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.

SUMMARY The Toll/interleukin-1 receptor (TIR) domain occurs in animal and plant immune receptors. In the animal Toll-like receptors, homodimerization of the intracellular TIR domain is required for initiation of signaling cascades leading to innate immunity. By contrast, the role of the TIR domain in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant immune resistance proteins is poorly understood. L6 is a TIR-NB-LRR resistance protein from flax (Linum usitatissimum) that confers resistance to the flax rust phytopathogenic fungus (Melampsora lini). We determine the crystal structure of the L6 TIR domain and show that, although dispensable for pathogenic effector protein recognition, the TIR domain alone is both necessary and sufficient for L6 immune signaling. We demonstrate that the L6 TIR domain self-associates, most likely forming a homodimer. Analysis of the structure combined with site-directed mutagenesis suggests that self-association is a requirement for immune signaling and reveals distinct surface regions involved in self-association, signaling, and autoregulation.

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.

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.

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.

Race-specific resistance genes protect the global wheat crop from stem rust disease caused by f. sp. () but are often overcome owing to evolution of new virulent races of the pathogen. To understand virulence evolution in , we identified the protein ligand (AvrSr50) recognized by the Sr50 resistance protein. A spontaneous mutant of virulent to contained a 2.5 mega-base pair loss-of-heterozygosity event. A haustorial secreted protein from this region triggers-dependent defense responses in planta and interacts directly with the Sr50 protein. Virulence alleles of 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.

Bacterial wilt, a disease impacting cultivated crops worldwide, is caused by the pathogenic bacterium Ralstonia solanacearum. PopP2 (for Pseudomonas outer protein P2) is an R. solanacearum type III effector that belongs to the YopJ/AvrRxv protein family and interacts with the Arabidopsis thaliana RESISTANT TO RALSTONIA SOLANACEARUM 1-R (RRS1-R) resistance protein. RRS1-R contains the Toll/Interleukin1 receptor-nucleotide binding site-Leu-rich repeat domains found in several cytoplasmic R proteins and a C-terminal WRKY DNA binding domain. In this study, we identified the Arabidopsis Cys protease RESPONSIVE TO DEHYDRATION19 (RD19) as being a PopP2-interacting protein whose expression is induced during infection by R. solanacearum. An Arabidopsis rd19 mutant in an RRS1-R genetic background is compromised in resistance to the bacterium, indicating that RD19 is required for RRS1-R-mediated resistance. RD19 normally localizes in mobile vacuole-associated compartments and, upon coexpression with PopP2, is specifically relocalized to the plant nucleus, where the two proteins physically interact. No direct physical interaction between RRS1-R and RD19 in the presence of PopP2 was detected in the nucleus as determined by Fö rster resonance energy transfer. We propose that RD19 associates with PopP2 to form a nuclear complex that is required for activation of the RRS1-R-mediated resistance response.

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 TIRdomain 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. Selfassociation 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.plant immunity | NLR | TIR domain | plant disease resistance | signaling by cooperative assembly formation P lants have evolved a sophisticated innate immune system to detect pathogens, in which plant resistance (R) proteins recognize pathogen proteins (effectors) in a highly specific manner. This recognition leads to the effector-triggered immunity (ETI) response that often induces a localized cell death known as the hypersensitive response (1). Most R proteins belong to the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family. NLRs are prevalent in the immune systems of plants and animals and provide resistance to a broad range of pathogens, including fungi, oomycetes, bacteria, viruses, and insects (2, 3). NLRs contain a central nucleotide-binding (NB) domain, often referred to as the nucleotide-binding adaptor shared by APAF-1, resistance proteins, and CED-4 (NB-ARC domain) (4) and a C-terminal leucine-rich repeat (LRR) domain. Plant NLRs can be further classified into two main subfamilies, depending on the presence of either a Toll/interleukin-1 receptor domain (TIR-NLR) or a coiled-coil domain (CC-NLR) at their N termini (5).The CC and TIR domains of many plant NLRs can autonomously signal cell-death responses when expressed ectopically in planta, and mutations in these domains within full-length proteins also compromise signaling, suggesting that these domains are responsible for propagating the resistance signal after activation of the receptor (6-14). Self-association of both TIR (8, 9, 11, 15) and CC (10,13,16,17) domains has been shown to be important for the signaling function. In animal NLRs, the formation of postactivation oligomeric complexes, such as the NLRC4/NAIP inflammasome or...