NLR signaling in plants: from resistosomes to second messengers

Pathogen effector-induced assembly of resistosomes has been established as an important event for nucleotide binding and leucine-rich repeat-containing receptor (NLR) signaling in plants.The pentameric coiled-coil domain-containing NLR (CNL) resistosomes act as Ca2+-permeable channels, whereas the tetrameric Toll-interleukin 1-like receptor (TIR) NLR (TNL) resistosomes are NADase holoenzymes.TNL resistosomes catalyze the production of nucleotide-derived second messengers to activate the downstream helper NLRs activated disease resistance 1 (ADR1) and N requirement gene 1 (NRG1) of the CNL class. Thus, CNLs and TNLs converge on Ca2+ signals to trigger plant immunity.NLR signaling cross-talks with pattern-triggered immunity (PTI) signaling pathways.NLR signaling pathways in plants are negatively regulated by both hosts and pathogens. Nucleotide binding and leucine-rich repeat-containing receptors (NLRs) have a critical role in plant immunity through direct or indirect recognition of pathogen effectors. Recent studies have demonstrated that such recognition induces formation of large protein complexes called resistosomes to mediate NLR immune signaling. Some NLR resistosomes activate Ca2+ influx by acting as Ca2+-permeable channels, whereas others function as active NADases to catalyze the production of nucleotide-derived second messengers. In this review we summarize these studies on pathogen effector-induced assembly of NLR resistosomes and resistosome-mediated production of the second messengers of Ca2+ and nucleotide derivatives. We also discuss downstream events and regulation of resistosome signaling. Nucleotide binding and leucine-rich repeat-containing receptors (NLRs) have a critical role in plant immunity through direct or indirect recognition of pathogen effectors. Recent studies have demonstrated that such recognition induces formation of large protein complexes called resistosomes to mediate NLR immune signaling. Some NLR resistosomes activate Ca2+ influx by acting as Ca2+-permeable channels, whereas others function as active NADases to catalyze the production of nucleotide-derived second messengers. In this review we summarize these studies on pathogen effector-induced assembly of NLR resistosomes and resistosome-mediated production of the second messengers of Ca2+ and nucleotide derivatives. We also discuss downstream events and regulation of resistosome signaling. Plants rely on multiple receptors to detect invading microbial pathogens and mount immune responses [1.Ngou B.P.M. et al.Thirty years of resistance: zig-zag through the plant immune system.Plant Cell. 2022; 34: 1447-1478Crossref PubMed Scopus (45) Google Scholar,2.Zhou J.M. Zhang Y. Plant immunity: danger perception and signaling.Cell. 2020; 181: 978-989Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar]. One subfamily of plant immune receptors are pattern-recognition receptors (PRRs) (see Glossary) at the cell surface [1.Ngou B.P.M. et al.Thirty years of resistance: zig-zag through the plant immune system.Plant Cell. 2022; 34: 1447-1478Crossref PubMed Scopus (45) Google Scholar, 2.Zhou J.M. Zhang Y. Plant immunity: danger perception and signaling.Cell. 2020; 181: 978-989Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 3.DeFalco T.A. Zipfel C. Molecular mechanisms of early plant pattern-triggered immune signaling.Mol. Cell. 2021; 81: 3449-3467Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. PRRs recognize pathogen-associated molecular patterns (PAMPs) or host-derived damage-associated molecular patterns (DAMPs), leading to pattern-triggered immunity (PTI). PTI constitutes the first line of inducible plant defense against pathogens. Some pathogens can breach this layer of defense by secreting effector proteins into plant cells to dampen PTI. To counteract the virulence activity of the pathogen effectors, plants have evolved a second subfamily of immune receptors: intracellular NLRs. NLRs specifically recognize effector proteins, inducing effector-triggered immunity (ETI) and confer race-specific resistance to pathogens at the site of pathogen entry [1.Ngou B.P.M. et al.Thirty years of resistance: zig-zag through the plant immune system.Plant Cell. 2022; 34: 1447-1478Crossref PubMed Scopus (45) Google Scholar,2.Zhou J.M. Zhang Y. Plant immunity: danger perception and signaling.Cell. 2020; 181: 978-989Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar,4.Jones J.D.G. et al.Intracellular innate immune surveillance devices in plants and animals.Science. 2016; 354aaf6395Crossref Google Scholar]. PRRs and NLRs have different structures and subcellular localizations but mediate conserved downstream immune responses, including Ca2+ influx, bursts of reactive oxygen species (ROS), production of phytocytokines and defense phytohormones, and transcriptional reprogramming [1.Ngou B.P.M. et al.Thirty years of resistance: zig-zag through the plant immune system.Plant Cell. 2022; 34: 1447-1478Crossref PubMed Scopus (45) Google Scholar,2.Zhou J.M. Zhang Y. Plant immunity: danger perception and signaling.Cell. 2020; 181: 978-989Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar]. Probably for this reason, PTI and ETI are tightly connected [5.Yuan M. et al.Pattern-recognition receptors are required for NLR-mediated plant immunity.Nature. 2021; 592: 105-109Crossref PubMed Scopus (351) Google Scholar,6.Ngou B.P.M. et al.Mutual potentiation of plant immunity by cell-surface and intracellular receptors.Nature. 2021; 592: 110-115Crossref PubMed Scopus (329) Google Scholar]. However, PTI and ETI differ in timing, amplitude, and duration of defense, which could be important in determining their different physiological outcomes. In addition to these responses, ETI also includes a hypersensitive response (HR), a form of rapid localized programmed cell death at the site of infection [7.Jones J.D. Dangl J.L. The plant immune system.Nature. 2006; 444: 323-329Crossref PubMed Scopus (8693) Google Scholar]. NLRs are the largest intracellular immune receptors with hundreds of distinct members in different plant species [4.Jones J.D.G. et al.Intracellular innate immune surveillance devices in plants and animals.Science. 2016; 354aaf6395Crossref Google Scholar]. NLRs have two conserved domains: a central nucleotide-binding and oligomerization domain (NOD), and a C-terminal leucine-rich repeat (LRR) domain. A variable Toll-interleukin 1-like receptor (TIR) or coiled-coil (CC) domain is attached at the N terminus, resulting in TIR-NLR (TNL) or CC-NLR (CNL), respectively [8.Maruta N. et al.Structural basis of NLR activation and innate immune signalling in plants.Immunogenetics. 2022; 74: 5-26Crossref PubMed Scopus (25) Google Scholar,9.Hu Z. Chai J. Assembly and architecture of NLR resistosomes and inflammasomes.Annu. Rev. Biophys. 2022; 52: 8.1-8.22Google Scholar]. In addition to pathogen-sensing NLRs, there are some helper (h) NLRs which function to translate signals from pathogen-sensing NLRs into ETI responses [10.Feehan J.M. et al.Plant NLRs get by with a little help from their friends.Curr. Opin. Plant Biol. 2020; 56: 99-108Crossref PubMed Scopus (18) Google Scholar,11.Jubic L.M. et al.Help wanted: helper NLRs and plant immune responses.Curr. Opin. Plant Biol. 2019; 50: 82-94Crossref PubMed Scopus (139) Google Scholar]. Examples of hNLRs include activated disease resistance 1 (ADR1) and N requirement gene 1 (NRG1) of the Resistance to Powdery Mildew 8 (RPW8) CNL family [12.Wu Z. et al.Differential regulation of TNL-mediated immune signaling by redundant helper CNLs.New Phytol. 2019; 222: 938-953Crossref PubMed Scopus (118) Google Scholar, 13.Castel B. et al.Diverse NLR immune receptors activate defence via the RPW8-NLR NRG1.New Phytol. 2019; 222: 966-980Crossref PubMed Scopus (144) Google Scholar, 14.Qi T. et al.NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in Nicotiana benthamiana.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E10979-E10987Crossref PubMed Scopus (121) Google Scholar, 15.Collier S.M. et al.Cell death mediated by the N-terminal domains of a unique and highly conserved class of NB-LRR protein.Mol. Plant Microbe Interact. 2011; 24: 918-931Crossref PubMed Scopus (230) Google Scholar] (Figure 1) and NLRs required for cell death (NRCs) [16.Adachi H. Kamoun S. NLR receptor networks in plants.Essays Biochem. 2022; 66: 541-549Crossref PubMed Scopus (4) Google Scholar,17.Wu C.H. et al.NLR network mediates immunity to diverse plant pathogens.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 8113-8118Crossref PubMed Scopus (211) Google Scholar]. More recent studies have demonstrated that ETI signaling, mediated by many sensor NLRs in members of the Solanaceae, depends on NRCs which form resistosomes upon activation [18.Ahn H.K. et al.Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1.EMBO J. 2023; 42e111484Crossref Scopus (5) Google Scholar,19.Contreras M.P. et al.Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens.EMBO J. 2022; 42e111519PubMed Google Scholar]. In this review we summarize the activation and assembly of NLR resistosomes and discuss their downstream second messengers, including calcium ion and nucleotide derivations. We also review the crosstalk between ETI and PTI signaling pathways and negative regulation of NLR signaling by pathogen effectors and host regulators. NLRs can recognize effectors directly or indirectly. For example, direct interaction of the Nicotiana benthamiana TNL Roq1 (recognition of XopQ 1) with its recognized effector XopQ (Xanthomonas outer protein Q) [20.Schultink A. et al.Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1.Plant J. 2017; 92: 787-795Crossref PubMed Scopus (82) Google Scholar,21.Martin R. et al.Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ.Science. 2020; 370eabd9993Crossref Scopus (174) Google Scholar], the arabidopsis TNL RPP1 (recognition of Peronospora parasitica 1) with effector ATR1 (Arabidopsis thaliana recognized 1) [22.Ma S. et al.Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme.Science. 2020; 370abe3069Crossref Scopus (169) Google Scholar,23.Krasileva K.V. et al.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. 2010; 22: 2444-2458Crossref PubMed Scopus (220) Google Scholar], and the wheat CNL Sr35 (stem rust resistance gene 35) with effector AvrSr35 [24.Zhao Y.-B. et al.Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism.Sci. Adv. 2022; 8eabq5108Crossref Scopus (13) Google Scholar, 25.Forderer A. et al.A wheat resistosome defines common principles of immune receptor channels.Nature. 2022; 610: 532-539Crossref PubMed Scopus (0) Google Scholar, 26.Salcedo A. et al.Variation in the AvrSr35 gene determines Sr35 resistance against wheat stem rust race Ug99.Science. 2017; 358: 1604-1606Crossref PubMed Scopus (117) Google Scholar] confer resistance against Xanthomonas spp, Hyaloperonospora parasitica, and Puccinia graminis tritici, respectively. Some NLRs recognize their cognate effectors by monitoring effector-mediated perturbations of host targets. One example of this is the arabidopsis CNL ZAR1 (HOPZ-ACTIVATED RESISTANCE 1) [27.Lewis J.D. et al.Allele-specific virulence attenuation of the Pseudomonas syringae HopZ1a type III effector via the Arabidopsis ZAR1 resistance protein.PLoS Genet. 2010; 6e1000894Crossref Scopus (111) Google Scholar], which exists in a preformed complex with a host kinase RKS1 (resistance-related kinase 1) in normal conditions and recognizes the Xanthomonas effector AvrAC indirectly through uridylylation of another host kinase PBL2 (PBS1-like protein 2) [28.Wang J. et al.Ligand-triggered allosteric ADP release primes a plant NLR complex.Science. 2019; 364aav5868Crossref Scopus (231) Google Scholar,29.Wang G. et al.The decoy substrate of a pathogen effector and a pseudokinase specify pathogen-induced modified-self recognition and immunity in plants.Cell Host Microbe. 2015; 18: 285-295Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar]; AvrAC-uridylylated PBL2 associates with RKS1 and consequently activates ZAR1-mediated immunity. Alternatively, in some cases, NLRs may not be necessarily activated by pathogen effectors. For example, the arabidopsis TNL CHS3 (CHILLING SENSITIVE 3)/CSA1 (CONSTITUTIVE SHADE-AVOIDANCE 1) pair detects perturbations of the PRR coreceptor BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) [30.Yang Y. et al.Allelic variation in the Arabidopsis TNL CHS3/CSA1 immune receptor pair reveals two functional cell-death regulatory modes.Cell Host Microbe. 2022; 30: 1701-1716Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar], whereas the malectin-like receptor-like kinase LET1 activates autoimmunity by the CNL SUMM2 (SUPPRESSOR OF MKK1 MKK2) via MEKK2 (MAP/ERK kinase kinase-2) scaffolding [31.Liu J. et al.The malectin-like receptor-like kinase LETUM1 modulates NLR protein SUMM2 activation via MEKK2 scaffolding.Nat. Plants. 2020; 6: 1106-1115Crossref PubMed Scopus (26) Google Scholar]. Effector recognition leads to NLR oligomerization and the formation of large protein complexes termed resistosomes (Figure 1). Cryoelectron microscopy (cryoEM) analyses show that the ZAR1 resistosome containing ZAR1, RKS1, and uridylylated PBL2 (PBL2UMP) forms a wheel-like pentameric complex [32.Wang J. et al.Reconstitution and structure of a plant NLR resistosome conferring immunity.Science. 2019; 364aav5870Crossref Scopus (368) Google Scholar] (Figure 1, left). Pentamerization of the ZAR1 resistosome is mediated mainly by the NOD module of ZAR1, with RKS1 and PBL2UMP being presented at the rim of the wheel (Figure 2). In contrast to ZAR1, Sr35 directly recognizes its cognate effector AvrSr35, but the resulting Sr35 resistosome is also a pentameric complex with structure remarkably similar to that of the ZAR1 resistosome [24.Zhao Y.-B. et al.Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism.Sci. Adv. 2022; 8eabq5108Crossref Scopus (13) Google Scholar,25.Forderer A. et al.A wheat resistosome defines common principles of immune receptor channels.Nature. 2022; 610: 532-539Crossref PubMed Scopus (0) Google Scholar]. A more recent study showed that oligomeric NRG1 resistosomes are likely to be formed at the plasma membrane (PM) [33.Feehan J.M. et al.Oligomerization of a plant helper NLR requires cell-surface and intracellular immune receptor activation.Proc. Natl. Acad. Sci. U. S. A. 2023; 120e2210406120Crossref PubMed Scopus (3) Google Scholar]. Interestingly, PTI signaling is required for the formation of NRG1 resistosomes, but the underlying mechanisms await further elucidation [33.Feehan J.M. et al.Oligomerization of a plant helper NLR requires cell-surface and intracellular immune receptor activation.Proc. Natl. Acad. Sci. U. S. A. 2023; 120e2210406120Crossref PubMed Scopus (3) Google Scholar]. By comparison, direct binding of ATR1 to the C-terminal end of RPP1 results in the formation of a tetrameric RPP1 resistosome [22.Ma S. et al.Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme.Science. 2020; 370abe3069Crossref Scopus (169) Google Scholar] (Figure 1, right). A similar assembly of the Roq1 resistosome is induced by direct XopQ binding to Roq1 [21.Martin R. et al.Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ.Science. 2020; 370eabd9993Crossref Scopus (174) Google Scholar]. As seen in the CNL resistosomes, oligomerization of the two TNL resistosomes is primarily mediated by the NOD module (Figure 2). Structural and modeling studies showed that NOD is sequestered from oligomerization in inactive NLRs [22.Ma S. et al.Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme.Science. 2020; 370abe3069Crossref Scopus (169) Google Scholar,24.Zhao Y.-B. et al.Pathogen effector AvrSr35 triggers Sr35 resistosome assembly via a direct recognition mechanism.Sci. Adv. 2022; 8eabq5108Crossref Scopus (13) Google Scholar,25.Forderer A. et al.A wheat resistosome defines common principles of immune receptor channels.Nature. 2022; 610: 532-539Crossref PubMed Scopus (0) Google Scholar,32.Wang J. et al.Reconstitution and structure of a plant NLR resistosome conferring immunity.Science. 2019; 364aav5870Crossref Scopus (368) Google Scholar], suggesting that recognition of pathogens results in release of the NOD module for NLR oligomerization. However, it remains unknown whether oligomerization of all plant NLRs leads to assembly of resistosomes. Many NLRs act in pairs, with the sensor NLR recognizing pathogen effectors and the executor NLR initiating immune signaling [34.Xi Y. et al.Insight into the structure and molecular mode of action of plant paired NLR immune receptors.Essays Biochem. 2022; 66: 513-526Crossref PubMed Scopus (2) Google Scholar]. Whether and how paired NLRs form resistosomes represent a challenge for understanding their signaling mechanisms. In the cryoEM structure of the ZAR1 resistosome, the five N-terminal α1-helices of ZAR1 form a solvent-exposed structure shaped like a channel or pore [32.Wang J. et al.Reconstitution and structure of a plant NLR resistosome conferring immunity.Science. 2019; 364aav5870Crossref Scopus (368) Google Scholar] (Figure 2). Functional data support a critical role for the solvent-exposed structure in ZAR1-mediated ETI. Importantly, electrophysiological evidence supports the ZAR1 resistosome function as a Ca2+-permeable channel to mediate immune response [35.Bi G. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; 184: 3528-3541Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar] (Figure 1, bottom left). Single molecule imaging showed that the ZAR1 resistosome forms hours before the loss of PM integrity [35.Bi G. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; 184: 3528-3541Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar]. These results suggest that Ca2+ influx mediated by the ZAR1 resistosome acts as a trigger for ZAR1 signaling. ZAR1 α1 is conserved in many CNLs from distantly related plant species [36.Adachi H. et al.An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species.eLife. 2019; 8e49956Crossref Scopus (92) Google Scholar], suggesting that Ca2+-channel activity may be conserved among CNL resistosomes. Indeed, similar activity has been demonstrated for the wheat CNL Sr35 resistosome, which bears a highly similar structure to that of the ZAR1 resistosome [25.Forderer A. et al.A wheat resistosome defines common principles of immune receptor channels.Nature. 2022; 610: 532-539Crossref PubMed Scopus (0) Google Scholar]. However, in contrast to that in the ZAR1 resistosome, the functionally essential α1 helix is not well-defined in the Sr35 resistosome. It may be that a membrane environment is required for Sr35 to form a funnel-shaped structure in the Sr35 resistosome. PM localization has been shown for the arabidopsis CNLs RPM1 (resistance to Pseudomonas syringae pv. maculicola 1) [37.El Kasmi F. et al.Signaling from the plasma-membrane localized plant immune receptor RPM1 requires self-association of the full-length protein.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E7385-E7394Crossref PubMed Scopus (70) Google Scholar] and RPS2 (resistance to P. syringae 2) [38.Axtell M.J. Staskawicz B.J. Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4.Cell. 2003; 112: 369-377Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar], and the N. bethamiana CNL Tm-22 [39.Wang J. et al.Plant NLR immune receptor Tm-22 activation requires NB-ARC domain-mediated self-association of CC domain.PLoS Pathog. 2020; 16e1008475Crossref Scopus (20) Google Scholar], but whether these CNLs can form ZAR1-like resistosomes remains to be examined. The TNL-activated hNLRs, NRG1s, and ADR1s can also form resistosomes at the PM and display similar Ca2+-permeable channel activity in their autoactive forms [33.Feehan J.M. et al.Oligomerization of a plant helper NLR requires cell-surface and intracellular immune receptor activation.Proc. Natl. Acad. Sci. U. S. A. 2023; 120e2210406120Crossref PubMed Scopus (3) Google Scholar,40.Jacob P. et al.Plant “helper” immune receptors are Ca2+-permeable nonselective cation channels.Science. 2021; 373: 420-425Crossref PubMed Scopus (125) Google Scholar], indicating that CNLs and TNLs converge on Ca2+ signals (Figure 2). A large domain called Solanaceae domain (SD) before the CC domain is found in many noncanonical CNLs in members of the Solanaceae [41.Seong K. et al.Evolution of NLR resistance genes with noncanonical N-terminal domains in wild tomato species.New Phytol. 2020; 227: 1530-1543Crossref PubMed Scopus (36) Google Scholar], suggesting that these CNLs themselves may not form oligomeric structures. Recent studies showed that some of these noncanonical CNLs function to activate NRC resistosomes [18.Ahn H.K. et al.Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1.EMBO J. 2023; 42e111484Crossref Scopus (5) Google Scholar,19.Contreras M.P. et al.Sensor NLR immune proteins activate oligomerization of their NRC helpers in response to plant pathogens.EMBO J. 2022; 42e111519PubMed Google Scholar]. However, it remains undetermined whether these NRC resistosomes have Ca2+-permeable channel activity. Multiple lines of functional evidence support extracellular Ca2+ influx as a trigger of ETI signaling [42.Xu G. et al.A tale of many families: calcium channels in plant immunity.Plant Cell. 2022; 34: 1551-1567Crossref PubMed Scopus (19) Google Scholar,43.Kim N.H. et al.Con-Ca2+-tenating plant immune responses via calcium-permeable cation channels.New Phytol. 2022; 234: 813-818Crossref PubMed Scopus (8) Google Scholar]. Elevation in intracellular Ca2+ concentrations is one of the earliest events during ETI. Gain-of-function mutations of CNGC19/20 (CYCLIC NUCLEOTIDE GATED CHANNEL19/20) with increased Ca2+ influx activity constitutively activate EDS1 (enhanced disease susceptibility 1)- and SA (salicylic acid)-dependent arabidopsis immunity [44.Yu X. et al.The receptor kinases BAK1/SERK4 regulate Ca2+ channel-mediated cellular homeostasis for cell death containment.Curr. 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Although Ca2+ released from internal pools can contribute to ETI signaling, pharmacological study showed that blocking of Ca2+ release from intracellular compartments by ruthenium red (RR) is less efficient for inhibition of HR cell death than by the Ca2+ influx blocker LaCl3 [46.Gao F. et al.A heat-activated calcium-permeable channel Arabidopsis cyclic nucleotide-gated ion channel 6 is involved in heat shock responses.Plant J. 2012; 70: 1056-1069Crossref PubMed Scopus (0) Google Scholar], supporting the notion that Ca2+ influx is a major trigger of ETI responses. Nuclear localization is required for the disease resistance activity of many NLRs [47.Ludke D. et al.NLR we there yet? Nucleocytoplasmic coordination of NLR-mediated immunity.New Phytol. 2022; 236: 24-42Crossref PubMed Scopus (0) Google Scholar], but the mechanism of how the cellular localization of NLRs is associated with this activity remains enigmatic. Several CNLs have been shown to interact with transcriptional factors [48.Wang J. et al.Diversity, structure and function of the coiled-coil domains of plant NLR immune receptors.J. Integr. Plant Biol. 2021; 63: 283-296Crossref PubMed Scopus (11) Google Scholar], suggesting that CNLs may directly regulate transcriptional programming in the nucleus. PTI and ETI signaling activate transcription of a similar set of genes, suggesting that other mechanisms can also be involved in NLR-mediated transcriptional reprogramming. NRG1A is both PM- and nucleus-localized upon activation, but only the PM-resident NRG1A forms oligomers [33.Feehan J.M. et al.Oligomerization of a plant helper NLR requires cell-surface and intracellular immune receptor activation.Proc. Natl. Acad. Sci. U. S. A. 2023; 120e2210406120Crossref PubMed Scopus (3) Google Scholar], suggesting an oligomerization-independent NRG1 function. Increases in nuclear free Ca2+ concentrations have been reported in response to various stresses [49.Pauly N. et al.The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells.Cell Calcium. 2001; 30: 413-421Crossref PubMed Scopus (0) Google Scholar, 50.van Der Luit A.H. et al.Distinct calcium signaling pathways regulate calmodulin gene expression in tobacco.Plant Physiol. 1999; 121: 705-714Crossref PubMed Google Scholar, 51.Xiong T.C. et al.Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling.Plant J. 2004; 40: 12-21Crossref PubMed Scopus (0) Google Scholar]. It should be kept in mind that spatial distribution of Ca2+ in a cell is not uniform, and concentrations of Ca2+ can have steep gradients a few nanometers away from the Ca2+ channel [52.Pangršič T. et al.EF-hand protein Ca2+ buffers regulate Ca2+ influx and exocytosis in sensory hair cells.Proc. Natl. Acad. Sci. U. S. 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Assembly of the ATR1-induced RPP1 resistosome significantly enhances RPP1 NADase activity, indicating that the resistosome acts as an NADase holoenzyme [22.Ma S. et al.Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme.Science. 2020; 370abe3069Crossref Scopus (169) Google Scholar]. The enzymatic activity is encoded in the N-terminal TIR domain of TNLs [54.Wan L. et al.TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death.Science. 2019; 365: 799-803Crossref PubMed Scopus (226) Google Scholar,55.Horsefield S. et al.NAD+ cleavage activity by animal and plant TIR domains in cell death pathways.Science. 2019; 365: 793-799Crossref PubMed Scopus (244) Google Scholar], which is required for immune signaling mediated by TNLs [22.Ma S. et al.Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme.Science. 2020; 370abe3069Crossref Scopus (169) Google Scholar] and TIR-only proteins such as response to the bacterial type III effector protein HopBA1 (RBA1) in arabidopsis [56.Nishimura M.T. et al.TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E2053-E2062Crossref PubMed Scopus (93) Google Scholar]. Tetramerization results in the formation of two composite active sites in the TIR domains of TNL resistosomes [21.Martin R. et al.Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ.Science. 2020; 370eabd9993Crossref Scopus (174) Google Scholar,22.Ma S. et al.Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme.Science. 2020; 370abe3069Crossref Scopus (169) Google Scholar] (Figure 2). A similar mechanism has also been demonstrated for activation of TIR domain proteins from other species [57.Shi Y. et al.Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules.Mol. 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