Plant TIR domains physically interact with EDS1 family proteins to propagate immune signalling

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. TIR signalling domains possess NADase activity, producing NAM and v-cADPR from NAD+ in vitro . However, after TIR activation different small non-cyclic signalling molecules have been detected bound to EDS1/SAG101 and EDS1/PAD4 heterodimers. These molecules have not been detected in in vitro assays or as free molecules in planta and it is not clear how they are delivered to the EDS1 complexes. Here we investigate physical and functional interactions between TIR signalling domains, EDS1 family proteins and helper NLRs to clarify these signalling transduction pathways. We show that the Nb EDS1- Nb SAG101b- Nb NRG1 signalling pathway in N. benthamiana is necessary and sufficient for cell death signalling induced by six different TIR-containing NLRs from a range of plant species, suggesting this module is likely a universal requirement for TIR-NLR mediated cell death in N. benthamiana . We also find that TIR domains physically interact with Nb EDS1, Nb PAD4 and Nb SAG101 in planta , independently of each other. We also find evidence for direct interaction of Nb NRG1 with Nb SAG101b via its C-terminal EP domain, but not with other EDS1 family members. These data suggest a model in which physical interaction between activated TIRs and EDS1 signalling complexes facilitates efficient transfer of low abundance products of TIR catalytic activity directly to EDS1 heterocomplexes. The interaction could also alter TIR catalytic activity to favor production of the ligands recognised by EDS1/SAG101 and EDS1/PAD4. ### Competing Interest Statement The authors have declared no competing interest.