Toll/interleukin-1 Receptor Domain Research Articles

Structural insights into the role of deleterious mutations at the dimeric interface of Toll-like receptor interferon-β related adaptor protein.

Toll-like receptors (TLRs) are major players in the innate immune system-recognizing pathogens and differentiating self/non-self components of immunity. These proteins are present either on the plasma membrane or endosome and recognize pathogens at their extracellular domains. They are characterized by a single transmembrane helix and an intracellular toll-interleukin-1 receptor (TIR) domain. Few TIRs directly invoke downstream signaling, while others require other TIR domains of adaptors like TIR domain-containing adaptor-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM). On recognizing pathogenic lipopolysaccharides, TLR4 dimerises and interacts with the intracellular TRAM dimer through the TIR domain to recruit a downstream signaling adaptor (TRIF). We have performed an in-depth study of the structural effect of two mutations (P116H and C117H) at the dimeric interface of the adaptor TRAM, which are known to abrogate downstream signaling. We modeled the structure and performed molecular dynamics studies in order to decipher the structural basis of this effect. We observed that these mutations led to an increased radius of gyration of the complex and resulted in several changes to the interaction energy values when compared against the wild type (WT) and positive control mutants. We identified highly interacting residues as hubs in the WT dimer, and a few such hubs that were lost in the mutant dimers. Changes in the protein residue path, hampering the information flow between the crucial A86/E87/D88/D89 and T155/S156 sites, were observed for the mutants. Overall, we show that such residue changes can have subtle but long-distance effects, impacting the signaling path allosterically.

Substrate-induced condensation activates plant TIR domain proteins

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR–NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Toll/interleukin-1 receptor (TIR) domain-containing proteins have NAD-RNA decapping activity

The occurrence of NAD+ as a non-canonical RNA cap has been demonstrated in diverse organisms. TIR domain-containing proteins present in all kingdoms of life act in defense responses and can have NADase activity that hydrolyzes NAD+. Here, we show that TIR domain-containing proteins from several bacterial and one archaeal species can remove the NAM moiety from NAD-capped RNAs (NAD-RNAs). We demonstrate that the deNAMing activity of AbTir (from Acinetobacter baumannii) on NAD-RNA specifically produces a cyclic ADPR-RNA, which can be further decapped in vitro by known decapping enzymes. Heterologous expression of the wild-type but not a catalytic mutant AbTir in E. coli suppressed cell propagation and reduced the levels of NAD-RNAs from a subset of genes before cellular NAD+ levels are impacted. Collectively, the in vitro and in vivo analyses demonstrate that TIR domain-containing proteins can function as a deNAMing enzyme of NAD-RNAs, raising the possibility of TIR domain proteins acting in gene expression regulation.

O-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

CAMTA3 repressor destabilization triggers TIR domain protein TN2-mediated autoimmunity in the Arabidopsis exo70B1 mutant.

Calcium-dependent protein kinases (CPKs) can decode and translate intracellular calcium signals to induce plant immunity. Mutation of the exocyst subunit gene EXO70B1 causes autoimmunity that depends on CPK5 and the Toll/interleukin-1 receptor (TIR) domain resistance protein TIR-NBS2 (TN2), where direct interaction with TN2 stabilizes CPK5 kinase activity. However, how the CPK5-TN2 interaction initiates downstream immune responses remains unclear. Here, we show that, besides CPK5 activity, the physical interaction between CPK5 and functional TN2 triggers immune activation in exo70B1 and may represent reciprocal regulation between CPK5 and the TIR domain functions of TN2 in Arabidopsis (Arabidopsis thaliana). Moreover, we detected differential phosphorylation of the calmodulin-binding transcription activator 3 (CAMTA3) in the cpk5 background. CPK5 directly phosphorylates CAMTA3 at S964, contributing to its destabilization. The gain-of-function CAMTA3A855V variant that resists CPK5-induced degradation rescues immunity activated through CPK5 overexpression or exo70B1 mutation. Thus, CPK5-mediated immunity is executed through CAMTA3 repressor degradation via phosphorylation-induced and/or calmodulin-regulated processes. Conversely, autoimmunity in camta3 also partially requires functional CPK5. While the TIR domain activity of TN2 remains to be tested, our study uncovers a TN2-CPK5-CAMTA3 signaling module for exo70B1-mediated autoimmunity, highlighting the direct embedding of a calcium-sensing decoder element within resistance signalosomes.

Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

The oncogenic human B-cell lymphoma MYD88 L265P mutation genocopies activation by phosphorylation at the Toll/interleukin-1 receptor (TIR) domain

MYD88 is the key signaling adaptor-protein for Toll-like and interleukin-1 receptors. A somatic L265P mutation within the Toll/interleukin-1 receptor (TIR) domain of MYD88 is found in 90% of Waldenström macroglobulinemia cases and in a significant subset of diffuse large B-cell lymphomas. MYD88-L265P strongly promotes NF-κB pathway activation, JAK-STAT signaling and lymphoma cell survival. Previous studies have identified other residues of the TIR-domain crucially involved in NF-κB activation, including serine 257 (S257), indicating a potentially important physiological role in the regulation of MYD88 activation. Here, we demonstrate that MYD88 S257 is phosphorylated in B-cell lymphoma cells and that this phosphorylation is required for optimal TLR-induced NF-κB activation. Furthermore, we demonstrate that a phosphomimetic MYD88-S257D mutant promotes MYD88 aggregation, IRAK1 phosphorylation, NF-κB activation and cell growth to a similar extent as the oncogenic L265P mutant. Lastly, we show that expression of MYD88-S257D can rescue cell growth upon silencing of endogenous MYD88-L265P expression in lymphoma cells addicted to oncogenic MYD88 signaling. Our data suggest that the L265P mutation promotes TIR domain homodimerization and NF-κB activation by copying the effect of MY88 phosphorylation at S257, thus providing novel insights into the molecular mechanism underlying the oncogenic activity of MYD88-L265P in B-cell malignancies.

Plasma membrane association and resistosome formation of plant helper immune receptors

Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation.

Characterization and genomic analysis of a novel lytic phage vB_PstM_ZRG1 infecting Stutzerimonas stutzeri, representing a new viral genus, Elithevirus

Stutzerimonas stutzeri is an opportunistic pathogen widely distributed in the environment and displays diverse metabolic capabilities. In this study, a novel lytic S. stutzeri phage, named vB_PstM_ZRG1, was isolated from the seawater in the East China Sea (29°09’N, 123°39’E). vB_PstM_ZRG1 was stable at temperatures ranging from -20°C to 65°C and across a wide range of pH values from 3 to 10. The genome of vB_PstM_ZRG1 was determined to be a double-stranded DNA with a genome size of 52,767 bp, containing 78 putative open reading frames (ORFs). Three auxiliary metabolic genes encoded by phage vB_PstM_ZRG1 were predicted, including Toll/interleukin-1 receptor (TIR) domain, proline-alanine-alanine-arginine (PAAR) protein and SGNH (Ser-Gly-Asn-His) family hydrolase, especially TIR domain is not common in isolated phages. Phylogenic and network analysis showed that vB_PstM_ZRG1 has low similarity to other phage genomes in the GenBank and IMG/VR database, and might represent a novel viral genus, named Elithevirus. Additionally, the distribution map results indicated that vB_PstM_ZRG1 could infect both extreme colds- and warm-type hosts in the marine environment. In summary, our finding provided basic information for further research on the relationship between S. stutzeri and their phages, and expanded our understanding of genomic characteristics, phylogenetic diversity and distribution of Elithevirus.

Characterization and Computational Investigation of Polymorphisms in the TIR Domain of the TLR4 Protein in Holsteins Raised in Turkey

Increasing environmental temperatures due to climate change adversely affect livestock welfare and health. Moreover, temperatures increase the distribution and survival of parasites and infectious agents. Livestock diseases that cause significant economic losses are a worldwide concern. Toll-like receptor 4 (TLR4) is an ideal marker gene candidate, due to its critical role it plays in initiating the immune response against pathogens. In this study, the toll-interleukin-1 receptor (TIR) domain polymorphisms were investigated in the Holsteins raised in Turkey. by DNA sequencing. The effects of polymorphisms on the protein structure and function were evaluated by computational tools (I-Mutant Suite, Project Hope and PyMOL). The rs8193069 C>T polymorphism was detected in the TIR domain. The SNP causes Threonine to Isoleucine substitution at position 674 of the TLR4 protein which is the second amino acid of the TIR domain. The I-Mutant Suite predicted that Thr674Ile substitution could decrease protein stability (DDG= -0.40 Kcal/mol). Project Hope results showed that mutant and wild-type amino acids have different properties and that this can disturb the TIR domain. The Thr674Ile polymorphism observed in the TIR domain of TLR4 protein in Holsteins raised in Turkey might affect the function of the TLR4 by causing physicochemical changes.

TcpC affects the grazing resistance of Escherichia coli to the social amoeba Dictyostelium discoideum

Abstract Uropathogenic Escherichia coli (UPEC) is among the most common causative agents of urinary tract infections (UTIs). UPECs survive in hosts using various mechanisms, including by secreting a protein known as TcpC, which is a Toll/interleukin-1 receptor (TIR) domain containing protein. In mammals, TIR domains are important components of Toll like receptor (TLR) signaling pathways that activate innate immune responses. TcpC interacts with components of TLR signaling, inhibiting activation of the immune system and preventing the detection and destruction of the bacteria in mammalian hosts. We hypothesize that TcpC also affects the survival of UPECs in model systems, including the social amoeba Dictyostelium discoideum and planaria flatworms. To test this hypothesis, we are using mutant strains of the UPEC CFTO73 that have altered expression of TcpC to analyze the effects of TcpC on bacterial growth and virulence in model organisms. Our initial tests with D. discoideum indicate that the presence of TcpC in UPEC strains may increase their resistance to grazing and phagocytosis by the social amoeba D. discoideum. If TcpC affects survival of UPECs in model systems such as D. discoideum, this may open new avenues for characterization of the molecular mechanisms used by bacterial TIR domains to evade innate immune cells. VA was supported by NSF LSAMP Award #1619676 and CP was supported by the NIH Bridges to Doctorate Scholars Program, NIH- R25 GM119970.

Identification and characterization of Toll-like receptor 14d in Northeast Chinese lamprey (Lethenteron morii).

Toll-like receptors (TLRs) play an important role in innate immunity of defense against bacterial or viral pathogens. To study the biological characteristics and functions of the TLR genes, TLR14d was identified from Northeast Chinese lamprey (Lethenteron morii) and named LmTLR14d. LmTLR14d coding sequence (cds) is 3285 bp in length and encodes 1094 amino acids (aa). The results showed that LmTLR14d has the typical structure of TLR molecule, which contains the extracellular domain of leucine-rich repeats (LRR), transmembrane domain, and intracellular domain of Toll/interleukin-1 receptor (TIR). The phylogenetic tree showed that LmTLR14d is a homologous gene of TLR14/18 in bony fish. Quantitative real-time PCR (qPCR) revealed that LmTLR14d was expressed in various healthy tissues, including immune and non-immune tissues. Pseudomonas aeruginosa infection up-regulated LmTLR14d in the supraneural body (SB), gill, and kidney tissues of infected Northeast Chinese lamprey. Immunofluorescence results showed that LmTLR14d was located in the cytoplasm of HEK 293T cells in clusters, and its subcellular localization was determined by the TIR domain. The immunoprecipitation results showed that LmTLR14d could recruit L.morii MyD88 (LmMyD88) but not L.morii TRIF (LmTRIF). Dual luciferase reporter results showed that LmTLR14d significantly enhanced the activity of L.morii NF-κβ (LmNF-κβ) promoter. Furthermore, co-transfection of LmTLR14d with MyD88 significantly enhanced the L.morii NF-κβ (LmNF-κβ) promoter activity. LmTLR14d can induce the expression of inflammatory cytokine genes il-6 and tnf-α downstream of NF-κB signal. This study suggested that LmTLR14d might play an important role in the innate immune signal transduction process of lamprey and revealed the origin and function of teleost-specific TLR14.

Subcellular localization requirements and specificities for plant immune receptor Toll-interleukin-1 receptor signaling.

Recent work shed light on how plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLR) family are activated upon pathogen effector recognition to trigger immune responses. Activation of Toll-interleukin-1 receptor (TIR) domain-containing NLRs (TNLs) induces receptor oligomerization and close proximity of the TIR domain, which is required for TIR enzymatic activity. TIR-catalyzed small signaling molecules bind to EDS1 family heterodimers and subsequently activate downstream helper NLRs, which function as Ca2+ permeable channel to activate immune responses eventually leading to cell death. Subcellular localization requirements of TNLs and signaling partners are not well understood, although they are required to understand fully the mechanisms underlying NLR early signaling. TNLs show diverse subcellular localization while EDS1 shows nucleocytosolic localization. Here, we studied the impact of TIR and EDS1 mislocalization on the signaling activation of different TNLs. In Nicotiana benthamiana, our results suggest that close proximity of TIR domains isolated from flax L6 and Arabidopsis RPS4 and SNC1 TNLs drives signaling activation from different cell compartments. Nevertheless, both Golgi-membrane anchored L6 and nucleocytosolic RPS4 have the same requirements for EDS1 subcellular localization in Arabidopsis thaliana. By using mislocalized variants of EDS1, we found that autoimmune L6 and RPS4 TIR domain can induce seedling cell death when EDS1 is present in the cytosol. However, when EDS1 is restricted to the nucleus, both induce a stunting phenotype but no cell death. Our data point out the importance of thoroughly investigating the dynamics of TNLs and signaling partners subcellular localization to understand TNL signaling fully.

Plant and prokaryotic TIR domains generate distinct cyclic ADPR NADase products

Toll/interleukin-1 receptor (TIR) domain proteins function in cell death and immunity. In plants and bacteria, TIR domains are often enzymes that produce isomers of cyclic adenosine 5'-diphosphate-ribose (cADPR) as putative immune signaling molecules. The identity and functional conservation of cADPR isomer signals is unclear. A previous report found that a plant TIR could cross-activate the prokaryotic Thoeris TIR-immune system, suggesting the conservation of plant and prokaryotic TIR-immune signals. Here, we generate autoactive Thoeris TIRs and test the converse hypothesis: Do prokaryotic Thoeris TIRs also cross-activate plant TIR immunity? Using in planta and in vitro assays, we find that Thoeris and plant TIRs generate overlapping sets of cADPR isomers and further clarify how plant and Thoeris TIRs activate the Thoeris system via producing 3'cADPR. This study demonstrates that the TIR signaling requirements for plant and prokaryotic immune systems are distinct and that TIRs across kingdoms generate a diversity of small-molecule products.

Structural modelling and dynamics of full-length of TLR10 sheds light on possible modes of dimerization, ligand binding and mechanism of action.

Toll like receptors (TLRs) play a pivotal role in innate and adaptive immunity. There are 10 TLRs in the human genome, of which TLR10 is the least characterized. Genetic polymorphism of TLR10 has been shown to be associated with multiple diseases including tuberculosis and rheumatoid arthritis. TLR10 consists of an extracellular domain (ECD), a single-pass transmembrane (TM) helix and intracellular TIR (Toll/Interleukin-1 receptor) domain. ECD is employed for ligand recognition and the intracellular domain interacts with other TIR domain-containing adapter proteins for signal transduction. Experimental structure of ECD or TM domain is not available for TLR10. In this study, we have modelled multiple forms of TLR10-ECD dimers, such as closed and open forms, starting from available structures of homologues. Subsequently, multiple full-length TLR10 homodimer models were generated by utilizing homology modelling and protein-protein docking. The dynamics of these models in membrane-aqueous environment revealed the global motion of ECD and TIR domain towards membrane bilayer. The TIR domain residues exhibited high root mean square fluctuation compared to ECD. The 'closed form' model was observed to be energetically more favorable than 'open form' model. The evaluation of persistent interchain interactions, along with their conservation score, unveiled critical residues for each model. Further, the binding of dsRNA to TLR10 was modelled by defined and blind docking approaches. Differential binding of dsRNA to the protomers of TLR10 was observed upon simulation that could provide clues on ligand disassociation. Dynamic network analysis revealed that the 'open form' model can be the functional form while 'closed form' model can be the apo form of TLR10.

Integrated approaches for the recognition of small molecule inhibitors for Toll-like receptor 4

Toll-like receptors (TLRs) are pattern recognition receptors present on the surface of cells playing a crucial role in innate immunity. One of the TLRs, TLR4, recognizes LPS (Lipopolysaccharide) as its ligand leading to the release of anti-inflammatory mediators as well as pro-inflammatory cytokines through signal transduction and domain recruitment. TLR4 homodimerizes at its intracellular TIR (Toll/interleukin-1 receptor) domain that helps in the recruitment of the TRAM/TICAM2 (TIR domain-containing adaptor molecule 2) molecule. TRAM also contains TIR domain which in turn, dimerizes and functions as an adapter protein to further recruit TRIF/TICAM1 (TIR domain-containing adaptor molecule 1) protein for mediating downstream signaling. Apart from LPS, TLR4 also recognizes endogenous ligands like fibrinogen, HMGB1, and hyaluronan in autoimmune conditions and sepsis. We employed computational approaches to target TRAM and recognize small molecule inhibitors from small molecules of natural origin, as contained in the Super Natural II database. Finally, cell reporter assays and NMR studies enabled the identification of promising lead compounds. Hence, this study aims to attenuate the signaling of the TLR4-TRAM-TRIF cascade in these auto-inflammatory conditions.

A conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1

Sterile alpha (SAM) and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1) is an autoinhibitory NAD-consuming enzyme that is activated by the accumulation of nicotinamide mononucleotide (NMN) during axonal injury. Its activation mechanism is not fully understood. Here, we generate a nanobody, Nb-C6, that specifically recognizes NMN-activated SARM1. Nb-C6 stains only the activated SARM1 in cells stimulated with CZ-48, a permeant mimetic of NMN, and partially activates SARM1 in vitro and in cells. Cryo-EM of NMN/SARM1/Nb-C6 complex shows an octameric structure with ARM domains bending significantly inward and swinging out together with TIR domains. Nb-C6 binds to SAM domain of the activated SARM1 and stabilized its ARM domain. Mass spectrometry analyses indicate that the activated SARM1 in solution is highly dynamic and that the neighboring TIRs form transient dimers via the surface close to one BB loop. We show that Nb-C6 is a valuable tool for studies of SARM1 activation.

The intracellular immune receptor like gene SNC1 is an enhancer of effector-triggered immunity in Arabidopsis.

Plants contain many nucleotide-binding leucine-rich repeat (NLR) proteins that are postulated to function as intracellular immune receptors but do not yet have an identified function during plant-pathogen interactions. SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1) is one such NLR protein of the Toll-interleukin 1 receptor (TIR) type, despite its well-characterized gain-of-function activity and its involvement in autoimmunity in Arabidopsis (Arabidopsis thaliana). Here, we investigated the role of SNC1 in natural plant-pathogen interactions and genetically tested the importance of the enzymatic activities of its TIR domain for its function. The SNC1 loss-of-function mutants were more susceptible to avirulent bacterial pathogen strains of Pseudomonas syringae containing specific effectors, especially under constant light growth condition. The mutants also had reduced defense gene expression induction and hypersensitive responses upon infection by avirulent pathogens under constant light growth condition. In addition, genetic and biochemical studies supported that the TIR enzymatic activity of SNC1 is required for its gain-of-function activity. In sum, our study uncovers the role of SNC1 as an amplifier of plant defense responses during natural plant-pathogen interactions and indicates its use of enzymatic activity and intermolecular interactions for triggering autoimmune responses.

Variation in plant Toll/Interleukin-1 receptor domain protein dependence on ENHANCED DISEASE SUSCEPTIBILITY 1.

Toll/Interleukin-1 receptor (TIR) domains are integral to immune systems across all kingdoms. In plants, TIRs are present in nucleotide-binding leucine-rich repeat (NLR) immune receptors, NLR-like, and TIR-only proteins. Although TIR-NLR and TIR signaling in plants require the ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) protein family, TIRs persist in species that have no EDS1 members. To assess whether particular TIR groups evolved with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algal and land plant species, we identified 4 TIR families that are shared by several plant orders. One group occurred in TIR-NLRs of eudicots and another in TIR-NLRs across eudicots and magnoliids. Two further groups were more widespread. A conserved TIR-only group co-occurred with EDS1 and members of this group elicit EDS1-dependent cell death. In contrast, a maize (Zea mays) representative of TIR proteins with tetratricopeptide repeats was also present in species without EDS1 and induced EDS1-independent cell death. Our data provide a phylogeny-based plant TIR classification and identify TIRs that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity.

Viruses inhibit TIR gcADPR signalling to overcome bacterial defence.

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.