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Plant cell surface and intracellular immune receptors recognizing pathogen attack utilize the same defense machineries to mobilize resistance. New genetic, protein structural and biochemical information on receptor activation and signaling is transforming understanding of how their shared defense network operates. We discuss the biochemical activities of two classes of intracellular nucleotide-binding/leucine-rich repeat (NLR) receptor - one forming a Ca

In Nicotiana benthamiana, the expression of the Xanthomonas effector XANTHOMONAS OUTER PROTEIN Q (XopQ) triggers RECOGNITION OF XOPQ1 (ROQ1)-dependent effector-triggered immunity (ETI) responses accompanied by the accumulation of plastids around the nucleus and the formation of stromules. Both plastid clustering and stromules were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here, we utilized transient expression experiments to determine whether XopQ-triggered plastid reactions are a result of XopQ perception by the immune receptor ROQ1 or a consequence of XopQ virulence activity. We found that N. benthamiana mutants lacking ROQ1, ENHANCED DISEASE SUSCEPTIBILITY 1, or the helper NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT IMMUNE RECEPTORS (NLRs) N-REQUIRED GENE 1 (NRG1) and ACTIVATED DISEASE RESISTANCE GENE 1 (ADR1), fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the nrg1_adr1 double mutant. This analysis aligns XopQ-triggered stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast perinuclear dynamics, is an integral part of the N. benthamiana ETI response and that both NRG1 and ADR1 hNLRs play a role in this ETI response.

Plant root-associated bacteria can confer protection against pathogen infection. By contrast, the beneficial effects of root endophytic fungi and their synergistic interactions with bacteria remain poorly defined. We demonstrate that the combined action of a fungal root endophyte from a widespread taxon with core bacterial microbiota members provides synergistic protection against an aggressive soil-borne pathogen inArabidopsis thalianaand barley. We additionally reveal early inter-kingdom growth promotion benefits which are host and microbiota composition dependent. Using RNA-sequencing, we show that these beneficial activities are not associated with extensive host transcriptional reprogramming but rather with the modulation of expression of microbial effectors and carbohydrate-active enzymes.

Plant pathogen-activated immune signaling by nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/Interleukin-1 receptor (TIR) domain converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TIR-encoded NADases produce signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR sub-classes. Here we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) via ADPR polymerase-like and NADase activity, forming ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), respectively. Specific binding of di-ADPR or ADPr-ATP allosterically promotes EDS1-SAG101 interaction with helper NLR N requirement gene 1A (NRG1A)in vitroandin planta. Our data reveal an enzymatic activity of TIRs that enables specific activation of the EDS1-SAG101-NRG1 immunity branch.

Plant nucleotide-binding leucine-rich repeat-containing (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to enable TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) activity for immune signaling. TIR-NLR signaling requires the helper NLRs N requirement gene 1 (NRG1), Activated Disease Resistance 1 (ADR1), and Enhanced Disease Susceptibility 1 (EDS1), which forms a heterodimer with each of its paralogs Phytoalexin Deficient 4 (PAD4) and Senescence-Associated Gene 101 (SAG101). Here, we show that TIR-containing proteins catalyze the production of 2′-(5′′-phosphoribosyl)-5′-adenosine monophosphate (pRib-AMP) and diphosphate (pRib-ADP) in vitro and in planta. Biochemical and structural data demonstrate that EDS1-PAD4 is a receptor complex for pRib-AMP and pRib-ADP, which allosterically promote EDS1-PAD4 interaction with ADR1-L1 but not NRG1A. Our study identifies TIR-catalyzed pRib-AMP and pRib-ADP as a missing link in TIR signaling through EDS1-PAD4 and as likely second messengers for plant immunity.

Plant disease resistance involves both detection of microbial molecular patterns by cell-surface pattern recognition receptors and detection of pathogen effectors by intracellular NLR immune receptors. NLRs are classified as sensor NLRs, involved in effector detection, or helper NLRs required for sensor NLR signaling. TIR-domain-containing sensor NLRs (TNLs) require helper NLRs NRG1 and ADR1 for resistance, and helper NLR activation of defense requires the lipase-domain proteins EDS1, SAG101, and PAD4. Previously, we found that NRG1 associates with EDS1 and SAG101 in a TNL activation-dependent manner [X. Sun et al. , Nat. Commun. 12 , 3335 (2021)]. We report here how the helper NLR NRG1 associates with itself and with EDS1 and SAG101 during TNL-initiated immunity. Full immunity requires coactivation and mutual potentiation of cell-surface and intracellular immune receptor-initiated signaling [B. P. M. Ngou, H.-K. Ahn, P. Ding, J. D. G. Jones, Nature 592 , 110–115 (2021), M. Yuan et al. , Nature 592 , 105–109 (2021)]. We find that while activation of TNLs is sufficient to promote NRG1–EDS1–SAG101 interaction, the formation of an oligomeric NRG1–EDS1–SAG101 resistosome requires the additional coactivation of cell-surface receptor-initiated defense. These data suggest that NRG1–EDS1–SAG101 resistosome formation in vivo is part of the mechanism that links intracellular and cell-surface receptor signaling pathways.

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1–PAD4–ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity. The authors provide mechanistic insights into the crosstalk between signalling components of pattern-triggered immunity and effector-triggered immunity and their molecular linkers.

In plants, a first layer of inducible immunity is conferred by pattern recognition receptors (PRRs) that bind microbe- and damage-associated molecular patterns to activate pattern-triggered immunity (PTI). PTI is strengthened or followed by another potent form of immunity when intracellular receptors recognize pathogen effectors, termed effector-triggered immunity. Immunity signaling regulators have been reported to influence abiotic stress responses as well, yet the governing principles and mechanisms remain ambiguous. Here, we report that PRRs of a leucine-rich repeat ectodomain also confer salt tolerance in Arabidopsis thaliana, following recognition of cognate ligands such as bacterial flagellin (flg22 epitope) and elongation factor Tu (elf18 epitope), and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires authentic PTI signaling components; namely, the PRR-associated kinases BAK1 and BIK1 and the NADPH oxidase RBOHD. Exposure to salt stress induces the release of Pep precursors, pointing to the involvement of the endogenous immunogenic peptides in developing plant tolerance to high salinity. Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following a preexposure to immunogenic patterns. In good accordance, plants challenged with nonpathogenic bacteria also acquired salt tolerance in a manner dependent on PRRs. Our findings provide insight into signaling plasticity underlying biotic or abiotic stress cross-tolerance in plants conferred by PRRs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The EDS1 family of structurally unique lipase-like proteins EDS1, SAG101, and PAD4 evolved in seed plants, on top of existing phytohormone and nucleotide-binding–leucine-rich-repeat (NLR) networks, to regulate immunity pathways against host-adapted biotrophic pathogens. Exclusive heterodimers between EDS1 and SAG101 or PAD4 create essential surfaces for resistance signaling. Phylogenomic information, together with functional studies in Arabidopsis and tobacco, identify a coevolved module between the EDS1–SAG101 heterodimer and coiled-coil (CC) HET-S and LOP-B (CCHELO) domain helper NLRs that is recruited by intracellular Toll-interleukin1-receptor (TIR) domain NLR receptors to confer host cell death and pathogen immunity. EDS1–PAD4 heterodimers have a different and broader activity in basal immunity that transcriptionally reinforces local and systemic defenses triggered by various NLRs. Here, we consider EDS1 family protein functions across seed plant lineages in the context of networking with receptor and helper NLRs and downstream resistance machineries. The different modes of action and pathway connectivities of EDS1 family members go some way to explaining their central role in biotic stress resilience.

Plants have evolved mechanisms to protect themselves against pathogenic microbes and insect pests. In Arabidopsis, the immune regulator PAD4 functions with its cognate partner EDS1 to limit pathogen growth. PAD4, independently of EDS1, reduces infestation by green peach aphid (GPA). How PAD4 regulates these defense outputs is unclear. By expressing the N-terminal PAD4 lipase-like domain (PAD4LLD) without its C-terminal EDS1-PAD4 (EP) domain, we interrogated PAD4 functions in plant defense. Here, we show that transgenic expression of PAD4LLD in Arabidopsis is sufficient for limiting GPA infestation but not for conferring basal and effector-triggered pathogen immunity. This suggests that the C-terminal PAD4 EP domain is necessary for EDS1-dependent immune functions but is dispensable for aphid resistance. Moreover, PAD4LLD is not sufficient to interact with EDS1, indicating the PAD4-EP domain is required for stable heterodimerization. These data provide molecular evidence that PAD4 has domain-specific functions.

SummaryHeterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, inA. thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Biochemical activities underlying these mechanistic frameworks remain unknown.We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes inN. benthamiana.We do not detect impaired PRR signaling inN. benthamianalines deficient in EDS1 complexes or RNLs. Intriguingly, mutations within the catalytic triad ofSolanaceaeEDS1 can abolish or enhance TNL immunity inN. benthamiana. Furthermore, nuclear EDS1 accumulation is sufficient forN. benthamianaTNL (Roq1) immunity.Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Dependency ofSolanaceaebut notA. thalianaEDS1 on catalytic triad residues raises the possibility that a TNL-derived small molecule binds to theSolanaceaeEDS1 lipase-like domain, and that EDS1 lipase-like domain pocket contributions to TNL immune responses vary between lineages. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown.

In Nicotiana benthamiana, expression of the Xanthomonas effector XopQ triggers ROQ1-dependent ETI responses and in parallel accumulation of plastids around the nucleus and the formation of stromules. Both processes were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here we utilized transient expression experiments to determine whether XopQ-mediated plastid reactions are a result of XopQ perception by ROQ1 or a consequence of XopQ virulence activity. We find that N. benthamiana mutants lacking ROQ1, both RNLs (NRG1 and ADR1) or EDS1, fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the RNL double mutant. This analysis aligns XopQ-induced stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast peri-nuclear dynamics, is an integral part of the N. benthamiana ETI response and that both RNL sub-types play a role in this ETI response.One sentence summaryGenetic analysis aligns effector triggered immunity (ETI) induced stromule formation with the ETI signaling cascade but not programmed cell death and questions stromule guided peri-nuclear plastid clustering.

Toll/interleukin-1 Receptor (TIR) domains are integral to immune systems across all domains of life. TIRs exist as single-domain and as larger receptor or adaptor proteins. In plants, TIRs constitute N-terminal domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors. Although TIR-NLR and TIR signaling requires the Enhanced disease susceptibility 1 (EDS1) protein family, TIR domains persist in species that have incomplete or no EDS1 members. To assess whether particular TIR groups appear with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algae and land plant species, we identify four conserved TIR groups, two of which are TIR-NLRs present in eudicots and two are more widespread. Presence of one TIR-only protein group is highly correlated with EDS1 and members of this group elicit EDS1-dependent cell death. By contrast, a more widely represented TIR group of TIR-NB-WD40/TPR (TNP) proteins (formerly called XTNX) has at least one member which can induce EDS1-independent cell death. Our data provide a new phylogeny-based plant TIR classification and identify TIR groups that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity.One sentence summaryLand plants have evolved four conserved TIR groups

Iron is critical for host-pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remain unknown. We identified a receptor kinase, SRF3 coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthase activity. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domain which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.

SummaryTranscriptional corepressors of the Topless family are important regulators of plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of transcriptional regulation in plant immune responses. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed on GFP-tagged Topless-related 1 (TPR1) expressed inArabidopsis thalianalines with and without constitutive immunity dependent onEnhanced Disease Susceptibility 1(EDS1). RNA-seq profiling of pathogen-infectedtpl/tprmutants and assessments of growth and physiological parameters were employed to determine TPL/TPR roles in transcriptional immunity and defense homeostasis. TPR1 bound to promoter regions of ~1,400 genes and ~10% of the detected binding requiredEDS1immunity signaling. Atpr1 tpl tpr4(t3) mutant displayed mildly enhanced defense-related transcriptional reprogramming upon bacterial infection but not increased bacterial resistance. Bacteria or pep1 phytocytokine-challengedt3plants exhibited, respectively, photosystem II dysfunction and exacerbated root growth inhibition. Transgenic expression ofTPR1restored thet3physiological defects. We propose that TPR1 and TPL-family proteins function inArabidopsisto reduce detrimental effects associated with activated transcriptional immunity.

Iron is critical for host–pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remains unknown. Here we report a receptor kinase SRF3, with a role in coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthases. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domains which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.

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.

SummaryIron is critical for host-pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remain unknown. We identified a receptor kinase, SRF3 coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthase activity. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domain which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.

Plant root-associated bacteria can confer protection against pathogen infection. By contrast, the beneficial effects of root endophytic fungi and their synergistic interactions with bacteria remain poorly defined. We demonstrate that the combined action of a fungal root endophyte from a widespread taxon with core bacterial microbiota members provides synergistic protection against an aggressive soil-borne pathogen in Arabidopsis thaliana and barley. We additionally reveal early inter-kingdom growth promotion benefits which are host and microbiota composition dependent. Using RNA-sequencing, we show that these beneficial activities are not associated with extensive host transcriptional reprogramming but rather with the modulation of expression of microbial effectors and carbohydrate-active enzymes.

Plant root-associated bacteria can confer protection against pathogen infection. By contrast, the beneficial effects of root endophytic fungi and their synergistic interactions with bacteria remain poorly defined. We demonstrate that the combined action of a fungal root endophyte from a widespread taxon with core bacterial microbiota members provides synergistic protection against an aggressive soil-borne pathogen inArabidopsis thalianaand barley. We additionally show early inter-kingdom growth promotion benefits which are host and microbiota composition dependent.HighlightsThe root endophytic fungusSerendipita vermiferacan functionally replace core bacterial microbiota members in mitigating pathogen infection and disease symptoms.S. vermiferaadditionally stabilizes and potentiates the protective activities of root-associated bacteria and mitigates the negative effects of a non-native bacterial community inA. thaliana.Inter-kingdom synergistic beneficial effects do not require extensive host transcriptional reprogramming nor high levels ofS. vermiferacolonisation.Inter-kingdom protective benefits are largely independent of the host while synergism leading to early inter-kingdom growth promotion is driven by host species and microbiota composition.

Temperature impacts plant immunity and growth but how temperature intersects with endogenous pathways to shape natural variation remains unclear. Here we uncover variation between Arabidopsis thaliana natural accessions in response to two non-stress temperatures (22°C and 16°C) affecting accumulation of the thermoresponsive stress hormone salicylic acid (SA) and plant growth. Analysis of differentially responding A. thaliana accessions shows that pre-existing SA provides a benefit in limiting infection by Pseudomonas syringae pathovar tomato DC3000 bacteria at both temperatures. Several A. thaliana genotypes display a capacity to mitigate negative effects of high SA on growth, indicating within-species plasticity in SA—growth tradeoffs. An association study of temperature x SA variation, followed by physiological and immunity phenotyping of mutant and over-expression lines, identifies the transcription factor bHLH059 as a temperature-responsive SA immunity regulator. Here we reveal previously untapped diversity in plant responses to temperature and a way forward in understanding the genetic architecture of plant adaptation to changing environments., Author summary Temperature has a profound effect on plant innate immune responses but little is known about the mechanisms underlying natural variation in transmission of temperature signals to defence pathways. Much of our understanding of temperature effects on plant immunity and tradeoffs between activated defences and growth has come from analysis of the common Arabidopsis thaliana genetic accession, Col-0. Here we examine A. thaliana genetic variation in response to temperature (within the non-stress range—22 oC and 16 oC) at the level of accumulation of the thermoresponsive biotic stress hormone salicylic acid (SA), bacterial pathogen resistance, and plant biomass. From analysis of 105 genetically diverse A. thaliana accessions we uncover plasticity in temperature-modulated SA homeostasis and in the relationship between SA levels and plant growth. We find that high SA amounts prior to infection provide a robust benefit of enhancing bacterial resistance. In some accessions this benefit comes without compromised plant growth, suggestive of altered defence–growth tradeoffs. Based on a temperature x SA association study we identify the transcription factor gene, bHLH059, and show that it has features of a temperature-sensitive immunity regulator that are unrelated to PIF4, a known thermosensitive coordinator of immunity and growth.

International audience; HeLo domain-containing mixed lineage kinase domain-like protein (MLKL), a pseudokinase, mediates necroptotic cell death in animals. Here, we report the discovery of a conserved protein family across seed plants that structurally resembles vertebrate MLKL. The Arabidopsis genome encodes three MLKLs (AtMLKLs) with overlapping functions in disease resistance mediated by Toll-interleukin 1-receptor domain intracellular immune receptors (TNLs). The HeLo domain of AtMLKLs confers cell death activity but is dispensable for immunity. Cryo-EM structures reveal a tetrameric configuration, in which the HeLo domain is buried, suggestive of an auto-repressed complex. The mobility of AtMLKL1 along microtubules is reduced by chitin, a fungal immunity-triggering molecule. An AtMLKL1 phosphomimetic variant exhibiting reduced mobility enhances immunity. Coupled with the predicted presence of HeLo domains in plant helper NLRs, our data reveal the importance of HeLo domain proteins for TNL-dependent immunity and argue for a cell death-independent immune mechanism mediated by MLKLs.

Plants use both cell surface and intracellular immune receptors with leucine rich-repeat (LRRs) to detect pathogens. LRR receptor kinases (LRR-RKs) and LRR receptor-like proteins (LRR-RPs) recognize extracellular microbe-derived molecules to confer pattern-triggered immunity (PTI), while nucleotide-binding LRR (NLR) proteins detect microbial effectors inside the cell to confer effector-triggered immunity (ETI). Despite PTI and ETI signaling being initiated in different compartments, both rely on the transcriptional activation of similar sets of genes, suggesting convergence in signaling upstream of nuclear events. Here we report that two sets of molecules, helper NLRs from the ADR1 (ACTIVATED DISEASE RESISTANCE 1) family as well as lipase-like proteins EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and PAD4 (PHYTOALEXIN DEFICIENT 4), are required not only for ETI, but also for PTI. A further similarity is seen in the evolutionary patterns of some PTI and ETI receptor genes, with both often being highly polymorphic, and with nevertheless distinct roles of LRR-RK and LRR-RP receptors in immunity. We find that the LRR-RK SOBIR1 directly links LRR-RPs with the ADR1 helper NLR as well as EDS1 and PAD4, suggesting the formation of constitutive supramolecular signalosome complexes at the inner side of the plasma membrane. We propose that the EDS1-PAD4-ADR1 node is an essential component and convergence point for immune signaling cascades activated by both surface-resident LRR-RP receptors and intracellular NLR receptors.

Hyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen causing Arabidopsis downy mildew. Effector proteins secreted from the pathogen into the plant play key roles in promoting infection by suppressing plant immunity and manipulating the host to the pathogen’s advantage. One class of oomycete effectors share a conserved ‘RxLR’ motif critical for their translocation into the host cell. Here we characterize the interaction between an RxLR effector, HaRxL21 (RxL21), and the Arabidopsis transcriptional co-repressor Topless (TPL). We establish that RxL21 and TPL interact via an EAR motif at the C-terminus of the effector, mimicking the host plant mechanism for recruiting TPL to sites of transcriptional repression. We show that this motif, and hence interaction with TPL, is necessary for the virulence function of the effector. Furthermore, we provide evidence that RxL21 uses the interaction with TPL, and its close relative TPL-related 1, to repress plant immunity and enhance host susceptibility to both biotrophic and necrotrophic pathogens., Author summary Disease in plants depends on the outcome of interaction between the pathogen and the host plant. Pathogens use effector proteins to aid colonization by suppression of the host immune system. Identification of effector targets can pinpoint key components of plant defence in addition to aiding our understanding of how pathogens manipulate their hosts. Here, we see a downy mildew effector mimicking a plant transcriptional repression motif and interacting with a transcriptional co-repressor in order to manipulate the plant immunity transcriptional response and susceptibility to both a biotrophic and a necrotrophic pathogen.

Pattern recognition receptors (PRRs) bind microbe- and damage-associated molecular patterns (MAMPs/DAMPs, respectively) to enhance host immunity in animals and plants. Here, we report that PRRs also confer salt tolerance in the model plant Arabidopsis thaliana following recognition of cognate ligands, such as bacterial flagellin and EF-Tu and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires the PRR-associated kinases BAK1 and BIK1, and the NADPH oxidase RBOHD. Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following an exposure to immunogenic patterns. In their regulatory DNA sequences, specific binding sites for a subset of WRKY transcription factors are over-represented. Accordingly, PTST requires WRKY40 and WRKY18, which activate salt tolerance-related genes but attenuate pathogen defense-related genes, including the EDS1 immunity activator. PRR signaling leads to sustained WRKY40/WRKY18 accumulation under salt stress and utilizes both WRKYs for salt tolerance. The PRR-WRKY40/WRKY18 module also confers salt tolerance after challenge with non-pathogenic bacteria. Our findings give molecular insight into signaling plasticity underlying biotic-abiotic stress cross-tolerance in plants conferred by PRRs.

The aerial portion of a plant, namely the leaf, is inhabited by pathogenic and non-pathogenic microbes. The leaf’s physical and chemical properties, combined with fluctuating and often challenging environmental factors, create surfaces that require a high degree of adaptation for microbial colonization. As a consequence, specific interactive processes have evolved to establish a plant leaf niche. Little is known about the impact of the host immune system on phyllosphere colonization by non-pathogenic microbes. These organisms can trigger plant basal defenses and benefit the host by priming for enhanced resistance to pathogens. In most disease resistance responses, microbial signals are recognized by extra- or intracellular receptors. The interactions tend to be species specific and it is unclear how they shape leaf microbial communities. In natural habitats, microbe–microbe interactions are also important for shaping leaf communities. To protect resources, plant colonizers have developed direct antagonistic or host manipulation strategies to fight competitors. Phyllosphere-colonizing microbes respond to abiotic and biotic fluctuations and are therefore an important resource for adaptive and protective traits. Understanding the complex regulatory host–microbe–microbe networks is needed to transfer current knowledge to biotechnological applications such as plant-protective probiotics., Microbial colonization of above-ground parts of plants is a dynamic and interactive process that requires a high degree of adaptation. Understanding complex host–microbe–microbe interactions is key to new strategies for plant protection.

Timely and specific regulation of gene expression is critical for plant responses to environmental and developmental cues. Transcriptional coregulators have emerged as important factors in gene expression control, although they lack DNA-binding domains and the mechanisms by which they are recruited to and function at the chromatin are poorly understood. Plant Topless-related 1 (TPR1), belonging to a family of transcriptional corepressors found across eukaryotes, contributes to immunity signaling in Arabidopsis thaliana and wild tobacco. We performed chromatin immunoprecipitation and sequencing (ChIP-seq) on an Arabidopsis TPR1-GFP expressing transgenic line to characterize genome-wide TPR1-chromatin associations. The analysis revealed ∼1400 genes bound by TPR1, with the majority of binding sites located at gene upstream regions. Among the TPR1 bound genes, we find not only regulators of immunity but also genes controlling growth and development. To support further analysis of TPR1-chromatin complexes and other transcriptional corepressors in plants, we provide two ways to access the processed ChIP-seq data and enable their broader use by the research community.

Calcium (Ca2+ ) is a second messenger for plant cell surface and intracellular receptors mediating pattern-triggered and effector-triggered immunity (respectively, PTI and ETI). Several CYCLIC NUCLEOTIDE-GATED CHANNELS (CNGCs) were shown to control transient cytosolic Ca2+ influx upon PTI activation. The contributions of specific CNGC members to PTI and ETI remain unclear. ENHANCED DISEASE SUSCEPTIBLITY1 (EDS1) regulates ETI signaling. In an Arabidopsis genetic screen for suppressors of eds1, we identify a recessive gain-of-function mutation in CNGC20, denoted cngc20-4, which partially restores disease resistance in eds1. cngc20-4 enhances PTI responses and ETI hypersensitive cell death. A cngc20-4 single mutant exhibits autoimmunity, which is dependent on genetically parallel EDS1 and salicylic acid (SA) pathways. CNGC20 self-associates, forms heteromeric complexes with CNGC19, and is phosphorylated and stabilized by BOTRYTIS INDUCED KINASE1 (BIK1). The cngc20-4 L371F exchange on a predicted transmembrane channel inward surface does not disrupt these interactions but leads to increased cytosolic Ca2+ accumulation, consistent with mis-regulation of CNGC20 Ca2+ -permeable channel activity. Our data show that ectopic Ca2+ influx caused by a mutant form of CNGC20 in cngc20-4 affects both PTI and ETI responses. We conclude that tight control of the CNGC20 Ca2+ ion channel is important for regulated immunity.

ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid–dependent and independent pathways. Here, we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogen Golovinomyces cichoracearum. The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss-of-function mutations in the protein kinase EDR1 (Enhanced Disease Resistance1), and previous work has shown that loss of PAD4 or EDS1 suppresses edr1-mediated phenotypes, placing these proteins downstream of EDR1. Here, we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet-unknown PAD4 signaling function.

International audience; Pattern recognition receptors (PRRs) and nucleotide-binding domain and leucine-rich repeat (LRR)-containing proteins (NLRs) initiate pattern-triggered immunity (PTI) and effector- triggered immunity (ETI), respectively, each associated with the activation of an overlapping set of defence genes. The regulatory mechanism behind this convergence of PTI- and ETI-mediated defence gene induction remains elusive. We generated transgenic Arabidopsis plants that enable conditional NLR activation without pathogen infection to dissect NLR- and PRR-mediated transcriptional signals. A comparative analysis of over 40 transcriptome datasets linked calmodulin-binding transcription activators (CAMTAs) to the activation of overlapping defence genes in PTI and ETI. We used a dominant camta3 mutant (camta3-D) to assess CAMTA functions in the corresponding transcriptional regulation. Transcriptional regulation by NLRs, although highly similar to PTI responses, can be established independently of pathogen-associated molecular pattern (PAMP) perception, defence phytohormones and host cell death. Conditional expression of the N-terminal coiled-coil domain of the barley MLA (Mildew resistance locus A) NLR is sufficient to trigger similar transcriptional reprogramming as full-length NLRs. CAMTA-binding motifs are overrepresented in the 5' regulatory regions of the identified primary immune response genes, consistent with their altered expression and disease resistance responses in camta3-D plants. We propose that CAMTA-mediated transcriptional regulation defines an early convergence point in NLR-and PRR-mediated signalling.

The conserved lipase-like protein EDS1 transduces signals from pathogen-activated intracellular nucleotide-binding leucine-rich repeat (NLR) receptors to transcriptional defences and host cell death. In this pivotal NLR signalling role, EDS1 works as a heterodimer with each of its partners, SAG101 and PAD4. Different properties of EDS1-SAG101 and EDS1-PAD4 complexes and functional relationships to sensor and helper NLRs have emerged. EDS1-SAG101 dimers confer effector-triggered immunity mediated by intracellular TNL receptors. In contrast, EDS1-PAD4 dimers have a broader role promoting basal immune responses that can be initiated inside cells by TNL- or CNL-type NLRs, and at the cell surface by LRR-receptor proteins. Characterizing the essential elements of these two EDS1 modules will help to connect intracellular and surface receptor signalling networks in the plant immune system.

Plants have evolved mechanisms to protect themselves against pathogenic microbes and insect pests. In

ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid-dependent and independent pathways. Here we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogenGolovinomyces cichoracearum.The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss of function mutations in the protein kinase Enhanced Disease Resistance1 (EDR1), and previous work has shown that loss ofPAD4orEDS1suppressesedr1-mediated phenotypes, placing these proteins downstream ofEDR1. Here we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet unknown PAD4 signaling function.

Temperature impacts plant immunity and growth but how temperature intersects with endogenous pathways remains unclear. Here we uncover variation between Arabidopsis thaliana natural accessions in response to two non-stress temperatures (22°C and 16°C) affecting accumulation of the thermoresponsive stress hormone salicylic acid (SA) and plant growth. Analysis of differentially responding A. thaliana accessions shows that pre-existing SA provides a benefit in limiting bacterial pathogen infection at both temperatures. Several A. thaliana genotypes display a capacity to mitigate negative effects of high SA on growth, indicating within-species plasticity in SA - growth tradeoffs. An association study of temperature x SA variation, followed by physiological and immunity phenotyping of mutant and over-expression lines, identifies the transcription factor unfertilized embryo sac 12 (UNE12) as a temperature-responsive SA immunity regulator. Here we reveal previously untapped diversity in plant responses to temperature and a way forward in understanding the genetic architecture of plant adaptation to changing environments.

Plants have evolved mechanisms to attract beneficial microbes and insects while protecting themselves against pathogenic microbes and pests. In Arabidopsis, the immune regulator PAD4 functions with its cognate partner EDS1 to limit pathogen growth. PAD4, independently of EDS1, reduces infestation by Green Peach Aphid (GPA). How PAD4 regulates these defense outputs is unclear. By expressing the N-terminal PAD4-lipase-like domain (LLD) without its C-terminal ‘EDS1-PAD4’ (EP) domain, we interrogated PAD4 functions in plant defense. Here we show that transgenic expression of PAD4LLDin Arabidopsis is sufficient for limiting GPA infestation, but not for conferring basal and effector-triggered pathogen immunity. This suggests that the C-terminal PAD4-EP domain is necessary for EDS1-dependent immune functions. Moreover, PAD4LLDis not sufficient to interact with EDS1, indicating the PAD4-EP domain is required for heterodimerisation. These data provide molecular evidence that PAD4 has domain specific functions.

Mixed lineage kinase domain-like (MLKL) protein mediates necroptotic cell death in vertebrates. We report here the discovery of a conserved protein family across seed plants that is structurally homologous to vertebrate MLKL. TheArabidopsis thalianagenome encodes three MLKLs with overlapping functions in limiting growth of obligate biotrophic fungal and oomycete pathogens. Although displaying a cell death activity mediated by N-terminal helical bundles, termed HeLo domain,AtMLKL-dependent immunity can be separated from host cell death. Cryo-electron microscopy structures ofAtMLKLs reveal a tetrameric configuration, in which the pseudokinase domain and brace region bury the HeLo-domains, indicative of an auto-repressed complex. We also show the association of twoAtMLKLs with microtubules. These findings, coupled with resistance-enhancing activity and altered microtubule association of a phosphomimetic mutation in the pseudokinase domain ofAtMLKL1, point to a cell death-independent immunity mechanism.One Sentence SummaryPlants have a protein family that is structurally homologous to vertebrate mixed lineage kinase domain-like protein, which induces necroptotic cell death, but these plant proteins can confer immunity without host cell death.

Plant intracellular NLR receptors recognise pathogen interference to trigger immunity but how NLRs signal is not known. Enhanced disease susceptibility1 (EDS1) heterodimers are recruited by Toll-interleukin1-receptor domain NLRs (TNLs) to transcriptionally mobilise resistance pathways. By interrogating the Arabidopsis EDS1 ɑ-helical EP-domain we identify positively charged residues lining a cavity that are essential for TNL immunity signalling, beyond heterodimer formation. Mutating a single, conserved surface arginine (R493) disables TNL immunity to an oomycete pathogen and to bacteria producing the virulence factor, coronatine. Plants expressing a weakly active EDS1R493A variant have delayed transcriptional reprogramming, with severe consequences for resistance and countering bacterial coronatine repression of early immunity genes. The same EP-domain surface is utilised by a non-TNL receptor RPS2 for bacterial immunity, indicating that the EDS1 EP-domain signals in resistance conferred by different NLR receptor types. These data provide a unique structural insight to early downstream signalling in NLR receptor immunity., CC and TIR-domain type NLR receptors trigger plant immunity in response to pathogen effectors. Here, Bhandari et al. show that the EP-domain surface of EDS1 heterodimers signals downstream of both receptor types and ensures a rapid transcriptional response in TIR-NLR immunity to bacteria.

International audience; Repressive epigenetic marks, such as DNA and histone methylation, are sometimes located within introns. In Arabidopsis (Arabidopsis thaliana), INCREASE IN BONSAI METHYLATION2 (IBM2), an RNA-binding protein containing a bromo-adjacent homology domain, is required to process functional transcript isoforms of genes carrying intronic heterochromatin. In a genetic screen for suppressors of the ibm2 mutation, we identified FPA, an RNA-binding protein that promotes use of proximal polyadenylation sites in genes targeted by IBM2, including IBM1 encoding an essential H3K9 histone demethylase and the disease resistance gene RECOGNITION OF PERONOSPORA PARASITICA7. Both IBM2 and FPA are involved in the processing of their common mRNA targets: Transcription of IBM2 target genes is restored when FPA is mutated in ibm2 and impaired in transgenic plants overexpressing FPA. By contrast, transposons targeted by IBM2 and localized outside introns are not under this antagonistic control. The DNA methylation patterns of some genes and transposons are modified in fpa plants, including the large intron of IBM1, but these changes are rather limited and reversed when the mutant is complemented, indicating that FPA has a restricted role in mediating silencing. These data reveal a complex regulation by IBM2 and FPA pathways in processing mRNAs of genes bearing heterochromatic marks.