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.

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.

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.

~200 words) 28 Plant intracellular nucleotide-binding/leucine-rich repeat (NLR) immune receptors are 29 activated by pathogen effectors to trigger host defenses and cell death. Toll-30 Interleukin1-receptor (TIR)-domain NLRs (TNLs) converge on the Enhanced Disease 31 Susceptibility1 (EDS1) family of lipase-like proteins for all resistance outputs. In 32 Arabidopsis TNL immunity, AtEDS1 heterodimers with Phytoalexin Deficient4 33 (AtPAD4) transcriptionally boost basal defense pathways. AtEDS1 uses the same 34 surface to interact with PAD4-related Senescence-Associated Gene101 (AtSAG101), 35 but the role of AtEDS1-AtSAG101 heterodimers was unclear. We show that AtEDS1-36 AtSAG101 function together with AtNRG1 coiled-coil domain helper NLRs as a 37 coevolved TNL cell death signaling module. AtEDS1-AtSAG101-AtNRG1 cell death 38 activity is transferable to the solanaceous species, Nicotiana benthamiana, and 39 cannot be substituted by AtEDS1-AtPAD4 with AtNRG1 or AtEDS1-AtSAG101 with 40 endogenous NbNRG1. Analysis of EDS1-family evolutionary rate variation and 41 heterodimer structure-guided phenotyping of AtEDS1 variants or AtPAD4-AtSAG101 42 chimeras identify closely aligned ɑ-helical coil surfaces in the AtEDS1-AtSAG101 43 partner C-terminal domains that are necessary for TNL cell death signaling. Our data 44 suggest that TNL-triggered cell death and pathogen growth restriction are determined 45 by distinctive features of EDS1-SAG101 and EDS1-PAD4 complexes and that these 46 signaling machineries coevolved with further components within plant species or 47 clades to regulate downstream pathways in TNL immunity. 48 49