Broader functions of TIR domains in Arabidopsis immunity.

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 Ca2+ 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 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.

Shared TIR enzymatic functions regulate cell death and immunity across the tree of life.

In the 20th century, researchers studying animal and plant signaling pathways discovered a protein domain that is shared across diverse innate immune systems: the Toll/interleukin-1/resistance gene (TIR) domain. The TIR domain is found in several protein architectures and was defined as an adaptor that mediates protein-protein interactions in animal innate immunity and developmental signaling pathways. However, studies of nerve degeneration in animals-and subsequent breakthroughs in plant, bacterial, and archaeal systems-revealed that TIR domains possess enzymatic activities. We provide a synthesis of TIR functions and the role of various related TIR enzymatic products in evolutionarily diverse immune systems. These studies may ultimately guide interventions that would span the tree of life, from treating human neurodegenerative disorders and bacterial infections to preventing plant diseases.

Arabidopsis ADR1 helper NLR immune receptors localize and function at the plasma membrane in a phospholipid dependent manner.

Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM.

Enzymatic Functions for Toll/Interleukin-1 Receptor Domain Proteins in the Plant Immune System.

Rationally engineered improvements to crop plants will be needed to keep pace with increasing demands placed on agricultural systems by population growth and climate change. Engineering of plant immune systems provides an opportunity to increase yields by limiting losses to pathogens. Intracellular immune receptors are commonly used as agricultural disease resistance traits. Despite their importance, how intracellular immune receptors confer disease resistance is still unknown. One major class of immune receptors in dicots contains a Toll/Interleukin-1 Receptor (TIR) domain. The mechanisms of TIR-containing proteins during plant immunity have remained elusive. The TIR domain is an ancient module found in archaeal, bacterial and eukaryotic proteins. In animals, TIR domains serve a structural role by generating innate immune signaling complexes. The unusual animal TIR-protein, SARM1, was recently discovered to function instead as an enzyme that depletes cellular NAD+ (nicotinamide adenine dinucleotide) to trigger axonal cell death. Two recent reports have found that plant TIR proteins also have the ability to cleave NAD+. This presents a new paradigm from which to consider how plant TIR immune receptors function. Here, we will review recent reports of the structure and function of TIR-domain containing proteins. Intriguingly, it appears that TIR proteins in all kingdoms may use similar enzymatic mechanisms in a variety of cell death and immune pathways. We will also discuss TIR structure-function hypotheses in light of the recent publication of the ZAR1 resistosome structure. Finally, we will explore the evolutionary context of plant TIR-containing proteins and their downstream signaling components across phylogenies and the functional implications of these findings.

mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage.

Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription, thereby presenting an opportunity to study mechanisms of post-transcriptional regulatory control. We observed that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage) and P-bodies (associated with RNA processing). The subcellular distribution of transcripts differed in their dependence on 3'UTRs and RNA binding proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling translation from mRNA localization, we untangled a long-standing question: Are mRNAs directed to P granules to be translationally repressed, or do they accumulate there as a consequence of this repression? We found that translational repression preceded P granule localization and could occur independently of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. These results implicate transcriptional repression as a means to deliver essential maternal transcripts to the progenitor germ lineage for later translation.

TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death.

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.

A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana.

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.

Structural, Functional, and Genomic Diversity of Plant NLR Proteins: An Evolved Resource for Rational Engineering of Plant Immunity.

Plants employ a diverse intracellular system of NLR (nucleotide binding-leucine-rich repeat) innate immune receptors to detect pathogens of all types. These receptors represent valuable agronomic traits that plant breeders rely on to maximize yield in the face of devastating pathogens. Despite their importance, the mechanistic underpinnings of NLR-based disease resistance remain obscure. The rapidly increasing numbers of plant genomes are revealing a diverse array of NLR-type immune receptors. In parallel, mechanistic studies are describing diverse functions for NLR immune receptors. In this review, we intend to broadly describe how the structural, functional, and genomic diversity of plant immune receptors can provide a valuable resource for rational engineering of plant immunity.

Concerted Action of Evolutionarily Ancient and Novel SNARE Complexes in Flowering-Plant Cytokinesis.

Membrane vesicles delivered to the cell-division plane fuse with one another to form the partitioning membrane during plant cytokinesis, starting in the cell center. In Arabidopsis, this requires SNARE complexes involving the cytokinesis-specific Qa-SNARE KNOLLE. However, cytokinesis still occurs in knolle mutant embryos, suggesting contributions from KNOLLE-independent SNARE complexes. Here we show that Qa-SNARE SYP132, having counterparts in lower plants, functionally overlaps with the flowering plant-specific KNOLLE. SYP132 mutation causes cytokinesis defects, knolle syp132 double mutants consist of only one or a few multi-nucleate cells, and SYP132 has the same SNARE partners as KNOLLE. SYP132 and KNOLLE also have non-overlapping functions in secretion and in cellularization of the embryo-nourishing endosperm resulting from double fertilization unique to flowering plants. Evolutionarily ancient non-specialized SNARE complexes originating in algae were thus amended by the appearance of cytokinesis-specific SNARE complexes, meeting the high demand for membrane-fusion capacity during endosperm cellularization in angiosperms.

A Bacterial Type III Effector Targets the Master Regulator of Salicylic Acid Signaling, NPR1, to Subvert Plant Immunity.

Most plant bacterial pathogens rely on type III effectors to cause diseases. Although it is well known that the plant hormone salicylic acid (SA) plays an essential role in defense, whether the master regulator of SA signaling, NPR1, is targeted by any plant pathogen effectors is unknown. SA facilitates the reduction of cytosolic NPR1 oligomers into monomers, which enter the nucleus and function as transcriptional coactivators of plant defense genes. We show that SA promotes the interaction between the Pseudomonas syringae type III effector AvrPtoB and NPR1. In the presence of SA, AvrPtoB mediates the degradation of NPR1 via the host 26S proteasome in a manner dependent on AvrPtoB's E3 ligase activity. Intriguingly, we found that NPR1 plays an important role in MAMP-triggered immunity (MTI), inducing the expression of MTI marker genes. Thus, this work uncovers a strategy in which AvrPtoB targets NPR1 and represses NPR1-dependent SA signaling, thereby subverting plant innate immunity.

TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis.

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll-interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR-TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. Our identification of RBA1 demonstrates that "truncated" NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system.

Genome-Wide Assessment of Efficiency and Specificity in CRISPR/Cas9 Mediated Multiple Site Targeting in Arabidopsis.

Simultaneous multiplex mutation of large gene families using Cas9 has the potential to revolutionize agriculture and plant sciences. The targeting of multiple genomic sites at once raises concerns about the efficiency and specificity in targeting. The model Arabidopsis thaliana is widely used in basic plant research. Previous work has suggested that the Cas9 off-target rate in Arabidopsis is undetectable. Here we use deep sequencing on pooled plants simultaneously targeting 14 distinct genomic loci to demonstrate that multiplex targeting in Arabidopsis is highly specific to on-target sites with no detectable off-target events. In addition, chromosomal translocations are extremely rare. The high specificity of Cas9 in Arabidopsis makes this a reliable method for clean mutant generation with no need to enhance specificity or adopt alternate Cas9 variants.

Treasure your exceptions: unusual domains in immune receptors reveal host virulence targets.

A mechanistic understanding of how plant pathogens modulate their hosts is critical for rationally engineered disease resistance in agricultural systems. Two new studies show that genomically paired plant immune receptors have incorporated decoy domains that structurally mimic pathogen virulence targets to monitor attempted host immunosuppression.

A truncated NLR protein, TIR-NBS2, is required for activated defense responses in the exo70B1 mutant.

During exocytosis, the evolutionarily conserved exocyst complex tethers Golgi-derived vesicles to the target plasma membrane, a critical function for secretory pathways. Here we show that exo70B1 loss-of-function mutants express activated defense responses upon infection and express enhanced resistance to fungal, oomycete and bacterial pathogens. In a screen for mutants that suppress exo70B1 resistance, we identified nine alleles of TIR-NBS2 (TN2), suggesting that loss-of-function of EXO70B1 leads to activation of this nucleotide binding domain and leucine-rich repeat-containing (NLR)-like disease resistance protein. This NLR-like protein is atypical because it lacks the LRR domain common in typical NLR receptors. In addition, we show that TN2 interacts with EXO70B1 in yeast and in planta. Our study thus provides a link between the exocyst complex and the function of a 'TIR-NBS only' immune receptor like protein. Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery. TN2 could monitor EXO70B1 integrity as part of an immune receptor complex.

Pseudomonas syringae CC1557: a highly virulent strain with an unusually small type III effector repertoire that includes a novel effector.

Both type III effector proteins and nonribosomal peptide toxins play important roles for Pseudomonas syringae pathogenicity in host plants, but whether and how these pathways interact to promote infection remains unclear. Genomic evidence from one clade of P. syringae suggests a tradeoff between the total number of type III effector proteins and presence of syringomycin, syringopeptin, and syringolin A toxins. Here, we report the complete genome sequence from P. syringae CC1557, which contains the lowest number of known type III effectors to date and has also acquired genes similar to sequences encoding syringomycin pathways from other strains. We demonstrate that this strain is pathogenic on Nicotiana benthamiana and that both the type III secretion system and a new type III effector, hopBJ1, contribute to pathogenicity. We further demonstrate that activity of HopBJ1 is dependent on residues structurally similar to the catalytic site of Escherichia coli CNF1 toxin. Taken together, our results provide additional support for a negative correlation between type III effector repertoires and the potential to produce syringomycin-like toxins while also highlighting how genomic synteny and bioinformatics can be used to identify and characterize novel virulence proteins.