Molecular innovations in plant TIR-based immunity signaling.

A protein domain (Toll and Interleukin-1 receptor [TIR]-like) with homology to animal TIRs mediates immune signaling in prokaryotes and eukaryotes. Here, we present an overview of TIR evolution and the molecular versatility of TIR domains in different protein architectures for host protection against microbial attack. Plant TIR-based signaling emerges as being central to the potentiation and effectiveness of host defenses triggered by intracellular and cell-surface immune receptors. Equally relevant for plant fitness are mechanisms that limit potent TIR signaling in healthy tissues but maintain preparedness for infection. We propose that seed plants evolved a specialized protein module to selectively translate TIR enzymatic activities to defense outputs, overlaying a more general function of TIRs.

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.

Bacterial effector targeting of a plant iron sensor facilitates iron acquisition and pathogen colonization.

Acquisition of nutrients from different species is necessary for pathogen colonization. Iron is an essential mineral nutrient for nearly all organisms, but little is known about how pathogens manipulate plant hosts to acquire iron. Here, we report that AvrRps4, an effector protein delivered by Pseudomonas syringae bacteria to plants, interacts with and targets the plant iron sensor protein BRUTUS (BTS) to facilitate iron uptake and pathogen proliferation in Arabidopsis thaliana. Infection of rps4 and eds1 by P. syringae pv. tomato (Pst) DC3000 expressing AvrRps4 resulted in iron accumulation, especially in the plant apoplast. AvrRps4 alleviates BTS-mediated degradation of bHLH115 and ILR3(IAA-Leucine resistant 3), two iron regulatory proteins. In addition, BTS is important for accumulating immune proteins Enhanced Disease Susceptibility1 (EDS1) at both the transcriptional and protein levels upon Pst (avrRps4) infections. Our findings suggest that AvrRps4 targets BTS to facilitate iron accumulation and BTS contributes to RPS4/EDS1-mediated immune responses.

The Role of Salicylic Acid in the Expression of RECEPTOR-LIKE PROTEIN 23 and Other Immunity-Related Genes.

The hormone salicylic acid (SA) plays a crucial role in plant immunity by activating responses that arrest pathogen ingress. SA accumulation also penalizes growth, a phenomenon visible in mutants that hyperaccumulate SA, resulting in strong growth inhibition. An important question, therefore, is why healthy plants produce basal levels of this hormone when defense responses are not activated. Here, we show that basal SA levels in unchallenged plants are needed for the expression of a number of immunity-related genes and receptors, such as RECEPTOR-LIKE PROTEIN 23 (RLP23). This was shown by depleting basal SA levels in transgenic Arabidopsis lines through the overexpression of the SA-inactivating hydroxylases DOWNY MILDEW-RESISTANT 6 (DMR6) or DMR6-LIKE OXYGENASE 1. RNAseq analysis revealed that the expression of a subset of immune receptor and signaling genes is strongly reduced in the absence of SA. The biological relevance of this was shown for RLP23: In SA-depleted and SA-insensitive plants, responses to the RLP23 ligand, the microbial pattern nlp24, were strongly reduced, whereas responses to flg22 remained unchanged. We hypothesize that low basal SA levels are needed for the expression of a subset of immune system components that enable early pathogen detection and activation of immunity.

Cavity surface residues of PAD4 and SAG101 contribute to EDS1 dimer signaling specificity in plant immunity.

Arabidopsis pathogen effector-triggered immunity (ETI) is controlled by a family of three lipase-like proteins (EDS1, PAD4, and SAG101) and two subfamilies of HET-S/LOB-B (HeLo)-domain "helper" nucleotide-binding/leucine-rich repeats (ADR1s and NRG1s). EDS1-PAD4 dimers cooperate with ADR1s, and EDS1-SAG101 dimers with NRG1s, in two separate defense-promoting modules. EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1 complexes were detected in immune-activated leaf extracts but the molecular determinants for specific complex formation and function remain unknown. EDS1 signaling is mediated by a C-terminal EP domain (EPD) surface surrounding a cavity formed by the heterodimer. Here we investigated whether the EPDs of PAD4 and SAG101 contribute to EDS1 dimer functions. Using a structure-guided approach, we undertook a comprehensive mutational analysis of Arabidopsis PAD4. We identify two conserved residues (Arg314 and Lys380) lining the PAD4 EPD cavity that are essential for EDS1-PAD4-mediated pathogen resistance, but are dispensable for the PAD4-mediated restriction of green peach aphid infestation. Positionally equivalent Met304 and Arg373 at the SAG101 EPD cavity are required for EDS1-SAG101 promotion of ETI-related cell death. In a PAD4 and SAG101 interactome analysis of ETI-activated tissues, PAD4R314A and SAG101M304R EPD variants maintain interaction with EDS1 but lose association, respectively, with helper nucleotide-binding/leucine-rich repeats ADR1-L1 and NRG1.1, and other immune-related proteins. Our data reveal a fundamental contribution of similar but non-identical PAD4 and SAG101 EPD surfaces to specific EDS1 dimer protein interactions and pathogen immunity.

Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity.

Transcriptional corepressors of the Topless (TPL) family regulate plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of the TPL family roles in transcriptional regulation. Chromatin immunoprecipitation with sequencing (ChIP-Seq) was performed on Arabidopsis thaliana lines expressing GFP-tagged Topless-related 1 (TPR1-GFP) with and without constitutive immunity via Enhanced Disease Susceptibility 1 (EDS1). RNA-Seq profiling of the TPR1-GFP lines and pathogen-infected tpl/tpr mutants, combined with measuring immunity, growth, and physiological parameters was employed to investigate TPL/TPR roles in immunity and defense homeostasis. TPR1 was enriched at promoter regions of c. 1400 genes and c. 10% of the detected binding required EDS1 immunity signaling. In a tpr1 tpl tpr4 (t3) mutant, resistance to bacteria was slightly compromised, and defense-related transcriptional reprogramming was weakly reduced or enhanced, respectively, at early (< 1 h) and late 24 h stages of bacterial infection. The t3 plants challenged with bacteria or pathogen-associated molecular pattern nlp24 displayed photosystem II dysfunctions. Also, t3 plants were hypersensitive to phytocytokine pep1 at the level of root growth inhibition. Transgenic expression of TPR1 rescued these t3 physiological defects. We propose that TPR1 and TPL family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity.

Downy Mildew effector HaRxL21 interacts with the transcriptional repressor TOPLESS to promote pathogen susceptibility.

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.

EDS1 complexes are not required for PRR responses and execute TNL-ETI from the nucleus in Nicotiana benthamiana.

Heterodimeric 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, in Arabidopsis thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in Nicotiana benthamiana. We did not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad could abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation was sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown.

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

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.

Obligate biotroph downy mildew consistently induces near-identical protective microbiomes in Arabidopsis thaliana.

Hyaloperonospora arabidopsidis (Hpa) is an obligately biotrophic downy mildew that is routinely cultured on Arabidopsis thaliana hosts that harbour complex microbiomes. We hypothesized that the culturing procedure proliferates Hpa-associated microbiota (HAM) in addition to the pathogen and exploited this model system to investigate which microorganisms consistently associate with Hpa. Using amplicon sequencing, we found nine bacterial sequence variants that are shared between at least three out of four Hpa cultures in the Netherlands and Germany and comprise 34% of the phyllosphere community of the infected plants. Whole-genome sequencing showed that representative HAM bacterial isolates from these distinct Hpa cultures are isogenic and that an additional seven published Hpa metagenomes contain numerous sequences of the HAM. Although we showed that HAM benefit from Hpa infection, HAM negatively affect Hpa spore formation. Moreover, we show that pathogen-infected plants can selectively recruit HAM to both their roots and shoots and form a soil-borne infection-associated microbiome that helps resist the pathogen. Understanding the mechanisms by which infection-associated microbiomes are formed might enable breeding of crop varieties that select for protective microbiomes.

Broad-spectrum resistance of Arabidopsis C24 to downy mildew is mediated by different combinations of isolate-specific loci.

Most natural Arabidopsis thaliana accessions are susceptible to one or more isolates of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). However, Arabidopsis C24 has proved resistant to all Hpa isolates tested so far. Here we describe the complex genetic basis of broad-spectrum resistance in C24. The genetics of C24 resistance to three Hpa isolates was analyzed by segregation analysis and quantitative trait locus (QTL) mapping on recombinant inbred and introgression lines. Resistance of C24 to downy mildew was found to be a multigenic trait with complex inheritance. Many identified resistance loci were isolate-specific and located on different chromosomes. Among the C24 resistance QTLs, we found dominant, codominant and recessive loci. Interestingly, none of the identified loci significantly contributed to resistance against all three tested Hpa isolates. Our study demonstrates that broad-spectrum resistance of Arabidopsis C24 to Hpa is based on different combinations of multiple isolate-specific loci. The identified quantitative resistance loci are particularly promising as they provide an important basis for the cloning of susceptibility- and immunity-related genes.

Bioengineered intestinal tubules as a tool to test intestinal biological efficacy of lettuce species.

Lettuce (Lactuca sativa) is one of the most consumed and cultivated vegetables globally. Its breeding is focused on the improvement of yield and disease resistance. However, potential detrimental or beneficial health effects for the consumer are often not targeted in the breeding programs. Here, a bioengineered intestinal tubule was used to assess the intestinal efficacy of extracts from five plant accessions belonging to four Lactuca species. These four species include the domesticated L. sativa, closely related wild species L. serriola, and phylogenetically more distant wild relatives L. saligna and L. virosa. We assessed the epithelial barrier integrity, cell viability, cell attachment, brush border enzyme activity, and immune markers. Extracts from L. sativa cv. Salinas decreased cell attachment and brush border enzyme activity. However, extracts from the non-edible wild species L. saligna and L. virosa reduced the epithelial barrier functions, cell attachment, cell viability, and brush border enzyme activity. Since wild species represent a valuable germplasm pool, the bioengineered intestinal tubules could open ways to evaluate the safety and nutritional properties of the lettuce breeding material originating from crosses with wild Lactuca species.

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity.

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 "helper" NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.

Origins and Immunity Networking Functions of EDS1 Family Proteins.

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.

Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme.

Direct or indirect recognition of pathogen-derived effectors by plant nucleotide-binding leucine-rich repeat (LRR) receptors (NLRs) initiates innate immune responses. The Hyaloperonospora arabidopsidis effector ATR1 activates the N-terminal Toll-interleukin-1 receptor (TIR) domain of Arabidopsis NLR RPP1. We report a cryo-electron microscopy structure of RPP1 bound by ATR1. The structure reveals a C-terminal jelly roll/Ig-like domain (C-JID) for specific ATR1 recognition. Biochemical and functional analyses show that ATR1 binds to the C-JID and the LRRs to induce an RPP1 tetrameric assembly required for nicotinamide adenine dinucleotide hydrolase (NADase) activity. RPP1 tetramerization creates two potential active sites, each formed by an asymmetric TIR homodimer. Our data define the mechanism of direct effector recognition by a plant NLR leading to formation of a signaling-active holoenzyme.

Discovery of a Family of Mixed Lineage Kinase Domain-like Proteins in Plants and Their Role in Innate Immune Signaling.

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.

An EDS1 heterodimer signalling surface enforces timely reprogramming of immunity genes in Arabidopsis.

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.

Susceptibility to plant disease: more than a failure of host immunity.

Susceptibility to infectious diseases caused by pathogens affects most plants in their natural habitat and leads to yield losses in agriculture. However, plants are not helpless because their immune system can deal with the vast majority of attackers. Nevertheless, adapted pathogens are able to circumvent or avert host immunity making plants susceptible to these uninvited guests. In addition to the failure of the plant immune system, there are other host processes that contribute to plant disease susceptibility. In this review, we discuss recent studies that show the active role played by the host in supporting disease, focusing mainly on biotrophic stages of infection. Plants attract pathogens, enable their entry and accommodation, and facilitate nutrient provision.