Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. In Arabidopsis, a subfamily of “helper” NLRs are required by many “sensor” NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta and conferred cytoplasmic Ca2+ influx in plant and human cells. NRG1.1-dependent Ca2+ influx and cell death were sensitive to Ca2+ channel blockers and were suppressed by mutations impacting oligomerization or plasma membrane enrichment. Ca2+ influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another “helper” NLR, required conserved negatively charged N-terminal residues. Whole-cell voltage-clamp recordings demonstrate that Arabidopsis “helper” NLRs form Ca2+-permeable cation channels to directly regulate cytoplasmic Ca2+ levels and consequent cell death. Thus, “helper” NLRs transduce cell death signals directly.

Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive.We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/ RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM.Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo-and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function.We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. RPW8 domain-containing 'helper' NLRs (RNLs) are required by many 'sensor' NLRs. Our crystal structure of the RNL N REQUIREMENT GENE 1.1 (NRG1.1) N-terminal signaling domain resembled that of the resting state plant resistosome-forming HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) and the animal MIXED-LINEAGE KINASE-LIKE (MLKL) cation channel. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta and conferred cytoplasmic Ca2+ influx in plant and human HeLa cells. NRG1.1-dependent Ca2+ influx and cell death were sensitive to Ca2+ channel blockers. Ca2+ influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another RNL, required conserved negatively charged N-terminal residues. Thus, RNLs apparently form influx channels to directly regulate cytoplasmic [Ca2+] and consequent cell death.

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