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Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to initiate immune signaling. TIR domains across different kingdoms have NADase activities and can produce phosphoribosyl adenosine monophosphate/diphosphate (pRib-AMP/ADP) or cyclic ADPR (cADPR) isomers. The lipase-like proteins EDS1 and PAD4 transduce immune signals from sensor TIR-NLRs to a helper NLR called ADR1, which executes immune function. We report the structure and function of anArabidopsisEDS1-PAD4-ADR1 (EPA) heterotrimer in complex with pRib-AMP/ADP activated by plant or bacterial TIR signaling. 2′cADPR can be hydrolyzed into pRib-AMP and thus activate EPA signaling. Bacterial TIR domains producing 2′cADPR also activate EPA function. Our findings suggest that 2′cADPR may be the storage form of the unstable signaling molecule pRib-AMP. 

BACKGROUND Diverse organisms, from archaea and bacteria to plants and humans, use receptor systems to recognize both pathogens and dangerous self-derived or environmentally derived stimuli. These intricate, well-coordinated immune systems, composed of innate and adaptive components, ensure host survival. In the late 20th century, researchers identified the Toll/interleukin-1/resistance gene (TIR) domain as an evolutionarily conserved component of animal and plant innate immune systems. Today, TIR-domain proteins are known to be broadly distributed across the tree of life. The TIR domain was first recognized as an adaptor for the assembly of macromolecular signaling complexes in mammalian innate immune pathways. Work on axon degeneration in animals—as well as on plant, archaeal, and bacterial immune systems—has uncovered additional enzymatic activities for TIR domains. ADVANCES Mammalian axons initiate a self-destruct program upon injury and during disease that is mediated by the sterile alpha and TIR motif containing 1 (SARM1) protein. The SARM1 TIR domain enzymatically consumes the essential metabolic cofactor nicotinamide adenine dinucleotide (NAD + ) to promote axonal death. Identification of the SARM1 NAD + -consuming enzyme (NADase) revealed that TIR domains can function as enzymes. Given the evolutionary conservation of TIR domains, studies investigated whether the SARM1 TIR NADase was also conserved. Indeed, bacteria, archaea, and plant TIR domains possess NADase activity. In prokaryotes, TIR NADase activity is found in an ancient antiphage immune system. In plants, identification of TIR NADase activity and linkage of TIR enzymatic products to downstream signaling components addressed the question of how nucleotide-binding, leucine-rich repeat (NLR) receptors trigger hypersensitive cell death during an immune response. Studies in plants show that their TIR domains can cleave nucleic acids and possess 2′,3′ cyclic adenosine monophosphate (2′,3′-cAMP) and 2′,3′ cyclic guanosine monophosphate (2′,3′-cGMP) synthetase activity that aids cell death programs in plant innate immunity. Thus, TIR domains constitute an ancient family of enzymes that are activated in immune and cell death pathways. OUTLOOK The discovery of TIR-domain enzyme activities carries implications for innate immunity and neurodegeneration. The identification of the SARM1 NADase defined a drug target for a wide number of neurodegenerative diseases that is being exploited in both preclinical and clinical studies. Hyperactive mutations in the SARM1 NADase have been discovered in amyotrophic lateral sclerosis (ALS) patients. Future work will seek to clarify the contribution of the SARM1 axon degeneration pathway to ALS pathogenesis. NAD + biology influences cellular processes from metabolism to DNA repair to aging. How TIR enzymes influence the NAD + metabolome and its associated pathways in bacteria, archaea, plants, and animals will be an exciting area for upcoming investigation. The discovery of the diversity of TIR enzymatic products is revealing signaling pathways across kingdoms. Discovery of TIR enzymatic function in plants and animals may yet inspire studies of enzymatic functions for Toll-like receptors in animals. We anticipate that cross-kingdom studies of TIR-domain function will guide interventions that will span the tree of life, from treating human neurodegenerative disorders and bacterial infections to preventing plant diseases. Conserved TIR-domain enzymatic activity. TIR-domain proteins from prokaryotes and eukaryotes cleave NAD + into nicotinamide (Nam), ADP-ribose (ADPR), cyclic ADP-ribose (cADPR), isomers of cyclic ADP-ribose (2′ or 3′cADPR), and related molecules [e.g., phosphoribosyl adenosine monophosphate (pRib-AMP)]. Plant TIR domains also possess a nuclease activity, can degrade DNA and RNA, and can function as a 2′,3′-cAMP or 2′,3′-cGMP synthetase. TIR enzymatic activity drives cell death and immune pathways across kingdoms. TIR activity can kill cells directly through NAD + depletion or indirectly using enzymatic products as signal molecules. The representative TIR domain structure shown here is Protein Data Bank ID 6O0Q. EDS1, enhanced disease susceptibility 1; ThsA, Thoeris A. 

Immune pathways of plants and prokaryotes are activated by distinct TIR-generated metabolites. 

TIR domains are NAD-degrading enzymes that function during immune signaling in prokaryotes, plants, and animals. In plants, most TIR domains are incorporated into intracellular immune receptors termed TNLs. In Arabidopsis, TIR-derived small molecules bind and activate EDS1 heterodimers, which in turn activate RNLs, a class of cation channel–forming immune receptors. RNL activation drives cytoplasmic Ca 2+ influx, transcriptional reprogramming, pathogen resistance, and host cell death. We screened for mutants that suppress an RNL activation mimic allele and identified a TNL, SADR1. Despite being required for the function of an autoactivated RNL, SADR1 is not required for defense signaling triggered by other tested TNLs. SADR1 is required for defense signaling initiated by some transmembrane pattern recognition receptors and contributes to the unbridled spread of cell death in lesion simulating disease 1 . Together with RNLs, SADR1 regulates defense gene expression at infection site borders, likely in a non-cell autonomous manner. RNL mutants that cannot sustain this pattern of gene expression are unable to prevent disease spread beyond localized infection sites, suggesting that this pattern corresponds to a pathogen containment mechanism. SADR1 potentiates RNL-driven immune signaling not only through the activation of EDS1 but also partially independently of EDS1. We studied EDS1-independent TIR function using nicotinamide, an NADase inhibitor. Nicotinamide decreased defense induction from transmembrane pattern recognition receptors and decreased calcium influx, pathogen growth restriction, and host cell death following intracellular immune receptor activation. We demonstrate that TIR domains can potentiate calcium influx and defense and are thus broadly required for Arabidopsis immunity. 

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors activate cell death and confer disease resistance by unknown mechanisms. We demonstrate that plant Toll/interleukin-1 receptor (TIR) domains of NLRs are enzymes capable of degrading nicotinamide adenine dinucleotide in its oxidized form (NAD + ). Both cell death induction and NAD + cleavage activity of plant TIR domains require known self-association interfaces and a putative catalytic glutamic acid that is conserved in both bacterial TIR NAD + -cleaving enzymes (NADases) and the mammalian SARM1 (sterile alpha and TIR motif containing 1) NADase. We identify a variant of cyclic adenosine diphosphate ribose as a biomarker of TIR enzymatic activity. TIR enzymatic activity is induced by pathogen recognition and functions upstream of the genes enhanced disease susceptibility 1 ( EDS1 ) and N requirement gene 1 ( NRG1 ), which encode regulators required for TIR immune function. Thus, plant TIR-NLR receptors require NADase function to transduce recognition of pathogens into a cell death response.