Final Report Summary - PLANT-DEF-MECH (Investigating how pathogen effector recognition by plant resistance proteins activates defence) Based on our experimental results we have several published and under submission manuscripts in various high quality journals. Below I will describe in brief our published and under submission works.Plants are constantly interpreting microbial signals from potential pathogens and potential commensals or mutualists. Because plants have no circulating cells dedicated to this task, every plant cell must, in principle, recognize any microbe as friend, foe, or irrelevant bystander. That tall order is mediated by an array of innate immune system receptors. Plant intracellular immune receptors contain nucleotide-binding and leucine-rich repeat domains, and resemble mammalian NOD-like receptor (NLR) proteins. When plant immune receptors were first isolated 20 years ago, it was surprising to find that structurally similar NLRs could individually confer resistance to strains of plant pathogens from all kingdoms—insects, fungi, oomycetes, bacteria, and viruses. The plant receptors belong to nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins and recognize specific “avirulent” pathogen effectors and activate immune responses. How NB-LRR and NLR proteins activate defense is still poorly understood. During infection, plant NB-LRR proteins activate effector-triggered immunity upon recognition of corresponding pathogen effectors. NB-LRR protein activation of defense mechanisms is ATP-dependent, causes defense gene induction and often culminates in the hypersensitive cell death response (hereafter referred to as cell death). Cytoplasmic plant immune receptors recognise specific pathogen effector proteins and initiate effector-triggered immunity. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), belong to Toll/interleukin-1 receptor/resistance (TIR) NB-LRR family of R proteins. They function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. My initial contribution into “RRS1/RPS4 immune-receptors” project started with the development of a transient expression assay of co-expression of both receptors in the heterologous system Nicotiana tabacum. The PopP2 and AvrRps4 effectors are not recognised in tobacco plant, so there is no defence activation. However, the transient co-expression of both RRS1 and RPS4 with PopP2 or AvrRps4 perfectly activate defence. This indicates that the transient expression system is functional. The transient expression system gave us the opportunity to study our concepts and theories, regarding the way of function of the RRS1/RPS4 system, effectively and in very short time, since we are able to avoid the time-consuming stable transformation of Arabidopsis plants, etc.In our first work, published in Science (Science 2014, vol. 344 no. 6181 pp. 299-303), we reported that RPS4 and RRS1 physically associate and this association is essential for the defence activation. Crystal structures of the N-terminal TIR domains of RPS4 and RRS1, individually and as a heterodimeric complex reveal a conserved TIR/TIR interaction interface. We show that TIR domain heterodimerization is required to form a functional RPS4/RRS1 effector recognition complex. The RPS4 TIR domain activates effector-independent defense (autoimmunity), which is inhibited by the RRS1 TIR domain, dependent on a wild-type interaction interface. Thus, RPS4 and RRS1 function as a receptor complex, in which the two components play distinct roles in recognition and signaling. Because TIR/TIR domain interactions have previously been difficult to define structurally, our data may have broad implications for understanding immune receptors’ TIR domain function across phyla. Current models of plant NB-LRR protein activation imply that effector perception leads to considerable domain reorganization and formation of oligomeric forms. Rather than effector-induced disassociation of RRS1 and RPS4 proteins, rearrangements within a preformed RRS1/RPS4 complex, culminating in stabilization of an RPS4 TIR domain homodimer, likely distinguish the pre-activation complex from its activated state. Nucleotide-binding or exchange by RPS4, but not RRS1, is required for a functional NLR resistance complex. Thus AvrRps4 or PopP2 recognition is accomplished by an RRS1/RPS4 complex, distinct from indirect recognition of effectors by other plant NB-LRR proteins. We propose that upon effector binding, defense activation requires the release of RPS4 TIR domain inhibition by the RRS1 TIR domain, allowing formation of a signaling-competent RPS4 TIR domain homodimer.In a second work, just accepted for publication in PLoS Genetics (accepted with minor revision,) we define the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion system. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation (autoimmunity) is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, indicating that RPS4 acts in a complex with RRS1, which is consistent with our previous data published in Science. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs can activate defense signalling, or are held inactive in the absence of a pathogen effector.In a third work of our group, which is under submission to Cell journal, we present our results regarding the role of a WRKY-DNA-binding domain of RRS1 in recognition and defence activation by two unrelated pathogen effectors (PopP2, AvrRps4). Post-translational modifications of RRS1-WRKY-domain, triggered by PopP2 acetyltransferase activity, or the acetyl-lysine mimics substitution, results in reduction of DNA binding and defence activation. Interestingly, these modifications alter the AvrRps4 recognition by RRS1. The AvrRps4/RRS1-WRKY-domain interaction and its disruption by PopP2, reveals the role of WRKY-domain in AvrRps4 dependent defence activation. Likewise, the interaction of both PopP2 and AvrRps4 with other WRKY proteins potentially indicates their virulence function(s). Our data suggest a recognition mechanism in which several NB-LRR receptors have been evolved to carry protein domains that give additional functions and also act as baits for pathogen effectors perception and defence activation. The vistas opened by cell and structural biology in a field rich with genetics and natural variation in both hosts and pathogens provide an exciting new view of plant immunology, 20 years after the first pathogen sensors were discovered.