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

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

Correction: Autoacetylation of the Ralstonia solanacearum Effector PopP2 Targets a Lysine Residue Essential for RRS1-R-Mediated Immunity in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.ppat.1001202.].

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants.

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.

Multiple functional self-association interfaces in plant TIR domains.

The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model.

NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling.

Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins.

Plants possess intracellular immune receptors designated "nucleotide-binding domain and leucine-rich repeat" (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively.

The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance.

Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.

Intramolecular interaction influences binding of the Flax L5 and L6 resistance proteins to their AvrL567 ligands.

L locus resistance (R) proteins are nucleotide binding (NB-ARC) leucine-rich repeat (LRR) proteins from flax (Linum usitatissimum) that provide race-specific resistance to the causal agent of flax rust disease, Melampsora lini. L5 and L6 are two alleles of the L locus that directly recognize variants of the fungal effector AvrL567. In this study, we have investigated the molecular details of this recognition by site-directed mutagenesis of AvrL567 and construction of chimeric L proteins. Single, double and triple mutations of polymorphic residues in a variety of AvrL567 variants showed additive effects on recognition strength, suggesting that multiple contact points are involved in recognition. Domain-swap experiments between L5 and L6 show that specificity differences are determined by their corresponding LRR regions. Most positively selected amino acid sites occur in the N- and C-terminal LRR units, and polymorphisms in the first seven and last four LRR units contribute to recognition specificity of L5 and L6 respectively. This further confirms that multiple, additive contact points occur between AvrL567 variants and either L5 or L6. However, we also observed that recognition of AvrL567 is affected by co-operative polymorphisms between both adjacent and distant domains of the R protein, including the TIR, ARC and LRR domains, implying that these residues are involved in intramolecular interactions to optimize detection of the pathogen and defense signal activation. We suggest a model where Avr ligand interaction directly competes with intramolecular interactions to cause activation of the R protein.

Plant Toll/interleukin-1 receptor/resistance protein domains physically associate with enhanced disease susceptibility1 family proteins in immune signaling.

Plant Toll/interleukin-1 receptor/resistance protein (TIR) type nucleotide-binding and leucine-rich repeat immune receptors (NLRs) require enhanced disease susceptibility 1 (EDS1) family proteins and the helper NLRs NRG1 and ADR1 for immune activation. We show that the NbEDS1-NbSAG101b-NbNRG1 signaling pathway in N. benthamiana is necessary for cell death signaling by TIR-NLRs from a range of plant species, suggesting a universal requirement for this module in TIR-NLR-mediated cell death in N. benthamiana. We also find that TIR domains physically associate with NbEDS1, NbPAD4, and NbSAG101 in planta, independently of each other. Furthermore, NbNRG1 associates with NbSAG101b, but not with other EDS1 family members, via its C-terminal EP domain. Physical interaction between activated TIRs and EDS1 signaling complexes may facilitate the transfer of low abundance products of TIR catalytic activity or alter TIR catalytic activity to favor the production of EDS1 heterodimer ligands.

Crystallization and preliminary X-ray diffraction analyses of the TIR domains of three TIR-NB-LRR proteins that are involved in disease resistance in Arabidopsis thaliana.

The Toll/interleukin-1 receptor (TIR) domain is a protein-protein interaction domain that is found in both animal and plant immune receptors. The N-terminal TIR domain from the nucleotide-binding (NB)-leucine-rich repeat (LRR) class of plant disease-resistance (R) proteins has been shown to play an important role in defence signalling. Recently, the crystal structure of the TIR domain from flax R protein L6 was determined and this structure, combined with functional studies, demonstrated that TIR-domain homodimerization is a requirement for function of the R protein L6. To advance the molecular understanding of the function of TIR domains in R-protein signalling, the protein expression, purification, crystallization and X-ray diffraction analyses of the TIR domains of the Arabidopsis thaliana R proteins RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1) and the resistance-like protein SNC1 (suppressor of npr1-1, constitutive 1) are reported here. RPS4 and RRS1 function cooperatively as a dual resistance-protein system that prevents infection by three distinct pathogens. SNC1 is implicated in resistance pathways in Arabidopsis and is believed to be involved in transcriptional regulation through its interaction with the transcriptional corepressor TPR1 (Topless-related 1). The TIR domains of all three proteins have successfully been expressed and purified as soluble proteins in Escherichia coli. Plate-like crystals of the RPS4 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.05 Šresolution, had the symmetry of space group P1 and analysis of the Matthews coefficient suggested that there were four molecules per asymmetric unit. Tetragonal crystals of the RRS1 TIR domain were obtained using ammonium sulfate as a precipitant; they diffracted X-rays to 1.75 Šresolution, had the symmetry of space group P4(1)2(1)2 or P4(3)2(1)2 and were most likely to contain one molecule per asymmetric unit. Crystals of the SNC1 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.20 Šresolution and had the symmetry of space group P4(1)2(1)2 or P4(3)2(1)2, with two molecules predicted per asymmetric unit. These results provide a good foundation to advance the molecular and structural understanding of the function of the TIR domain in plant innate immunity.

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance.

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.

Autoacetylation of the Ralstonia solanacearum effector PopP2 targets a lysine residue essential for RRS1-R-mediated immunity in Arabidopsis.

Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as "guards". The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity.

Connecting the dots between cell surface- and intracellular-triggered immune pathways in plants.

Plants can detect microbial molecules via surface-localized pattern-recognition receptors (PRRs) and intracellular immune receptors from the nucleotide-binding, leucine-rich repeat receptor (NLR) family. The corresponding pattern-triggered (PTI) and effector-triggered (ETI) immunity were long considered separate pathways, although they converge on largely similar cellular responses, such as calcium influx and overlapping gene reprogramming. A number of studies recently uncovered genetic and molecular interconnections between PTI and ETI, highlighting the complexity of the plant immune network. Notably, PRR- and NLR-mediated immune responses require and potentiate each other to reach an optimal immune output. How PTI and ETI connect to confer robust immunity in different plant species, including crops will be an exciting future research area.

In the trenches of plant pathogen recognition: Role of NB-LRR proteins.

As in nearly every discipline of plant biology, new insights are constantly changing our understanding of plant immunity. It is now clear that plant immunity is controlled by two layers of inducible responses: basal responses triggered by conserved microbial features and specific responses triggered by gene-for-gene recognition of pathogen effector proteins by host resistance (R) proteins. The nucleotide-binding domain leucine-rich repeat (NB-LRR) class of R proteins plays a major role in the combat against a wide range of plant pathogens. The variation that has been generated and is maintained within these conserved proteins has diversified their specificity, subcellular localisations, activation and recognition mechanisms, allowing them to specifically adapt to different plant-pathogen interaction systems. This review addresses recent advances in the molecular role of NB-LRR proteins in pathogen recognition and activation of plant defence responses.

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P.

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.

What Do We Know About NOD-Like Receptors in Plant Immunity?

The first plant disease resistance (R) genes were identified and cloned more than two decades ago. Since then, many more R genes have been identified and characterized in numerous plant pathosystems. Most of these encode members of the large family of intracellular NLRs (NOD-like receptors), which also includes animal immune receptors. New discoveries in this expanding field of research provide new elements for our understanding of plant NLR function. But what do we know about plant NLR function today? Genetic, structural, and functional analyses have uncovered a number of commonalities and differences in pathogen recognition strategies as well as how NLRs are regulated and activate defense signaling, but many unknowns remain. This review gives an update on the latest discoveries and breakthroughs in this field, with an emphasis on structural findings and some comparison to animal NLRs, which can provide additional insights and paradigms in plant NLR function.

Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat.

Race-specific resistance genes protect the global wheat crop from stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) but are often overcome owing to evolution of new virulent races of the pathogen. To understand virulence evolution in Pgt, we identified the protein ligand (AvrSr50) recognized by the Sr50 resistance protein. A spontaneous mutant of Pgt virulent to Sr50 contained a 2.5 mega-base pair loss-of-heterozygosity event. A haustorial secreted protein from this region triggers Sr50-dependent defense responses in planta and interacts directly with the Sr50 protein. Virulence alleles of AvrSr50 have arisen through DNA insertion and sequence divergence, and our data provide molecular evidence that in addition to sexual recombination, somatic exchange can play a role in the emergence of new virulence traits in Pgt.

A novel conserved mechanism for plant NLR protein pairs: the "integrated decoy" hypothesis.

Plant immunity is often triggered by the specific recognition of pathogen effectors by intracellular nucleotide-binding, leucine-rich repeat receptors (NLR). Plant NLRs contain an N-terminal signaling domain that is mostly represented by either a Toll-interleukin1 receptor (TIR) domain or a coiled coil (CC) domain. In many cases, single NLR proteins are sufficient for both effector recognition and signaling activation. However, many paired NLRs have now been identified where both proteins are required to confer resistance to pathogens. Recent detailed studies on the Arabidopsis thaliana TIR-NLR pair RRS1 and RPS4 and on the rice CC-NLR pair RGA4 and RGA5 have revealed for the first time how such protein pairs function together. In both cases, the paired partners interact physically to form a hetero-complex receptor in which each partner plays distinct roles in effector recognition or signaling activation, highlighting a conserved mode of action of NLR pairs across both monocotyledonous and dicotyledonous plants. We also describe an "integrated decoy" model for the function of these receptor complexes. In this model, a plant protein targeted by an effector has been duplicated and fused to one member of the NLR pair, where it acts as a bait to trigger defense signaling by the second NLR upon effector binding. This mechanism may be common to many other plant NLR pairs.

Structural basis for assembly and function of a heterodimeric plant immune receptor.

Cytoplasmic plant immune receptors recognize specific pathogen effector proteins and initiate effector-triggered immunity. In Arabidopsis, the immune receptors RPS4 and RRS1 are both required to activate defense to three different pathogens. We show that RPS4 and RRS1 physically associate. Crystal structures of the N-terminal Toll-interleukin-1 receptor/resistance (TIR) domains of RPS4 and RRS1, individually and as a heterodimeric complex (respectively at 2.05, 1.75, and 2.65 angstrom resolution), reveal a conserved TIR/TIR interaction interface. We show that TIR domain heterodimerization is required to form a functional RRS1/RPS4 effector recognition complex. The RPS4 TIR domain activates effector-independent defense, which is inhibited by the RRS1 TIR domain through the heterodimerization interface. Thus, RPS4 and RRS1 function as a receptor complex in which the two components play distinct roles in recognition and signaling.

New insights in plant immunity signaling activation.

Plant disease resistance can be triggered by specific recognition of microbial effectors by plant nucleotide binding-leucine rich repeat (NB-LRR) receptors. Over the last few years, many efforts have greatly improved the understanding of effector and NB-LRR function, but have left a lot of questions as to how effector perception activates NB-LRR induction of defense signaling. This review describes exciting new findings showing similarities and differences in function of diverse plant NB-LRR proteins in terms of pathogen recognition and where and how resistance proteins are activated. Localization studies have shown that some NB-LRRs can activate signaling from the cytosol while others act in the nucleus. Also, the structural determination of two NB-LRR signaling domains demonstrated that receptor oligomerization is fundamental for the activation of resistance signaling.