Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity

Abstract

Systemic acquired resistance (SAR) is a broad-spectrum plant immune response involving profound transcriptional changes that are regulated by the coactivator NPR1. Nuclear translocation of NPR1 is a critical regulatory step, but how the protein is regulated in the nucleus is unknown. Here, we show that turnover of nuclear NPR1 protein plays an important role in modulating transcription of its target genes. In the absence of pathogen challenge, NPR1 is continuously cleared from the nucleus by the proteasome, which restricts its coactivator activity to prevent untimely activation of SAR. Surprisingly, inducers of SAR promote NPR1 phosphorylation at residues Ser11/Ser15, and then facilitate its recruitment to a Cullin3-based ubiquitin ligase. Turnover of phosphorylated NPR1 is required for full induction of target genes and establishment of SAR. These in vivo data demonstrate dual roles for coactivator turnover in both preventing and stimulating gene transcription to regulate plant immunity.

Figure 1. Proteasome-mediated degradation of NPR1 monomer in the nucleus

(A) Total protein was extracted from wild-type Col-0 and 35S::NPR1-GFP (in npr1-1) plants in a buffer supporting proteolytic activity. Extracts were untreated (−) or treated with either 2% DMSO (vehicle), 40 μM MG132, 40 μM MG115, 4 mM PMSF, or 40 μM Leupeptin. After 2 hours, proteins were analyzed by reducing SDS-PAGE and Western blotting using anti-NPR1 and anti-GFP antibodies. Detection of a constitutively expressed calcium-sensing receptor (CAS) confirmed equal loading. (B) Wild-type Col-0 plants were treated for 24 hours with either water (control), 0.5 mM SA, 100 μM MG115, or 100 μM MG132. Total protein was separated by SDS-PAGE in the presence or absence of DTT (50 mM) and analyzed by Western blotting using an anti-NPR1 antibody. Detection of a constitutively expressed calcium-sensing receptor (CAS) confirmed equal loading. Molecular weight standards are indicated. O, oligomer; M, monomer; T, total. (C) 35S::NPR1-GFP plants were treated as in (B). Leaf tissue was examined using fluorescence microscopy. (D) 35S::NPR1-GFP plants were treated and analyzed as in (B), except an anti-GFP antibody was used. Simultaneously, mRNA was extracted and analyzed by Northern blotting using gene-specific probes against PR-1 and constitutively expressed Ubiquitin (UBQ). (E) Total protein was extracted from 35S::NPR1-GFP plants in a buffer supporting proteolytic activity. Extracts were pretreated with (+) or without (−) 5 mM DTT and incubated at room temperature for the time points indicated. Total NPR1-GFP protein was analyzed by reducing SDS-PAGE and Western blotting using an anti-GFP antibody. (F) 35S::NPR1-GFP (in npr1-1) and 35S::npr1-nls-GFP (in npr1-1) plants were treated with (+) or without (−) 100 μM cycloheximide (CHX) for 24 hours. Total protein was analyzed by reducing SDS-PAGE and Western blotting using an anti-GFP antibody. Detection of constitutively expressed Tubulin (TUB) confirmed equal loading. (G) Seedlings of 35S::NPR1-GR (in npr1-3) plants were treated for with (+) or without (−) 100 μM CHX and 5 μM dexamethasone (DEX) for 24 hours. Total protein was analyzed by reducing SDS-PAGE and Western blotting using an anti-GR antibody. A non-specific band (*) confirmed equal loading.

Figure 2. NPR1 is constitutively targeted for degradation by a CUL3-based ubiquitin ligase

(A) 35S::NPR1-GFP (in npr1-1) plants were treated with (+) or without (−) MG115 (100 μM) for 24 hours. Protein extracts were immunoprecipitated (IP) with an antibody against CUL3A. Total and immunoprecipitated proteins were analyzed by Western blotting using anti-GFP and anti-CUL3A antibodies. (B) Wild-type (WT) and cul3a cul3b plants in the absence (−) or presence of the 35S::NPR1-GFP transgene (NPR1-GFP) were treated with (+) or without (−) 100 μM CHX for 24 hours. Total protein was analyzed by reducing SDS-PAGE and Western blotting using an antibody against the endogenous NPR1 or GFP. Detection of a constitutively expressed calcium-sensing receptor (CAS) confirmed equal loading. (C) 35S::NPR1-GFP was expressed in wild-type (WT) and cop9 plants. Plants were treated with 100 μM CHX for the indicated hours. Total protein was analyzed by reducing SDS-PAGE and Western blotting using an anti-GFP antibody. Detection of constitutively expressed Tubulin (TUB) confirmed equal loading. (D) mRNA was extracted from the wild-type (WT), npr1, cul3a cul3b (cul3) double, and cul3a cul3b npr1 (c3n1) triple mutant plants. The expression of PR-1, PR-2, and PR-5 was analyzed using qPCR and normalized against constitutively expressed UBQ. Error bars represent SD (n = 3).

Figure 3. SA-induced transcription of NPR1 target genes requires the proteasome

(A) 35S::NPR1-GFP (in npr1-1) plants were treated with (+) or without (−) 0.5 mM SA for 24 hours. Protein extracts were immunoprecipitated (IP) using antibodies against CUL3, COP9, CSN4, and CSN5. Total and immunoprecipitated proteins were analyzed by Western blotting using an anti-GFP antibody. (B) 35S::NPR1-GFP (in npr1-2) plants were treated with (+) or without (−) 0.5 mM SA and 100 μM CHX for 24 hours. Leaf tissue was examined by fluorescence microscopy. (C) 35S::NPR1-GFP (in npr1-2) plants were treated as in (B). Total protein was analyzed by reducing SDS-PAGE and Western blotting using an anti-GFP antibody. Detection of a constitutively expressed calcium-sensing receptor (CAS) confirmed equal loading. (D) 35S::NPR1-GFP (in npr1-2) and npr1-2 plants were treated with (+) or without (−) 0.5 mM SA and 100 μM MG115 for 28 hours. The expression of three WRKY genes was analyzed using qPCR and normalized with constitutively expressed UBQ. Error bars represent SD (n = 3).

Figure 4. SA-induced transcription of NPR1 target genes requires CUL3

(A) Wild-type (WT) and cul3a cul3b (cul3) plants were treated with 0.5 mM SA for 16 hours. Subsequently, plants were SA-treated for an additional 4 hours in the absence (−) or presence (+) of 100 μM CHX. Total protein was analyzed by reducing SDS-PAGE and Western blotting using an anti-NPR1 antibody. Detection of a constitutively expressed calcium-sensing receptor (CAS) confirmed equal loading. (B) Wild-type (WT) and cul3a cul3b (cul3) plants carrying the 35S::NPR1-GFP transgene were treated with 0.5 mM SA and 100 μM MG115 for 8 hours. Protein extracts were immunoprecipitated (IP) using an anti-GFP antibody. Immunoprecipitated proteins were analyzed by Western blotting using anti-Ubiquitin (Ub) and anti-GFP antibodies. Molecular weight standards are indicated. (C) Wild-type, cul3a cul3b (cul3), and npr1-2 plants were treated with (+) or without (−) 0.5 mM SA and 100 μM MG115 for 28 hours. The expression of three WRKY genes was analyzed using qPCR and normalized with constitutively expressed UBQ. Error bars represent SD (n = 3). (D) Induction of SAR against Psm ES4326 in wrky18 (w18) plants. Cfu, colony-forming units. Error bars represent 95% confidence limits (n = 8). Asterisks indicate statistically significant differences between the control and SAR treatment in each genotype (Tukey–Kramer ANOVA test; α = 0.05, n = 8). See Supplemental Data for details. (E) Induction of SAR against Psm ES4326 disease symptoms and growth in wrky38 wrky62 (w38, 62) plants was carried out as in (D). (F) Induction of SAR against Psm ES4326 growth in cul3a cul3b plants was carried out as in (D).

Figure 5. NPR1 is phosphorylated at Ser11 and Ser15 in the nucleus

(A) 35S::NPR1-GFP (in npr1-1) plants were treated with (+) or without (−) 0.5 mM SA for 24 hours. Total protein was loaded onto a column that specifically binds phosphoproteins. Bound proteins were eluted and the amount of NPR1-GFP determined by Western blotting with an anti-GFP antibody. (B) 35S::NPR1-GFP plants were treated as in (A). Total protein was extracted and immunoprecipitated (IP) with an anti-GFP antibody. Immunoprecipitated proteins were analyzed by Western blotting using an antibody that recognizes phosphorylated serine and threonine residues (α-pS/pT) as well as an anti-GFP antibody. (C) Sequence alignment of NPR1 proteins from different plant species. The IκB-like phosphodegron motif (DSxxxS; x, any amino acid) is indicated. Le, Lycopersicum esculentum; Nt, Nicotiana tabacum; Bv, Beta vulgaris; Os, Oryza sativa; At, Arabidopsis thaliana. (D) Wild-type Col-0 plants were treated with (+) or without (−) 0.5 mM SA for 24 hours. Total protein was analyzed by reducing SDS-PAGE and Western blotting. NPR1 phosphorylation was specifically detected with an antibody against phosphorylated Ser11 and Ser15 residues (α-pSer11/15). Equal loading was verified using an anti-NPR1 and anti-Tubulin (TUB) antibody. (E) 35S::NPR1-GFP (in npr1-1) and 35S::npr1-nls-GFP (in npr1-1) and npr1-1 plants were treated with (+) or without (−) 0.5 mM SA for 24 hours. Total protein was analyzed by reducing SDS-PAGE and Western blotting using anti-pSer11/15 and anti-GFP antibodies. A non-specific band (*) indicated equal loading.

Figure 6. Phosphorylation stimulates NPR1 turnover and is required for SAR

(A) Total protein was extracted from 35S::NPR1-GFP (in npr1-2), 35S::npr1S11/15A-GFP (in npr1-2), and 35S::npr1S11/15D-GFP (in npr1-2) plants in a buffer supporting proteasome activity. Extracts were incubated at room temperature for the time points indicated. Proteins were analyzed by reducing SDS-PAGE and Western blotting using an anti-GFP antibody. (B) 35S::NPR1-GFP, 35S::npr1S11/15A-GFP, and 35S::npr1S11/15D-GFP plants were treated with a combination of 0.5 mM SA and 100 μM MG115. Protein extracts were immunoprecipitated (IP) with an anti-CUL3 antibody. Immunoprecipitated proteins were analyzed by Western blotting using an anti-GFP antibody. Molecular weight standards are indicated. Arrow indicates NPR1; asterisk indicates a cross-reacting IgG chain. (C) 35S::NPR1-GFP and 35S::npr1S11/15A-GFP plants were treated as in (B). Protein extracts were immunoprecipitated (IP) with an anti-GFP antibody. Immunoprecipitated proteins were analyzed by Western blotting using anti-Ubiquitin and anti-GFP antibodies. Molecular weight standards are indicated. (D) The leaf-halves of 35S::NPR1-GFP and 35S::npr1S11/15A-GFP plants were inoculated with avirulent Pst DC3000/avrRpt2 (OD₆₀₀=0.02). At the indicated time points protein was extracted from the uninoculated leaf halves and subjected to reducing SDS-PAGE and Western blot analysis using an anti-GFP antibody. A non-specific band (*) indicated equal loading. (E) 35S::NPR1-GFP, 35S::npr1S11/15A-GFP, and npr1-2 plants were treated as described in (D). mRNA was extracted from the uninoculated halves of the inoculated leaves and analyzed for the expression of WRKY18, WRKY38, WRKY62, and PR-1 using qPCR. Expression was normalized against constitutively expressed UBQ. Error bars represent SD (n = 3). (F) Induction of SAR against Psm ES4326 in 35S::NPR1-GFP, 35S::npr1S11/15A-GFP, 35S::npr1S11/15D-GFP, and npr1-2 plants. Cfu, colony-forming units. Error bars represent 95% confidence limits (n = 8). Asterisks indicate statistically significant differences compared with uninduced 35S::NPR1-GFP plants (Tukey–Kramer ANOVA test; α = 0.05, n = 8).

Figure 7. Working model for the dual role of the proteasome in preventing and stimulating NPR1 target gene transcription

In uninduced cells (left panel), NPR1 monomer constitutively translocates, at a low rate (dashed lines), to the nucleus where it is targeted to the proteasome by CUL3-based E3 ligase-mediated ubiquitinylation (Ub). This prevents the activation of NPR1 target genes. In SAR-induced cells (right panel), a large amount of NPR1 monomer translocates to the nucleus where it interacts with transcription factors (TF) to initiate target gene transcription by recruiting the transcription initiation complex (I.C.) and RNA polymerase II (PolII). As a consequence NPR1 may be phosphorylated (P) by a kinase (Kin) that is associated with the I.C. and PolII. A CUL3-based ligase with high affinity for phosphorylated NPR1, possibly distinct from the one involved in turnover of non-phosphorylated NPR1 in uninduced cells, rapidly ubiquitinylates and targets NPR1 for degradation by the proteasome. Clearance of “exhausted” phosphorylated NPR1 from the target gene promoter allows “fresh” non-phosphorylated NPR1 to reinitiate the transcription cycle, thereby directly linking the rate of NPR1 degradation to the amplitude of target gene transcription.