Formation of NPR1 Condensates Promotes Cell Survival during the Plant Immune Response

Abstract

In plants, pathogen effector-triggered immunity (ETI) often leads to programmed cell death, which is restricted by NPR1, an activator of systemic acquired resistance. However, the biochemical activities of NPR1 enabling it to promote defense and restrict cell death remain unclear. Here we show that NPR1 promotes cell survival by targeting substrates for ubiquitination and degradation through formation of salicylic acid-induced NPR1 condensates (SINCs). SINCs are enriched with stress response proteins, including nucleotide-binding leucine-rich repeat immune receptors, oxidative and DNA damage response proteins, and protein quality control machineries. Transition of NPR1 into condensates is required for formation of the NPR1-Cullin 3 E3 ligase complex to ubiquitinate SINC-localized substrates, such as EDS1 and specific WRKY transcription factors, and promote cell survival during ETI. Our analysis of SINCs suggests that NPR1 is centrally integrated into the cell death or survival decisions in plant immunity by modulating multiple stress-responsive processes in this quasi-organelle.

Keywords: NPR1; cell survival; cullin 3 RING E3 ligase; effector-triggered immunity; plant immunity; protein homeostasis; salicylic acid-induced NPR1 condensate (SINC); systemic acquired resistance; ubiquitination.

Figure 1.. NPR1 Is Required for Cell Survival and Accumulation of Ubiquitinated Proteins

(A-C) Half leaves (left side) of Col-0, npr1–2 and sid2–2 plants were infiltrated with MgSO₄ (mock) or Psm ES4326/AvrRpt2 (Avr). At 2 dpi, the adjacent leaf halves were infiltrated with the same pathogen. Cell death was assessed by tissue collapse at 1 dpi (A) and conductivity assay (B). Growth of Psm ES4326/AvrRpt2 (Tet^r) was measured in the adjacent leaf halves after the first inoculation with Psm ES4326/AvrRpm1 (Avr; Kan^r) (C). Data are presented as mean ± SD (B), and mean ± 95% confidence intervals (C). (D) Half leaves of plants expressing dex:AvrRpt2 in Col-0, npr1–2, and rps2 were inoculated as in (B). At 2 dpi, the adjacent leaf halves were infiltrated with 25 μM dexamethasone (dex) and cell death was assessed as in (B). Data are presented as mean ± SD. (E-G) Col-0, rps2 and npr1–2 plants were treated with water (mock) or 1 mM SA 24 hr before inoculation with Psm ES4326/AvrRpt2. Cell death was assessed by trypan blue staining (E) and conductivity assay (F). Bacterial growth was measured at 1 dpi (G). Data are presented as mean ± SD (F), and mean ± 95% confidence intervals (G). (H) Plants expressing dex:AvrRpt2 in Col-0, npr1–2 and rps2 were treated as in (E-G) before treatment with 25 μM dexamethasone (dex). Cell death was assessed as in (B). Data are presented as mean ± SD. (I-J) Ws-2 (I) or Col-0 and npr1–2 (J), were treated as in (E-G) before inoculation with Pf Pf0–1/AvrRps4 or Pf Pf0–1/AvrRps4^KRVY-AAAA. Cell death was assessed as in (B). Data are presented as mean ± SD. (K) Col-0 and npr1–2 plants were treated with SA for 6 hr. Total proteins were probed with α-Ub and α-NPR1. See also Figure S1.

Figure 2.. NPR1 Accumulates in the Cytoplasm and Forms Cysteine-Dependent Condensates

(A) Subcellular fractionation of Col-0 6 hr after SA treatment. Cytoplasmic (C), nuclear (N) and combined (C+N) fractions were probed with α-NPR1 and α-Ub antibodies. Band intensities (b.i.) are shown as percentages of the combined C+N levels. The middle blot (a longer exposure of the upper blot) shows the higher MW NPR1 bands. (B) Localization of NPR1-GFP and sim3-GFP in transient expression in Col-0 seedlings treated with SA for 2 hr. Scale bar = 20 μm. (C-E) Single-cell time-lapse imaging of NPR1-GFP condensation induced by 5 mM SA. Imaging was started 40 min after SA induction (time 0) for a duration of 2 hr. Images from selected time points (C), fluorescence intensity per body (D) and number of bodies (E) are shown. Scale bar = 20 μm. Data are presented as mean ± SE (D). (F) Predicted RDR regions of NPR1. Values represent differential IDR score. Dots indicate position of cysteine residues (red), and known point mutations and their alleles (black). (G and H) Localization of NPR1-GFP and rdr3-GFP after treatment with 5 mM SA for 2 hr (G). Scale bar = 20 μm. Insets show enlarged nuclei at lower exposure. (H) Total fluorescence intensity of bodies from SA-treated samples. Data are presented as mean ± SE. (I) Transactivation of the PR1 promoter by NPR1 and rdr3 after treatment with 2 mM SA for 24 hr. Values represent promoter activity plotted relative to free HA. Data are presented as mean ± SD. See also Figures S1, S2 and S3; Video S1; Tables S1 and S2.

Figure 3.. NPR1 Condensates Are Enriched with Stress Proteins

(A) Functional categorization of sim3-GFP interactome (SINC components). The sizes of functional groups (left) and the number of proteins at their intersection (right) are shown. (B) Representative SINC components from four major functional groups. Black dots indicate confirmed localization in cytoplasmic NPR1 condensates. (C) Co-localization of sim3-GFP with free mCherry or mCherry-fused SINC components, EDS1, BCS1, GSTU19 after treatment with 1 mM SA for 5 hr. Scale bar = 20 μm. (D-F) Col-0 and npr1–2 plants were treated with water (mock) or 1 mM SA 24 hr before inducing cell death with indicated stresses. Cell death was assessed by conductivity assay. Data are presented as mean ± SD. See also Figure S4; Table S3.

Figure 4.. NPR1 Recruits CUL3 to Its Cytoplasmic Condensates

(A) Interaction of Myc-CUL3 with HA-fused NPR1 or its variants in N. benthamiana treated with water (−) or 1 mM SA (+) for 5 hr. (B) Interaction of endogenous CUL3 with GFP-fused NPR1, sim3 or ΔBTB in transgenic Arabidopsis treated with 1 mM SA for 24 hr. (C) Interaction of Myc-CUL3 with GST-fused NPR1 or its variants in E. coli. (D) Inhibition of CUL3-BTB interaction by the CTD in N. benthamiana. Myc-CUL3, BTB-HA and CTD-GFP, were co-expressed at 1:1:0 (0), 1:1:0.25 (1/4), 1:1:0.5 (1/2) and 1:1:1 (1) ratios, respectively, before co-IP. (E and F) Interaction of CUL3 with NPR1, sim3 or S55/59D in the BiFC assay after treatment with 1 mM SA for 5 hr. Scale bar = 10 μm. BiFC signal intensity was quantified and values are plotted relative to CUL3/NPR1 (F). Data are presented as mean ± SE. (G) Localization of NPR1-GFP, sim3-GFP and S55/59D-GFP in N. benthamiana treated with water (mock) or 5 mM SA for 2 hr. Scale bar = 10 μm. (H) Interaction of Myc-CUL3 with HA-fused rdr mutants in N. benthamiana after treatment with 1 mM SA for 5 hr. (I-L) Localization of GFP-CUL3 in NbNPR1-silenced N. benthamiana after treatment with 5 mM SA for 2 hr (I). Scale bar = 20 μm. Total fluorescence intensity per body (J) and number of bodies per cell (K) were quantified relative to the E.V. control. Data are presented as mean ± SE. Expression levels were verified by GFP-CUL3 immunoblotting (L). See also Figures S5 and S6.

Figure 5.. NPR1-CUL3 Cytoplasmic Condensates Are Active Ubiquitination Complexes

(A) Co-localization of CUL3/sim3 BiFC bodies with mCherry-fused organelle and protein body markers after treatment with 1 mM SA for 5 hr. Scale bar = 20 μm. Representative co-localizations with Ubiquitin and NBR1 are shown (left). (B) Co-localization of sim3-GFP with mCherry-NBR1 in the presence of Myc-CUL3 or free Myc after treatment with 1 mM SA for 5 hr. Scale bar = 20 μm. (C) Interaction of Myc-CUL3 with HA-fused NPR1 or sim3 in N. benthamiana treated with SA for 5 hr (upper panels). Total ubiquitination was tested in the “input” fractions (lower panels). (D) Total ubiquitination in N. benthamiana expressing Myc-CUL3 or Myc-CUL3^ΔRBX1, sim3-GFP or GFP, and V5-Ub, after treatment with 1 mM SA for 5 hr. (E) Total ubiquitination in the NbCUL3-silenced N. benthamiana or in the E.V. control expressing sim3-GFP and V5-Ub, after treatment with 1 mM SA for 5 hr. See also Figure S6.

Figure 6.. Proteins Localized in SA-Induced NPR1 Condensates Are Targeted for Degradation by NPR1-CRL3

(A) Interaction of EDS1-mCherry with HA-fused NPR1 or sim3 in N. benthamiana treated with 1 mM SA for 5 hr. (B) Interaction of FLAG-EDS1 with GST or GST-NPR1 in E. coli. (C) Co-localization of NPR1/CUL3 or sim3/CUL3 BiFC bodies with EDS1-mCherry after treatment with 1 mM SA for 5 hr. Values quantified from free mCherry samples (not shown) are included in the quantification. Scale bar = 20 μm. (D) Stability of EDS1 in Col-0 or npr1–2 mutant. Seedlings were incubated in water (−) or 1 mM SA (+) for 4 hr, with (+) or without (−) subsequent addition of 100 μM cycloheximide (CHX). After 16 hours of co-incubation, total protein was probed with α-EDS1 and α-NPR1 antibodies. (E) Ubiquitination of EDS1 in Col-0, npr1–2 and eds1–2 mutants treated with water (Mock) or 1 mM SA for 6 hr. (F) Co-localization of sim3/CUL3 BiFC bodies with mCherry-fused WRKY54 or WRKY70 after treatment with 1 mM SA for 5 hr. Scale bar = 20 μm. (G) Stability of WRKY70-GFP in Col-0 or npr1–2 mutant. Seedlings were treated with 1 mM SA or 50 μM MG132 or in combination for 24 hr, and total protein was probed with α-GFP, α-NPR1 and α-TUB antibodies. (H) Ubiquitination of WRKY70-GFP in Col-0 or npr1–2 mutant treated with 1 mM SA for 24 h before immunoprecipitation of WRKY70-GFP under denaturing conditions (dn). (I) NPR1- and sim3-dependent ubiquitination of WRKY70 by the NPR1-CUL3 E3 ubiquitin ligase reconstituted in E. coli. FLAG-WRKY70 was immunoprecipitated under denaturing conditions (dn). See also Figure S7.

Figure 7.. NPR1 Promotes Survival during ETI by Targeting WRKY54 and WRKY70

(A-C) Col-0, rps2, npr1–2, wrky54 wrky70 (w54w70), and npr1 wrky54 wrky70 (n1w54w70) plants were treated with water (mock) or 1 mM SA 24 hr before inoculation with Psm ES4326/AvrRpt2. Cell death was assessed by trypan blue staining (A) and conductivity assay (B). Bacterial growth was measured at 1 dpi (C). Data are presented as mean ± SD (B), and mean ± 95% confidence intervals (C). (D) Proposed model for NPR1 function in promoting cell survival during ETI. See text for description. “P”, phosphorylation at S55/59; “S”, SUMOylation; “U”, ubiquitination; “W”, WRKY TFs; “NPR1^C”, NPR1 condensate.