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.2021 Jul 23;373(6553):420-425.
doi: 10.1126/science.abg7917. Epub 2021 Jun 17.

Plant "helper" immune receptors are Ca2+-permeable nonselective cation channels

Affiliations

Plant "helper" immune receptors are Ca2+-permeable nonselective cation channels

Pierre Jacob et al. Science..

Abstract

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. InArabidopsis, a subfamily of "helper" NLRs is required by many "sensor" NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta, and conferred cytoplasmic calcium ion (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 affecting 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 demonstrated thatArabidopsis 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.

Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1.
Fig. 1.. NRG1.1 CC-R resembles the ZAR1 and MLKL four-helical bundle.
(A) Structural overlay of 7K(R)E (PBD: 7L7V) and 2KE (7L7W) with 2KE in green, 7K(R)E four-helical bundle (4HB) in cyan, and 7K(R)E N-terminal region in yellow. (B) Structural overlay of 2KE in green and the ZAR1 4HB (6J5V) structure in light purple. (C) Structural overlay of 2KE in green and mMLKL 4HB (4BTF) in orange. (D) Structural overlay of 2KE in green and hMLKL 4HB (2MSV) in magenta. (E) Conserved F86/I75/I76 and L69/I57/L58 shown in sticks in the α3 helix of superimposed 2KE, mMLKL and hMLKL 4HB structures, respectively. (F andG) In planta (N. benthamiana) phenotypes at 2 days after induction and protein accumulation of WT NRG1.1 CC-R and structure-derived mutants. EV, empty vector.
Fig. 2.
Fig. 2.. Active NRG1.1 (D485V) oligomerizes on the PM and triggers cell death.
(A) In planta phenotypes of NRG1.1 variants inN. benthamiana (Nb) at 2 dpi. YFP, yellow fluorescent protein. (B) Oligomerization of NRG1.1 DV variants. Accumulation of YFP-tagged NRG1.1 variants was verified by SDS–polyacrylamide gel electrophoresis (PAGE) immunoblotting, and their oligomeric states were checked by blue native-PAGE immunoblotting inNb adr1 nrg1. (C) Activation promotes PM enrichment. Total proteins (T) extracted fromNb-expressing NRG1.1:YFP variants were fractionated into cytosolic (C), total membrane (TM), and PM fractions and verified by marker proteins: Cytosol, cytosolic ascorbic peroxidase; endoplasmic reticulum membrane (EM), sterol methyltransferase 1; PM, H+ATPase.
Fig. 3.
Fig. 3.. NRG1.1 forms ion channels permeable to Ca2+ and not Cl−.
(A) Quantification of apparent PM pores in HeLa cells expressing NRG1.1 variants. (B toD) Representative scanning electron micro-graphs of cells exhibiting apparent PM pores in cell lines expressing the NRG1.1 DV p-loop (B), NRG1.1 D485V (C) or the pore-forming protein GSDMN 1–275 L192D (D). White arrows indicate the visible PM pores. (E andF) Distribution of the diameter of PM pores visible after NRG1.1 D485V (E) or GSDMN L192D (F) expression. The average diameters are significantly different (t test,P < 0.0001). (G) [Ca2+]i in NRG1.1 D485V or NRG1.1 D485V p-loop–expressing HeLa cells. Black arrow indicates CaCl2 addition. (H) Ca2+ influx in HeLa cells expressing NRG1.1 variants. (I) Effect of the Ca2+ channel blockers LaCl3, GdCl3, and ruthenium red (RR) on NRG1.1 D485V–induced Ca2+ influx. (J) Intracellular [Cl−] accumulation in cells expressing the NRG1.1 D485V or NRG1.1 DV p-loop. (K) Representative time course experiment showing variation of [Cl−]. Black arrow indicates 200 mM NaCl addition. Letters indicate statistical significance (ANOVA with post hoc Tukey,P < 0.01).
Fig. 4.
Fig. 4.. Negatively charged residues in the conserved RNL motif are required for Ca2+ influx and cell death.
(A) In planta phenotypes (2 dpi) of plant RNLs in the presence of the Ca2+ channel blockers LaCl3 (La) and GdCl3 (Gd). Accumulation of YFP-tagged RNL variants was verified by SDS-PAGE and-immunoblotting, inNb adr1 nrg1. (B,C, andE) Time course RNL-induced Ca2+ influx measurements with GCaMP3-transgenicNb. (D andF) Time course conductivity measurement depicting RNL-triggered cell death inNb. (G toJ) [Ca2+]i in HeLa cells expressing NRG1.1 DV and variants (G and H) or ADR1 and variants (I and J). Black arrows indicate the addition of CaCl2. Letters indicate statistical significance (ANOVA with post hoc Tukey,P < 0.05). YFP-tagged RNL protein expression inNb and HeLa cells was verified (fig. S16).
Fig. 5.
Fig. 5.. NRG1.1 D485V forms nonselective cation channel.
(A) Whole-cell current recorded in voltage ramp (+50 to ‒200 mV) in a CaCl2 bath solution. (B) Whole-cell current recorded in voltage ramp (+50 to ‒200 mV) in a TEA-Cl bath solution. (C) Mean current recorded at ‒200 mV during experiments in (A) and (B). (D) Reversal potential (Erev) and relative ion permeability (PX/PCs) measured in voltage ramp experiments in CaCl2 or MgCl2 solutions in HEK293 cells expressing NRG1.1 D485V (N = 10 Ca2+ andN = 8 Mg2+). Letters indicate statistical difference (ANOVA with post hoc Tukey,P < 0.05).
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