. (2017). Calcium pumps and interacting
BON1 protein modulate calcium signature, stomatal closure, and
plant immunity. Plant Physiol. 175:424–437.
Yang, X., Lin, G., Han, Z., and Chai, J. (2019). Structural biology of NOD-
like receptors. Adv. Exp. Med. Biol. 1172:119–141.
Yang, X., Yang, F., Wang, W., Lin, G., Hu, Z., Han, Z., Qi, Y., Zhang, L.,
Wang, J., Sui, S.F., et al. (2018). Structural basis for specific flagellin
recognition by the NLR protein NAIP5. Cell Res. 28:35–47.
Yin, Q., Fu, T.M., Li, J., and Wu, H. (2015). Structural biology of innate
immunity. Annu. Rev. Immunol. 33:393–416.
Yu, D., Song, W., Tan, E.Y.J., Liu, L., Cao, Y., Jirschitzka, J., Li, E.,
Logemann, E., Xu, C., Huang, S., et al. (2022). TIR domains of plant
immune receptors are 2’,3’-cAMP/cGMP synthetases mediating cell
death. Cell 185:2370–2386.e2318.
Yu, X., Feng, B., He, P., and Shan, L. (2017). From chaos to harmony:
responses and signaling upon microbial pattern recognition. Annu.
Rev. Phytopathol. 55:109–137.
Yuan, M., Jiang, Z., Bi, G., Nomura, K., Liu, M., Wang, Y., Cai, B., Zhou,
J.M., He, S.Y., and Xin, X.F. (2021). Pattern-recognition receptors are
required for NLR-mediated plant immunity. Nature 592:105–109.
Zhang, J., Li, W., Xiang, T., Liu, Z., Laluk, K., Ding, X., Zou, Y., Gao, M.,
Zhang, X., Chen, S., et al. (2010a). Receptor-like cytoplasmic kinases
integrate signaling from multiple plant immune receptors and are
targeted by a Pseudomonas syringae effector. Cell Host Microbe
7:290–301.
Zhang, L., Chen, S., Ruan, J., Wu, J., Tong, A.B., Yin, Q., Li, Y., David,
L., Lu, A., Wang, W.L., et al. (2015). Cryo-EM structure of the activated
NAIP2-NLRC4 inflammasome reveals nucleated polymerization.
Science 350:404–409.
Zhang, Y., Dorey, S., Swiderski, M., and Jones, J.D. (2004). Expression
of RPS4 in tobacco induces an AvrRps4-independent HR that requires
EDS1, SGT1 and HSP90. Plant J. 40:213–224.
Zhang, Y., and Li, X. (2019). Salicylic acid: biosynthesis, perception, and
contributions to plant immunity. Curr. Opin. Plant Biol. 50:29–36.
Zhang, Y., Xu, S., Ding, P., Wang, D., Cheng, Y.T., He, J., Gao, M., Xu,
F., Li, Y., Zhu, Z., et al. (2010b). Control of salicylic acid synthesis and
systemic acquired resistance by two members of a plant-specific
family of transcription factors. Proc. Natl. Acad. Sci. USA 107:18220–
18225.
Zhao, C., Tang, Y., Wang, J., Zeng, Y., Sun, H., Zheng, Z., Su, R.,
Schneeberger, K., Parker, J.E., and Cui, H. (2021). A mis-regulated
cyclic nucleotide-gated channel mediates cytosolic calcium elevation
and activates immunity in Arabidopsis. New Phytol. 230:1078–1094.
Zhao, T., Rui, L., Li, J., Nishimura, M.T., Vogel, J.P., Liu, N., Liu, S.,
Zhao, Y., Dangl, J.L., and Tang, D. (2015). A truncated NLR protein,
TIR-NBS2, is required for activated defense responses in the
exo70B1 mutant. PLoS Genet. 11:e1004945.
Zhao, Y.-B., Liu, M.-X., Chen, T.-T., Ma, X., Li, Z.-K., Zheng, Z., Zheng,
S.-R., Chen, L., Li, Y.-Z., Tang, L.-R., et al. (2022). Pathogen effector
AvrSr35 triggers Sr35 resistosome assembly via a direct recognition
mechanism. Sci. Adv. 8:eabq5108.
Zhao, Y., Yang, J.L., Shi, J.J., Gong, Y.N., Lu, Q.H., Xu, H., Liu, L.P., and
Shao, F. (2011). The NLRC4 inflammasome receptors for bacterial
flagellin and type III secretion apparatus. Nature 477:596–U257.
Zhou, J.M., and Zhang, Y. (2020). Plant immunity: danger perception and
signaling. Cell 181:978–989.
Zhou, L., Lan, W., Jiang, Y., Fang, W., and Luan, S. (2014). A calcium-
dependent protein kinase interacts with and activates a calcium
channel to regulate pollen tube growth. Mol. Plant 7:369–376.
Zhou, M., Li, Y., Hu, Q., Bai, X.C., Huang, W., Yan, C., Scheres, S.H.,
and Shi, Y. (2015). Atomic structure of the apoptosome: mechanism
of cytochrome c- and dATP-mediated activation of Apaf-1. Genes
Dev. 29:2349–2361.
Zimmermann, S., Nurnberger, T., Frachisse, J.M., Wirtz, W., Guern, J.,
Hedrich, R., and Scheel, D. (1997). Receptor-mediated activation of a
plant Ca2+-permeable ion channel involved in pathogen defense.
Proc. Natl. Acad. Sci. USA 94:2751–2755.
Zonnchen, J., Gantner, J., Lapin, D., Barthel, K., Eschen-Lippold, L.,
Erickson, J.L., Villanueva, S.L., Zantop, S., Kretschmer, C.,
Joosten, M., et al. (2022). EDS1 complexes are not required for PRR
responses and execute TNL-ETI from the nucleus in Nicotiana
benthamiana. New Phytol. https://doi.org/10.1111/nph.18511.
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