Arabidopsis ADR1 helper NLR immune receptors localize and function at the plasma membrane in a phospholipid dependent manner.

Svenja C Saile,Li Wan,Yvon Jaillais,Vera Bonardi,Farid El Kasmi,Adam Bayless,Mehdi Doumane,Eva Stöbbe,Marc T Nishimura,Jeffery L Dangl,Jutta Keicher,Frank M Ackermann,Marie‐Cécile Caillaud,Sruthi Sunil,Claudia Oecking

doi:10.1111/nph.17788

Abstract

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.

Highlights

Plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat receptor (NLR) family mediate recognition of pathogen-derived effector proteins and the induction of a strong immune response
In Arabidopsis thaliana (Arabidopsis) the RPW8-like domain ‘helper’ NLR (RNL) subclass consists of two gene families, ACTIVATED DISEASE RESISTANCE 1 (ADR1) and N REQUIREMENT GENE 1 (NRG1), both being required for immune signalling and cell death induction of many other NLRs, TNLs, and are considered as ‘helper’ NLRs (Bonardi et al, 2011; Castel et al, 2019; Qi et al, 2018; Lapin et al, 2019; Wu et al, 2019; Saile et al, 2020)
The subcellular localization of two Arabidopsis full length RNLs, AtNRG1.1 and AtNRG1.2, was recently described. Both proteins localize to endoplasmic reticulum (ER) membranes, partially to the plasma membrane (PM) and in the cytosol when transiently expressed in Nicotiana benthamiana (N. benthamiana) and analyzed by confocal microscopy or subcellular fractionation experiments (Lapin et al, 2019; Wu et al, 2019; Jacob et al, 2021)

Summary

Introduction

Plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat receptor (NLR) family mediate recognition of pathogen-derived effector proteins and the induction of a strong immune response. NLR activation leads to the hypersensitive response, a type of programmed cell death of the infected cells (Jones & Dangl, 2006; Monteiro & Nishimura, 2018; Balint-Kurti, 2019) Based on their N-terminal domain architecture, three classes of NLRs have been described in plants: Toll/Interleukin-1 receptor (TIR) NLRs (TNLs), coiled-coil (CC) NLRs (CNLs) and the HeLo/RPW8-like coiled-coil (CCR) domain NLRs (RNLs) (Monteiro & Nishimura, 2018). The expression of activated AtNRG1.1DV and AtADR1 resulted in an increase of the intracellular calcium concentration and further electrophysiological analysis revealed that RNLs can act as potential PM-localized calciumpermeable channels (Jacob et al, 2021), similar as shown for the CNL AtZAR1 (Bi et al, 2021). The molecular determinants driving their localization and cell death function at the membrane are not identified

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Discussion

Conclusion