Cytoplasmic calcium influx mediated by plant MLKLs confers TNL-triggered immunity

The plant homolog of vertebrate necroptosis inducer mixed-lineage kinase domain-like (MLKL) contributes to downstream steps in Toll-interleukin-1 receptor domain NLR (TNL)-receptor-triggered immunity. Here, we show that Arabidopsis MLKL1 (AtMLKL1) clusters into puncta at the plasma membrane upon TNL activation and that this sub-cellular reorganization is dependent on the TNL signal transducer, EDS1. We find that AtMLKLs confer TNL-triggered immunity in parallel with RPW8-type HeLo-domain-containing NLRs (RNLs) and that the AtMLKL N-terminal HeLo domain is indispensable for both immunity and clustering. We show that the AtMLKL HeLo domain mediates cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) influx in plant and human cells, and AtMLKLs are responsible for sustained [Ca²⁺]_cyt influx during TNL-triggered, but not CNL-triggered, immunity. Our study reveals parallel immune signaling functions of plant MLKLs and RNLs as mediators of [Ca²⁺]_cyt influx and a potentially common role of the HeLo domain fold in the Ca²⁺-signal relay of diverse organisms.