SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

NAD+ cleavage activity by animal and plant TIR domains in cell death pathways

INTRODUCTION Plants and animals respond to pathogen invasion through intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly interact with pathogen proteins or indirectly detect pathogen-derived alterations in the host proteome. Upon recognition of pathogen invasion, NLRs trigger an immune response that resolves in a variety of ways depending on the type of NLR being activated. The overall architecture of NLRs is highly conserved, consisting of a C-terminal leucine-rich repeat (LRR) platform that determines substrate specificity and a central nucleotide-binding oligomerization domain. The N-terminal domain varies between NLRs and determines the mechanism used by the host to activate the immune response. Thus, NLRs in plants have been classified according to their N-terminal domain into Toll/interleukin 1 receptor (TIR) NLRs (TNLs), coiled-coil NLRs (CNLs), and RPW8-like coiled-coil NLRs (RNLs). Pathogen detection and oligomerization of the NLR activates these N-terminal domains by bringing them in close contact. In all three cases, association of the N-terminal domain leads to localized cell death and expression of disease resistance. The TIR domains of TNLs have been shown to have oligomerization-dependent NADase activity that is required for promoting cell death, but it is not understood how the interactions between TIR domains renders them catalytically active. RATIONALE The structure of the ROQ1 (recognition of XopQ 1)–XopQ (Xanthomonas outer protein Q) complex, an immune receptor bound to its pathogen substrate, was used as a model to study the mechanism of direct binding, oligomerization, and TIR domain activation of TNLs. ROQ1 has been shown to physically interact with the Xanthomonas effector XopQ, causing it to oligomerize and trigger a TIR-dependent hypersensitive cell death response. We coexpressed, extracted, and purified the assembled ROQ1-XopQ complex from ROQ1’s native host, Nicotiana benthamiana, and solved its structure by cryo–electron microscopy to 3.8-A resolution. The interactions described in our structure were further confirmed by in vivo mutational analysis. RESULTS Our structure reveals that ROQ1 forms a tetrameric resistosome upon recognizing XopQ. The LRR and a post-LRR domain named the C-terminal jelly-roll/Ig-like domain (C-JID), form a horseshoe-shaped scaffold that curls around the pathogen effector, thereby recognizing multiple regions of the substrate. Binding of the ROQ1 LRR to XopQ occurs through surface-exposed residues that make up the scaffold of the domain, as well as an elongated loop between two LRRs that forms a small amphipathic α-helix at the site of interaction. The mode of substrate recognition by the C-JID is reminiscent of that used by immunoglobulins to bind their antigen. Similar to the complementary-determining regions of antibodies, interconnecting loops emerging from the C-JID β-sandwich structure make substrate-specific contacts with XopQ. In particular, an extended loop of the C-JID dives into the active-site cleft of XopQ and interacts with conserved residues required for nucleoside binding, suggesting that ROQ1 not only recognizes its substrate but also inhibits its ligand-binding function. The nucleotide-binding domain (NBD), helical domain 1 (HD1) and the winged-helix domain (WHD), termed NB-ARC because of their presence in Apaf-1, R proteins, and CED-4 (ARC), are responsible for ROQ1 oligomerization in an ATP-bound state. Individual protomers intercalate in a similar fashion as found in other NLR structures, promoting association between the N-terminal TIR domains. The TIR domains bind to each other through two distinct interfaces (called AE and BE), causing them to form a dimer of dimers. BE-interface contacts cause a conformational rearrangement in a loop, called the BB-loop, at the periphery of the TIR domain active site that exposes the putative catalytic glutamate that is suggested to cleave NAD+. These results provide a rationale for the previously determined oligomerization dependence of TIR domain NADase activity. CONCLUSION We propose a step-by-step mechanism for ROQ1 immune signaling based on our structure of the activated complex and on previous biochemical studies. The LRR and C-JID of ROQ1 recognize the pathogen effector through direct contacts with its surface and active-site residues. Detection of the substrate releases autoinhibitory contacts between the NB-ARC domain and the LRR, allowing the NB-ARC domain to transition to an ATP-bound, oligomerization-prone state. Complex assembly brings the TIR domains in close contact, leading to opening of the NADase active site in an interface-dependent manner. Cleavage of NAD+ by the TIR domain results in the release of adenosine diphosphate ribose, a signaling molecule that triggers cytosolic Ca2+ influx, a widely used chemical cue in response to various biotic and abiotic stresses, leading to downstream activation of localized cell death and disease resistance.

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Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ.

Abstract Understanding the plant immune system is crucial for using genetics to protect crops from diseases. Plants resist pathogens via a two-tiered innate immune detection-and-response system. The first plant Resistance (R) gene was cloned in 1992 . Since then, many cell-surface pattern recognition receptors (PRRs) have been identified, and R genes that encode intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) have been cloned. Here, we provide a list of characterized PRRs and NLRs. In addition to immune receptors, many components of immune signaling networks were discovered over the last 30 years. We review the signaling pathways, physiological responses, and molecular regulation of both PRR- and NLR-mediated immunity. Recent studies have reinforced the importance of interactions between the two immune systems. We provide an overview of interactions between PRR- and NLR-mediated immunity, highlighting challenges and perspectives for future research.

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Thirty years of resistance: Zig-zag through the plant immune system

Plant nucleotide binding/leucine-rich repeat (NLR) immune receptors are activated by pathogen effectors to trigger host defenses and cell death. Toll-interleukin 1 receptor domain NLRs (TNLs) converge on the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) family of lipase-like proteins for all resistance outputs. In Arabidopsis (Arabidopsis thaliana) TNL-mediated immunity, AtEDS1 heterodimers with PHYTOALEXIN DEFICIENT4 (AtPAD4) transcriptionally induced basal defenses. AtEDS1 uses the same surface to interact with PAD4-related SENESCENCE-ASSOCIATED GENE101 (AtSAG101), but the role of AtEDS1-AtSAG101 heterodimers remains unclear. We show that AtEDS1-AtSAG101 functions together with N REQUIRED GENE1 (AtNRG1) coiled-coil domain helper NLRs as a coevolved TNL cell death-signaling module. AtEDS1-AtSAG101-AtNRG1 cell death activity is transferable to the Solanaceous species Nicotiana benthamiana and cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and heterodimer structure-guided phenotyping of AtEDS1 variants and AtPAD4-AtSAG101 chimeras identify closely aligned ɑ-helical coil surfaces in the AtEDS1-AtSAG101 partner C-terminal domains that are necessary for reconstituted TNL cell death signaling. Our data suggest that TNL-triggered cell death and pathogen growth restriction are determined by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these signaling machineries coevolved with other components within plant species or clades to regulate downstream pathways in TNL immunity.

/pdf/a-coevolved-eds1-sag101-nrg1-module-mediates-cell-death-36a10qvdxq.pdf

A Coevolved EDS1-SAG101-NRG1 Module Mediates Cell Death Signaling by TIR-Domain Immune Receptors.

Abstract Understanding the plant immune system is crucial for using genetics to protect crops from diseases. Plants resist pathogens via a two-tiered innate immune detection-and-response system. The first plant Resistance (R) gene was cloned in 1992 . Since then, many cell-surface pattern recognition receptors (PRRs) have been identified, and R genes that encode intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) have been cloned. Here, we provide a list of characterized PRRs and NLRs. In addition to immune receptors, many components of immune signaling networks were discovered over the last 30 years. We review the signaling pathways, physiological responses, and molecular regulation of both PRR- and NLR-mediated immunity. Recent studies have reinforced the importance of interactions between the two immune systems. We provide an overview of interactions between PRR- and NLR-mediated immunity, highlighting challenges and perspectives for future research. 

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OUP accepted manuscript

Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide-binding leucine-rich repeat (NLR) protein with a Toll-like interleukin-1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely-related Nicotiana sylvestris and the distantly-related beet plant (Beta vulgaris). Roq1 was found to co-immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.13715

Arielle Lee

Papers

Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1.