Identification, immune activation and signal regulation of plant NLR immune receptors

Abstract Higher plants have evolved a large number of membrane surface and intracellular immune receptors to sense various pathogenic signals and resist the invasion of pathogens
.

Among them, pattern recognition receptors on the cell surface activate basal immune responses after sensing pattern molecules, while nucleotide-binding and leucine-rich repeats (NLRs) activate specific immune responses by sensing effector proteins secreted by pathogenic microorganisms, resulting in hypersensitivity reactions.
with cell death
.

This review mainly reviews the latest research progress in the recognition of effector proteins by NLRs, the activation of plant immunity and the regulation of downstream signaling
.

To differentiate pathogenic microbes from commensal or beneficial microbes, and to moderately activate defense responses when attacked by pathogens, plants have evolved complex innate immune systems
.

There are a large number of immune receptors on the surface and inside of higher plant cells to sense various signals related to pathogen infection (Kourelis and Van Der Hoorn, 2018; Van De Weyer et al.
, 2019)
.

Cell surface immune receptors include receptor-like proteins (RLPs) and receptor-like kinases (RLKs), commonly referred to as pattern-recognition receptors (PRRs), which can sense Microbe-associated molecular patterns (MAMPs), pathogen-associated molecular patterns (PAMPs), or host-generated damage-associated molecular patterns (DAMPs)
.

After sensing the molecular pattern, PRR activates the basal immune response PTI (pattern-triggered immunity), including the rapid generation of reactive oxygen species (ROS), Ca2+ influx, and mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) cascade reactions, etc.
(Couto and Zipfel et al.
, 2016; Jones et al.
, 2016; Van Der Burgh and Joosten, 2019), thereby inhibiting the proliferation of pathogens
.

Pathogens use a variety of mechanisms to promote host infection, the most important of which is the secretion of effector proteins to interfere with the function of host PRR proteins and their immune-related processes
.

To overcome the interference caused by effector proteins and prevent disease occurrence, plants have evolved intracellular nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) to sense pathogen effectors and Initiates a strong and specific immune response ETI (effector-triggered immunity) (Dodds and Rathjen, 2010; Jones et al.
, 2016)
.

NLRs and PRR-mediated immune responses share similar but different molecular features, and are often accompanied by hypersensitive responses (HR) and cell death (Yuan et al.
, 2021; Ofir et al.
, 2021)
.

In recent years, a series of major breakthroughs have been made in the study of plant innate immunity, especially the plant immunity mediated by NLRs
.

This review focuses on the immune response mediated by NLRs, and summarizes the latest research progress in the recognition, immune activation and regulation of effector proteins
.

1 NLRs and their recognition with effector proteins 1.
1 Structure and types of NLRs In higher plants, NLRs are crucial for pathogen effector recognition and immune response initiation (Jones et al.
, 2016; Zhou and Zhang, 2020)
.

Although NLRs also exist in a variety of animals, including mammals, there is increasing evidence that both animals and plants have evolved similar receptors in different ways (Urbach and Ausubel, 2017)
.

Both plant and animal NLRs contain a central nucleotide-binding (NB) domain and a C-terminal leucine-rich repeats (LRR) region
.

Among them, the NB domain is necessary for the formation of higher-order complexes (such as disease resistance bodies in higher plants and inflammasomes in mammals) after receptor oligomerization to transmit signals; the highly variable LRR region is usually involved in Self-inhibition, protein-protein interactions and effector recognition (Saur et al.
, 2021)
.

Typical plant NLRs are divided into 2 main types according to the difference of the N-terminus: TIR type and CC type
.

TIR-type NLRs (TIR-type NLRs, TNL) are characterized by their N-terminal Toll/interleukin-1 receptor (TIR) ​​domain, while CC-type NLRs (CC-type NLRs, CNL) have Coiled-coil (CC) domain (Tamborski and Krasileva, 2020; Saur et al.
, 2021) (Figure 1A)
.

The number of NLRs in most animals is relatively small, while the number of NLRs in higher plants can reach hundreds.
Helper NLRs (hNLRs), such as NRG1 and hNLRs in the ADR1 protein family, act downstream of TNL (Peart et al.
, 2005; Bonar-di et al.
, 2011; Dong et al.
, 2016)
.

Since the N-terminus of ADR1 and NRG1 carry a RPW8 (Resistance to Powdery Mildew 8)-like CC domain instead of a typical CC domain (Jubic et al.
, 2019), they are also called RNLs (RPW8-type NLRs)
.

Figure 1 Structural composition of NLRs and their recognition patterns with effector proteins (modified from Duxbury et al.
, 2021) (A) The structural domains of plant NLRs are divided into three categories, including the nucleotide binding domain (NBD) in the middle and the C leucine-rich repeat (LRR) domains at the N-terminal and TIR, CC or RPW8-like CC domains at the N-terminal; (B) Different modes of plant NLRs recognizing effector proteins: some plant NLRs directly bind to the corresponding effector proteins, Or indirectly detect pathogen effector proteins through guard proteins or bait proteins; in addition, some plant NLRs have specific integration domains (IDs) that mediate the recognition of effector proteins
.

NLRs are widely distributed in subcellular structures such as cytoplasm, nucleus, plasma membrane (PM), tonoplast, and endoplasmic reticulum (Chiang and Coaker, 2014), such as barley (Hordeum vulgare) CNLMLA10 and Arabidopsis thaliana (Arabidopsis thaliana) TNLRPS4 is located in the nucleus and cytoplasm, and both subcellular localizations are required for resistance activation (Shen et al.
, 2007; Bai et al.
, 2012)
.

In contrast, Arabidopsis CNL (RPM1) constitutively binds to the plasma membrane and recognizes membrane-targeted Pseudomonas effector proteins (Nimchuk et al.
, 2000; Gao et al.
, 2011)
.

Recent studies have shown that there is a cell wall-localized NLR Rsc4 in soybean (Glycine max), which mediates soybean resistance to mosaic virus (Yin et al.
, 2021)
.

Therefore, NLRs not only localize to different subcellular structures, but also show diversity in pathogen-effector protein recognition mechanisms
.

1.
2 Multiple modes of NLRs recognition of effector proteins 1.
2.
1 Direct recognition mode The simplest and most intuitive mechanism for NLRs to perceive effector proteins is the direct binding recognition mode (Fig.
1B)
.

A typical example of direct recognition is the sensing of different variants of the Melampsora lini effector protein AvrL567 by TNL L5, L6 and L7 encoded by the Linum usitatissimum L locus (Dodds et al.
, 2006)
.

However, the identification of direct interactions may have driven the diversification of the AvrL567 locus, resulting in 12 variants, 5 of which have the ability to evade NLRs binding and hinder NLRs recognition (Dodds et al.
, 2006)
.

This coevolutionary "arms race" has also driven the diversification of the flax L locus, which contains 13 alleles, including three NLRs that recognize individual effector protein variants
.

At the same time, the high polymorphism of the LRR domain of the L protein endows it with recognition of multiple AvrL567 variants, so the substitution of 11 amino acids in the L6 LRR reduces the number of specific recognition effector proteins from multiple to 1 (Dodds et al.
, 2006; Rayamajhi et al.
, 2013)
.

The rice NLR Pik gene family senses multiple AVR-Pik effector proteins in M.
oryzae through direct binding
.

For example, Pikm can recognize three AVR-Pik effector proteins, while Pikp can only recognize one
.

By analyzing the interaction between Pik and AVR-Pik proteins, the researchers used structure-guided engineering to create a new Pik allele that combines a previously unidentified AVR-Pik (De La Concepcion et al.
, 2019)
.

It should be pointed out that a new allele has evolved in the barley CNL powdery mildew resistance locus A (mildew resistance locus a, Mla), which can directly identify the sequence-unrelated effectors of powdery mildew fungi.
) (Saur et al.
, 2019)
.

In addition, two recent cryo-electron microscopy structural analyses have shown that tobacco (Nicotiana benthamiana) TNL ROQ1 (Recognition of Xanthomonas outer protein Q1) and its effector protein XopQ, and Arabidopsis thaliana TNL RPP1 (Recognition of Peronosporaparasitica 1) and its effector protein ATR1, are Direct binding via the LRR domain
.

1.
2.
2 Indirect recognition mode Many plant NLRs specifically recognize not directly binding to effector proteins (Figure 1B), but in an indirect recognition mode, which can be divided into two types
.

One is the guard hypothesis model, which refers to the recognition of effector proteins through indirect ligand-receptor interactions (Dangl and Jones, 2001)
.

NLRs monitor the integrity of effector proteins (or protected target proteins) and activate immune responses upon sensing changes in the structure or function of protected proteins
.

In Arabidopsis, multiple effector proteins target RIN4 (RPM1-interacting protein 4), while RPS2 (Resistance to P.
syringae 2) and RPM1 (Resistance to P.
syringae pv.
maculicola 1) monitor RIN4 through protein interactions , to identify effector proteins
.

Pseudomonas syringae can secrete the protease AvrRpt2 through its type III secretion system (T3SS), which subsequently cleaves RIN4 to trigger an RPS2-dependent immune response (Axtell and Staskawicz, 2003; Mackey et al.
, 2003)
.

In addition, the Pseudomonas syringae effector AvrRpm1 induces adenosine diphosphate (ADP) ribosylation of RIN4, which in turn leads to phosphorylation of RIN4 by host kinases, triggering RPM1-mediated immune responses (Chung et al.
, 2011; Liu et al.
.
, 2011; Redditt et al.
, 2019)
.

Recent findings suggest that the pathogenic effector proteins HopZ5 and AvrBst can acetylate the conserved site of RIN4 T166 to trigger RPM1-dependent immune responses
.

It should be pointed out that the plant endogenous deacetylase SOBER1 can deacetylate RIN4 T166, thereby inhibiting the occurrence of immune responses
.

This may be the fine regulation of the immune response by plants to avoid excessive immunity (Choi et al.
, 2021)
.

The second is the decoy hypothesis model
.

This model is a derivation of the guard hypothesis model, which refers to the fact that the decoy proteins protected by NLRs do not have other functions in immunity except as effector proteins to recognize the decoy (Zhou and Chai, 2008; Van Der Hoorn and Kamoun, 2008)
.

Classical plant bait proteins, such as pseudokinase PBL2, are required for the activation of CNL ZAR1 (HopZ-activated resistance 1)
.

ZAR1 indirectly recognizes multiple effectors, including the effector protein AvrAC of Xanthomonas campestris pv.
campestris and the effector protein HopZ1a of Pseudomonas syringae (Lewis et al.
, 2013; Wang et al.
, 2015; Seto et al.
, 2017; Schultink et al.
, 2019; Laflamme et al.
, 2020)
.

During the invasion of pathogens, BIK1 is uridylated by secreted effector protein AvrAC, or MKK7 is acetylated by HopZ1a, which inhibits the PTI response of plants to enhance their pathogenicity (Feng et al.
, 2012; Wang et al.
, 2015; Rufián et al.
.
, 2021); while plants evolved the decoy protein PBL2, AvrAC uridylylates PBL2 (producing PBL2UMP) and activates ZAR1-mediated immune responses
.

However, PBL2 uridylylation does not enhance AvrAC-mediated pathogenicity, so these host proteins are considered to be decoy proteins rather than protected proteins (Lewis et al.
, 2013; Wang et al.
, 2015)
.

However, it is sometimes difficult to distinguish between bait and guard proteins due to functional redundancy
.

For example, RPS5 monitors the status of the protein kinase PBS1 at the plasma membrane (Shao et al.
, 2003; Ade et al.
, 2007), and PBS1 has partial function in plant PTIs when PBS1 is hydrolyzed by the plasma membrane-localized effector protein AvrPphB At the same time, RPS5 activates the immune response by recognizing the cleavage site (Zhang et al.
, 2010), indicating that PBS1 acts as a guard protein
.

However, AvrPphB can target and hydrolyze at least eight PBL proteins, for example, AvrPphB hydrolyzes BIK1, thereby inhibiting the PTI response of plants
.

Some studies speculate that the ortholog of soybean BIK1 may be the best substrate for AvrPphB
.

However, Arabidopsis PBS1 is only weakly functional in PTI (Zhang et al.
, 2010), suggesting that PBS1 can also act as a bait protein
.

Therefore, plants may flexibly respond to pathogen invasion through redundant regulatory modes
.

1.
2.
3 Modes of integration domain recognition Some NLRs contain other protein domains derived from effector protein targeting, termed integrated domains (IDs), which can recognize the corresponding pathogen effector proteins (Fig.
1B)
.

To this end, some IDs directly interact with their matching effector proteins, and are even modified by effector proteases
.

Similar to guard pattern recognition, the ID and NLR are like the fusion of guard protein and NLR
.

In fact, the genetic linkage resulting from the fusion of ID and NLR brings many benefits
.

For example, differences caused by mutations or alleles between protected proteins and their corresponding NLRs may lead to loss of compatibility during long-term evolution, but less likely in integrated NLR-IDs a lot
.

This indicates that the fusion of NLR and ID may occur through retrotranslocation or ectopic recombination (Bailey et al.
, 2018)
.

Currently, several mechanisms by which effector proteins are recognized by NLR-ID patterns have been revealed
.

One of the most well-studied examples is RRS1 (Resistance to Ralstonia solanacearum 1)-mediated R.
solanacearum resistance, which uses the C-terminal WRKY domain as the ID (Le Roux et al.
, 2015; Sarris et al.
, 2015)
.

The effector protein PopP2 secreted by R.
solanacearum is an acetyltransferase that binds and acetylates the WRKY transcription factor, disrupting its binding to DNA to increase pathogenicity (Le Roux et al.
, 2015; Sarris et al.
, 2015 )
.

However, in plants overexpressing RRS1, the WRKY domain of RRS1 is acetylated by PopP2, and WRKY domain acetylation is sufficient to activate the immune response mediated by RPS1 and RPS4 (Resistance to P.
syringae 4) (Le Roux et al.
, 2015; Sarris et al.
, 2015)
.

At the same time, RRS1 also has the function of recognizing other effector proteins
.

For example, the effector protein AvrRps4 is also able to bind the WRKY domain of RRS1 (Saris et al.
, 2015)
.

In addition, other known plant NLR-IDs, including the rice NLRs RGA5 and Pik-1, contain a heavy metal-associated (HMA) integration domain, which recognizes the rice blast effector protein AVR-PikD (Césari et al.
.
, 2014)
.

2 Immune activation and the formation of disease-resistant bodies 2.
1 CNL and disease-resistant bodies of plant NLRs are activated to form oligomeric complexes, which are similar to inflammasomes in animals (Davis et al.
, 2011; Wang et al.
, 2019; Ma et al.
, 2020; Martin et al.
, 2020)
.

For example, the CNL-type resistance body ZAR1 contains ZAR1, receptor-like cytoplasmic kinases (RLCKs) RKS1 and PBL2
.

The effector protein AvrAC secreted from Xanthomonas campestris converts PBL2 into PBL2UMP through its uridyltransferase function, ZAR1 forms a complex with RKS1 and recruits PBL2UMP to form an active PBLUMP-ZAR1-RKS1 complex, thereby completing the conversion of PBL2 to PBL2UMP.
Specific recognition of AvrAC
.

The complex assembles into a pentameric rot, and on its plane forms a funnel-like structure consisting of N-terminal α-helices of five CC domains (Wang et al.
, 2019), which serves as a channel pore to open the defense mechanism of plants.
Door (Figure 2A) (Wang et al.
, 2019; Xia Shitou and Li Xin, 2019)
.

However, whether native ZAR1 oligomers also adopt a pentameric configuration and form pore structures in plant cell membranes, how this pore structure regulates membrane permeability and substrate selectivity, and how this activity triggers cell death and immune responses are currently unclear
.

Two recent breakthrough studies have shown that ZAR1, NRG1a, and ADR1 can all form cation-selective channels in the plasma membrane for calcium permeation (Bi et al.
, 2021; Jacob et al.
, 2021)
.

When wild-type ZAR1 was expressed in Xenopus oocytes with AvrAC, RKS1, and PBL2, strong current signs after voltage application were detected in two-electrode voltage-clamp recording assays, suggesting that the activated ZAR1 complex has channel activity
.

Single-channel recording experiments further showed that the formed pore-like channel was permeable to cations including Na+, K+, Cs2+, Mg2+, and Ca2+, and that the activity of the channel depended on the conserved acidic residue E11 (Bi) located in the channel pore.
et al.
, 2021)
.

During ZAR1 disease-resistant body-induced cell death, plant cells undergo PM damage and rupture in a relatively short period of time, similar to mammalian pyroptosis and necroptosis (requiring the formation of pore-like channels) ( Bi et al.
, 2021)
.

However, osmotic swelling is often accompanied by necroptosis triggered by animal MLKL (mixed lineage kinase domain-like) cation channels (Xia et al.
, 2016), whereas no swelling or swelling was detected in ZAR1-mediated plant cell death.
PM highlights (Bi et al.
, 2021)
.

Therefore, cell death induced by ZAR1 disease-resistant bodies is not identical to pyroptosis and necroptosis
.

2.
2 TNL and disease-resistant bodies Most plant TNLs reported localize or function in the nucleus, and TIRs rarely induce cell death by the same mechanism as the CC domain
.

By studying the TIR domain-containing SARM1 (sterile alpha and TIR motif-containing 1) in animals, it was found that the TIR domain of SARM1 has oligomerization-dependent NADase activity
.

In vitro biochemical assays, the purified SARM1 TIR domain (SARM1TIR) cleaves NAD+ into adenosine diphosphate ribose (ADPR), cyclic adenosine diphosphate ribose (cADPR) and nicotinamide (NAM) (Essuman et al.
, 2017 ), which laid an important foundation for the study of plant TNL
.

Recently, a breakthrough study was to use the baculovirus expression system to co-express the TNL-like disease resistance protein RPP1 and its corresponding effector protein ATR1 in insect cells, purify it, and use cryo-electron microscopy single particle reconstitution technology to successfully analyze the disease resistance of RPP1.
The structure of the corpuscle, RPP1-ATR1 was found to form a tetramer (Ma et al.
, 2020) (Figure 2B)
.

At the same time, another research group solved the similar tetrameric structure of ROQ1-XopQ (Martin et al.
, 2020), indicating that TNL may have a common activation mechanism
.

The structural elucidation of these disease-resistant bodies lays the foundation for revealing the key mechanisms by which RPP1 and ROQ1 directly recognize their cognate effector proteins and tetramerize to enhance NADase activity, thereby activating downstream immune responses
.

Studies have shown that there is a different domain at the carboxy terminus of RPP1 and ROQ1, called C-JID (C-terminal jelly-roll and Ig-like domain) (Ma et al.
, 2020; Martin et al.
, 2020)
.

This domain cooperates with LRR to mediate specific binding to effector proteins.
Mutation of its residues will destroy the structural region of interaction with effector proteins and alleviate ETI-mediated host cell death
.

For RPP1 and ROQ1, direct binding of effectors to LRR and C-JID may release NBD, which changes conformation and oligomerizes into tetramers
.

The tetrameric loop brings the TIR domains into close contact to form TNL disease-resistant bodies with NADase holoenzyme activity
.

Figure 2 Activation mode of two types of NLRs (modified from Duxbury et al.
, 2021) (A) Schematic diagram of the formation of CNL (ZAR1) disease-resistant bodies
.

The Xanthomonas effector protein AvrAC uridylates the Arabidopsis kinase PBL2
.

Urylated PBL2 (PBL2UMP) binds to the intracellular pre-formed ZAR1-RKS1 dimer, resulting in a conformational change of ZAR1 and binding to adenosine triphosphate or deoxyadenosine triphosphate ((d)ATP) at the ZAR1 NBD nucleotide binding site Replaces adenosine diphosphate (ADP)
.

Finally, five ZAR1-RKS1-PBL2UMP monomers formed a pentameric rota-shaped ZAR1 disease-resistant body
.

(B) Schematic diagram of TNL (RPP1) disease-resistant body formation
.

The typical intracellular TIR-type NLR directly recognizes pathogen-free effectors through leucine-rich repeat (LRR) and carboxy-terminal domain (C-JID), forming a nicotinamide adenine dinucleotide glycohydrolase (NADase) ) active tetrameric structure
.

It should be pointed out that, unlike ZAR1-like disease-resistant bodies, which are activated by binding ATP/dATP to exchange ADP, RPP1-like disease-resistant bodies are activated by binding ADP to exchange ATP
.

In RPP1 and ROQ1 disease-resistant bodies, NBD tetramerization brings the four TIR domains close together to form two head-to-tail dimers, asymmetric TIR dimers form one predicted NAD+ binding site, and symmetric TIR dimers form one predicted NAD+ binding site.
TIR dimers stabilize the complex
.

After activation, with the assistance of the BB-loop, the two dimers appear in a head-to-tail manner, and act as a complete NADase to catalyze the hydrolysis of NAD+ to generate signaling substances to induce host cell death
.

It was found that only amino acid mutations within the BB-loop can alter RPP1 and ROQ1 NADase activities and their roles in immunity
.

Therefore, the tetramerization of the TIR domain of TNL to form a holoenzyme may represent a common activation mechanism for such plant NLRs
.

The structural analysis of RPP1 and ROQ1 revealed a novel TNL disease-resistant body different from CNL ZAR1, which provided help for understanding the activation mechanism of plant TNL
.

However, the specific mechanism by which TIR NADase activity activates downstream immunity remains unclear
.

Jubicet et al.
(2019) suggested that the NADase activity of TNL is activated by downstream key components and helper hNLRs (including ADR1 and NRG1), and conducts immune signal transduction in a currently unknown manner
.

Furthermore, unlike ZAR1, both RPP1 and ROQ1 disease-resistant bodies contain domains that directly recognize their effector proteins
.

Therefore, dissecting the atypical structures of disease-resistant bodies with different properties will help to better understand the different activation mechanisms of NLRs in higher plants
.

3 Immune execution and its signal regulation 3.
1 After immune execution CNL-type NLRs recognize effector proteins, oligomeric ZAR1 disease-resistant bodies bind to PM and form cation-selective channels for calcium permeation, leading to calcium influx and further activation Downstream defense responses, including cell death, etc.
(Bi et al.
, 2021)
.

Thus, ZAR1 disease-resistant bodies are both sensors of pathogen effectors and downstream executors of cell death and signal transduction
.

More importantly, ZAR1 disease-resistant bodies induce ROS production, disruption of PM integrity, and HR all require residue E11, suggesting a critical role for ZAR1 channel activity in leading to cell death and execution of downstream events in immunity (Bi et al.
, 2021)
.

However, whether all CNLs can act as Ca2+ influx channels needs further study
.

In the future, structural and functional analysis of other CNLs with different N-termini can further reveal whether there are alternative mechanisms for CNLs
.

The immune responses mediated by TIR-type NLRs depend on the NADase activity of oligomeric TIR complexes, lipase-like proteins of the EDS1 (enhanced disease susceptibility 1) family and helper hNLRs containing HeLo-like domains
.

Similar to ZAR1 E11, NRG1.
1 E14 and ADR1 D11 residues are also critical for calcium channel formation and induction of downstream signaling
.

Jacob et al.
(2021) found through the study of NRG1a variants that when the aspartic acid at position 485 is mutated to valine (D485V), immune self-activation occurs, and it is enriched and oligomerized in PM, thereby Triggers Ca2+ influx-dependent cell death
.

The X-ray crystal structure of NRG1a (D485V) (residues 1-124) is highly similar to the N-terminal 4-helical bundles (4HBs) of ZAR1 and MLKL (Jacob et al.
, 2021), suggesting that its N-terminal The formation of terminal holes may be similar to ZAR1
.

It is worth mentioning that although homo-oligomerization of NRG1a (D485V) was observed, native oligomers formed by hNLR usually require interaction with the lipase-like proteins EDS1-PAD4 (phytoalexin deficient 4) and EDS1-SAG101 (senescence).
-associated gene 101) interactions (Sun et al.
, 2021; Wu et al.
, 2021)
.

Therefore, TNL may acquire NADase activity through oligomerization to form disease-resistant bodies, generate signaling molecules, and induce the formation of complexes between lipase-like proteins of the EDS1 family and auxiliary NLR, thereby triggering cellular Ca influx and cell death
.

3.
2 Immune signaling regulates CNL and TNL activation, resulting in similar transcriptional expression and triggering local and systemic resistance (Bartsch et al.
, 2006; Mine et al.
, 2018; Saile et al.
, 2020; Zhou and Zhang, 2020 )
.

NLRs also act synergistically with PRR to enhance PTI, thereby generating a complete immune response (Ngou et al.
, 2021; Yuan et al.
, 2021)
.

CNL-like NLRs can act as cation channels on the plasma membrane to mediate Ca2+ influx, demonstrating that multiple Ca2+ permeable channels can conduct Ca2+ influx and activate membrane-localized receptors with kinase activity, thereby triggering immune responses (Tian et al.
, 2019; Thor et al.
, 2020)
.

Hu et al.
(2020) found that LaCl3 treatment can block calcium channel activity without affecting the formation of ZAR1 complexes, and inhibit cell death to a large extent, speculating that the rapid influx of Ca2+ caused by ZAR1 channels is mediated by ZAR1 The downstream signal of plant immune response, but it does not rule out that ZAR1 disease-resistant body also has other roles in the process of immune response
.

TNL-like NLRs cleaves NAD+ through NADase activity to produce a variety of products, including nicotinamide adenine mononucleotide (Wan et al.
, 2019), which can act as downstream activation EDS1-dependent defense signals
.

Extensive experiments confirmed that EDS1, PAD4 and SAG101 are required for TNL-mediated immune responses
.

Signaling downstream of EDS1 is transformed into two parallel pathways that are dependent on SAG101 and PAD4, respectively (Figure 3)
.

SAG101 and PAD4 share a lipase-like domain with EDS1, and they form distinct complexes with EDS1 to activate downstream defense responses (Cui et al.
, 2015)
.

Although the mode of action of these complexes is currently unclear, the cavities formed between heterodimers can serve as binding sites for unknown proteins or activating immune signaling ligands (Bhandari et al.
, 2019)
.

Figure 3 Working model of immune response mediated by NLRs in higher plants (modified from Liu et al.
, 2021) When plant cells are infected by pathogens, some pathogens can secrete effectors to break through the plant's immune defense line
.

During long-term evolution, plants have evolved many intracellular receptors to recognize these effectors, thereby triggering their resistance to pathogens
.

CNLs trigger their pentamerization and disease resistance body formation at the plasma membrane by sensing effector proteins (this model is exemplified by the indirect recognition pattern of the ZAR1 disease resistance body), and through the pore formed by the α1 helix in the N-terminal CC domain As a Ca2+ influx channel, it mediates an increase in cytoplasmic Ca2+ concentration, which initiates cell death and defense responses
.

TNLs sense effector proteins to form tetrameric disease-resistant bodies (this model is exemplified by RPP1 in direct recognition mode), and the formation of TNL disease-resistant bodies leads to activation of TIR NADase, triggering a trigger that may contain EDS1-PAD4-ADR1s or EDS1-SAG101-NRG1s Assembly of low polymers
.

The oligomerization of helper NLRs can form CNL-like pore channels, which serve as Ca2+ influx channels and mediate downstream immunity and cell death
.

Red arrows indicate CNL signals, blue arrows indicate TNL and RNL signals
.

Studies have shown that NRG1, together with EDS1 and SAG101, play a role in TNL-induced HR activation (Qi et al.
, 2018; Gantner et al.
, 2019; Lapin et al.
, 2019)
.

Arabidopsis has 2 redundant NRG1 orthologs, NRG1a and NRG1b, both of which are required for TNL-triggered HR and immunity (Castel et al.
, 2019; Wu et al.
, 2019) (Figure 3)
.

The Arabidopsis RNL ADR1, ADR1-L1 and ADR1-L2 act downstream of EDS1 and PAD4 to promote SA biosynthesis triggered by TNL and some CNLs (Bonardi et al.
, 2011; Dong et al.
, 2016; Wu et al.
, 2019) (Figure 3)
.

Different helper immune receptors form complexes with specific lipase-like protein families to transmit immune signals
.

Sun et al.
(2021) found that the activation of TNL immunoreceptors can induce the formation of protein complexes between EDS1-SAG101 and NRG1; Wu et al.
(2021) confirmed that TNL immunoreceptors can promote the formation of multimers between ADR1 and EDS1-PAD4
.

Therefore, it is inferred that TIR-type NLRs may generate signaling small molecules through the catalytic function of their TIR domains, these small molecules activate EDS1 and its family members and bind and activate auxiliary NLRs ADR1 or NRG1 (Figure 3), and possibly EDS1 and its family members Members provide other metabolic substrates to TIR-type NLRs, which are enzymatically converted into signaling molecules that activate ADR1 or NRG1
.

The above studies on CNL ZAR1, hNLRs NRG1a and ADR1 as Ca2+ influx channels enrich the NLR signal transduction pathway in plant immune response
.

Ca2+ influx as a trigger of cell death is consistent with previous findings that mis-upregulated Ca2+ accumulation can lead to cell death and autoimmunity (Yoshioka et al.
, 2006; Zhao et al.
, 2021), and the regulation of Ca2+ channels is in the The centrality of NLR-mediated plant immune responses (Xia et al.
, 2021)
.

4 Summary and outlook In recent years, major progress and landmark discoveries related to plant immunity mediated by NLRs have deepened our understanding of the plant immune system's perception and regulation of pathogens, and provided new ideas for crop disease resistance improvement
.

The analysis of the structure and function of NLRs also provides new clues for revealing the signal transduction mechanism of the CC and TIR domains, enabling people to better understand how NLRs specifically recognize effector proteins and design novel and specific NLRs
.

Nonetheless, there are still many unanswered questions that need to be further explored
.

For example, do other CNLs or TNLs form ZAR1, Roq1, or RPP1-like structures of disease resistance bodies? Could paired NLRs (such as RRS1/RPS4) or network-forming NLRs also form structures similar to disease resistance bodies or inflammasomes to Activates the immune response? Can other hNLRs be activated by TNL to form calcium channels like ADR1/NRG1 to mediate cell death and other downstream signals? Are there other overlooked mechanisms? Also, how does TNL-NADase activity interact with downstream signals such as transcriptional activation or auxiliary NLR activation (such as ADR1/NRG1) is also not well understood
.

TNL and CNL activation can lead to similar transcriptional reprogramming (Jacob et al.
, 2018; Ding et al.
, 2020; Saile et al.
, 2020), but how they converge and produce similar transcriptional programs is unclear
.

NLR-mediated disease resistance was recently found to be PRR-dependent, whereas NLR activation enhanced PRR-mediated immune responses (Ngou et al.
, 2021; Yuan et al.
, 2021), suggesting that both PRRs and NLRs are required for full activation of plant immune responses The synergistic effect of signaling (Wang Wei and Tang Dingzhong, 2021), therefore, PTI and ETI are not independent immune pathways
.

Future studies should focus on the general mechanisms of immune responses mediated between cell surface receptors and intracellular immune receptors
.

For example, how does the NLR-mediated immune pathway receive the PRR signal? Is the immune cooperation between PTI and ETI widespread in plant-pathogen interactions? The study of the underlying mechanisms of PTI and ETI immune cooperation will contribute to a comprehensive understanding of plant immune cooperation.
The immune system lays a theoretical foundation for crop disease resistance breeding engineering and green agriculture development
.

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