Structural basis of salicylic acid perception by Arabidopsis NPR proteins

Abstract

Salicylic acid (SA) is a plant hormone that is critical for resistance to pathogens^1-3. The NPR proteins have previously been identified as SA receptors^4-10, although how they perceive SA and coordinate hormonal signalling remain unknown. Here we report the mapping of the SA-binding core of Arabidopsis thaliana NPR4 and its ligand-bound crystal structure. The SA-binding core domain of NPR4 refolded with SA adopts an α-helical fold that completely buries SA in its hydrophobic core. The lack of a ligand-entry pathway suggests that SA binding involves a major conformational remodelling of the SA-binding core of NPR4, which we validated using hydrogen-deuterium-exchange mass spectrometry analysis of the full-length protein and through SA-induced disruption of interactions between NPR1 and NPR4. We show that, despite the two proteins sharing nearly identical hormone-binding residues, NPR1 displays minimal SA-binding activity compared to NPR4. We further identify two surface residues of the SA-binding core, the mutation of which can alter the SA-binding ability of NPR4 and its interaction with NPR1. We also demonstrate that expressing a variant of NPR4 that is hypersensitive to SA could enhance SA-mediated basal immunity without compromising effector-triggered immunity, because the ability of this variant to re-associate with NPR1 at high levels of SA remains intact. By revealing the structural mechanisms of SA perception by NPR proteins, our work paves the way for future investigation of the specific roles of these proteins in SA signalling and their potential for engineering plant immunity.

Extended Data Figure 1.. Mapping and refolding of the NPR4-SBC.

Related to Figs. 1 and 2. a, Domain arrangements of Arabidopsis thaliana NPR4 and different constructs used for mapping NPR4-SBC. b-f, Comparison of trypsin digestion profiles of truncated NPR4 proteins with or without 1 mM salicylic acid (SA) or 3-OH benzoic acid (BA). Negative controls of limited proteolytic digestion of NPR4 were conducted with bovine serum albumin (BSA) (b), SA-insensitive NPR4-R419Q mutant associated with npr4–4D (c), and NPR4–1-391 fragment (f). g, h, SA-dependent refolding of NPR4-SBC polypeptide affects its solubility. BA, benzoic acid, an inactive analogue of SA; Sup., supernatant; M, molecular weight marker. i, Superdex 75 size exclusion chromatography elution profile of the NPR4-SBC fragment refolded in the presence of SA. Excess SA was eluted after one column volume due to a weak interaction with the resin. The inset shows the final purified NPR4-SBC fragment analysed by SDS-PAGE with Coomassie staining. Experiments in (b-i) were repeated three times or more with similar results.

Extended Data Figure 2.. Deuterium exchange profiles of selected NPR4 peptides.

Related to Fig. 2. a, Deuterium uptake plots of representative peptides of NPR4-SBC derived from samples with (red) or without (blue) the presence of 0.1 mM SA. The SA-insensitive deuterium uptake plots of a BTB domain N-terminal peptide are shown on the left as a representative SA-insensitive region. n=3 independent samples. Error bars representing standard deviation of the mean (centre value) are shown, but often too small to be seen. The peptide sequences, amino acid numbers, and the structural domain they belong to are indicated on top of the plots. b, The SA-insensitive deuterium uptake plots of three different peptides containing residues that belong to the proposed EAR motif (underlined). c, The SA-free HDX profile is mapped on the NPR4-SBC crystal structure for the 4 time points with a colour ramp scheme indicative of percentage of exchange. Regions coloured in grey were outside of the peptide coverage.

Extended Data Figure 3.. Sequence alignment of the SBC regions in NPR proteins from several plant species and details of the SA-binding pocket and activity.

Related to Fig. 2. a, Structure-based sequence alignment of the SA binding core (SBC) regions of NPR4 and NPR1 orthologues. The secondary structure diagram of NPR4-SBC is shown above the sequences. Regions with no regular secondary structure are shown by lines, and α helices are represented by cylinders. The dashed lines indicate two disordered loops that are not resolved in the structure. Strictly conserved residues are coloured in blue. The rest of the residues are coloured with black (87.5%), brown (75%), or red (<75%) based on their degrees of conservation. The residues directly involved in SA binding are highlighted with asterisks. The putative EAR motif is labelled and indicated by a black bar. Six surface residues selected for mutagenesis analysis are labelled. At (Arabidopsis thaliana; NPR1-AT1G64280, NPR3-AT5G45110, and NPR4-AT4G19660); Os (Oriza sativa NH1-Os01g09800, NH2-Os01g56200, and NH3-Os03g46440); Nb (Nicotiana benthamiana NPR1- LOC107831756); Bn, (Brassica napa NPR1- LOC106389246). b, A close-up stereo view of the NPR4-SBC SA-binding pocket with the omit map electron density shown together with the residues in the stick model. SA is coloured in yellow and red and situated in the centre. Three selected SA-contacting residues in close proximity to the SA carboxyl group are indicated. c, Ligplot of the hydrophobic and polar interactions between SA and NPR4-SBC residues. d, A semi-transparent view of the SA-binding pocket with the SA analogue benzothiadiazole (BTH) (magenta-blue-red sticks) modelled onto SA (yellow-red sticks) situated in the centre and indicated by arrows. The view is related to the NPR4-SBC internal cavity shown in Fig. 2c by 180° vertical rotation. Ala434 is shown in yellow stick and indicated as A434. The internal cavity and surrounding surfaces of NPR4-SBC are shown in green surface representation. e, SA binding by wild type (WT) and R428A mutant of NPR3 as determined with radio-labelled ligand binding assay with 100 nM ³H-SA. n = 6 independent samples. Error bars indicate standard deviation from the mean (centre value).

Extended Data Figure 4.. Sequence comparison of Arabidopsis NPRs and characterization of His-MBP-NPR1.

Related to Fig. 3. a, Neighbour-joining tree of NPR C-terminal (CT) domains and pairwise comparisons of amino acid sequence identity within the CT and SA-binding core (SBC) regions. Bootstrap values are noted for the branching of each node. Numbers 1–6 correspond to the six Arabidopsis NPRs. NOTE: Despite featuring similar CT regions, NPR5 and NPR6 do not contain a regular SBC, reflected by the low sequence identity of their CT domains to that of NPR1–4. b, Size exclusion chromatography elution profile of His-MBP-AtNPR1, which was first purified by amylose affinity chromatography. The inset shows the final purified His-MBP-AtNPR1 protein analysed by SDS-PAGE with Coomassie staining. Experiments were repeated three times with similar results. c, Dose response curve of SA binding by NPR1. In the radio-labelled ligand binding assay, 5 μg of His-MBP-NPR1 protein was incubated with ³H-SA at different concentrations. Three replicates in a single experiment were used to calculate the kD of SA binding to NPR1. n=3 independent samples. Error bars represent the standard errors of the mean (centre value). cpm, counts per minute. c, Diagrams of NPR1 and NPR4 domain boundaries that are relevant to Fig. 3d.

Extended Data Figure 5.. NPR amino acid sequence homology in angiosperms.

Related to Fig. 3. a, Neighbour-joining tree depicting the divergence of the C-terminal (CT) domains of Arabidopsis thaliana NPRs and Oryza sativa NH proteins (highlighted), as well as relationship with other NPR-like proteins in angiosperms. Black, out groups; blue, NPR1/NPR2 clade; and orange, NPR3/NPR4 clade. b, c, Amino acid sequence alignments of NPR CTs indicating the amino acid conservation (black shade) at the position (arrow) of NPR4 residues R419 and F426 (b), as well as T459 and the putative EAR motif (c). The degree of conservation, alignment quality, and conservation strength are indicated by the histograms below the sequences.

Extended Data Figure 6.. NPR4 point mutant expression and their differential phenotypic effects.

Related to Fig. 4. a, Western blot analysis of transgenic npr3 npr4 (npr3/4) seedlings expressing similar amounts of the NPR4-GFP variants 24 hr after treatment with 0.1 mM SA. An antibody against GFP (α-GFP) was used. *, non-specific band. Experiments were repeated two times with similar results. b, Western blot depicting cell-free protein degradation assays comparing the rate of endogenous NPR1 degradation in protein extracts from (a) and quantifications of the data are shown in Fig. 4a. Arrows, endogenous NPR1; MG115/132, proteasome inhibitors. The ratios listed below each sample indicate NPR1 levels compared to 0 min for the degradation assay or 30 min for samples containing MG115/132. An antibody against NPR1 (α-NPR1) was used. Experiments were repeated three times with similar results. c, d, in planta protein degradation assays comparing the rate of endogenous NPR1 degradation in seedlings pre-treated with 0.1 mM SA for 24 hr. NPR1 was detected using an α-NPR1 antibody (c) and the relative band intensities were quantified (d). n=3 independent biological samples. Error bars indicate standard deviation from the mean (centre values). e, Western blot analysis of transgenic npr3/4 seedlings expressing NPR4-GFP or npr4^F426L-GFP after a 24-hr treatment with 0.1 mM SA. L1, 2, 6, 7, independent transgenic lines; TPE, total protein extract; CBB, Coomassie brilliant blue. An antibody against GFP (α-GFP) was used. *, non-specific band. f, Fold change of PR1 expression in seedlings from (e) 24 hr after 0.1 mM SA treatments. The data were normalized to UBQ5 expression, error bars indicate standard deviation from the mean (n = 3). Statistical significance was determined by 1-way ANOVA on log-transformed data followed by Tukey’s multiple comparison correction, letters indicate statistical significance, p < 0.05. g, Western blot analysis of mature leaves from transgenic npr3/4 plants expressing NPR4-GFP, npr4^F426L-GFP or npr4^{F426L T459G}-GFP after a 6-hr treatment with 0.5 mM SA spray. L8, 11, independent transgenic lines. An antibody against GFP (α-GFP) was used. *, non-specific band. h, Fold change of PR1 expression in leaves from (g) 6 hr after mock or 0.5 mM SA spray. The data were normalized to UBQ5 expression. n = 5 biologically independent samples. Error bars indicate standard deviation from the mean (centre values). Statistical significance was determined by 1-way ANOVA on log-transformed data followed by Tukey’s multiple comparison correction, letters indicate statistical significance, p < 0.05. i, j, SA protection against Psm ES4326 infection. Images of disease symptom development (i) and bacterial growth in infected leaves (j) were recorded 3 days after inoculation at OD_600nm = 0.001. Light grey bars, mock; dark grey bars, 0.1 mM SA. Colony forming units (cfu) were determined for three experiments and combined using linear mixed effect model (lme4) with experiment as random effects. n = three experiments each with eight biological repeats / genotype and treatment. Error bars indicate standard deviation from the mean (centre value). Statistical significance was determined by 2-way ANOVA on log-transformed data. ns p = 0.6, * p = 0.03; ** p = 0.008; *** p = 0.0004. Experiments in (i) were repeated three times with similar results. k, Relative band intensities were quantified after in planta protein degradation assays comparing the rate of endogenous NPR1 degradation in seedlings pre-treated with 1 mM SA for 24 hr as in (c, d). n = 5 biological independent samples. Error bars indicate standard error from the mean (centre values).

Figure 1.. Mapping the NPR4 salicylic acid (SA)-binding core domain.

a, Domain arrangements of Arabidopsis thaliana NPR1, NPR3 and NPR4. BTB, bric-a-brac, tramtrack, and broad-complex domain; ANK, ankyrin-repeat domain; CT, C terminal domain; SBC, SA binding core. b, Trypsin digestion profiles of full-length (FL) NPR4 with or without 1 mM SA. c, Mirror plots showing the percentage deuterium exchange after 3 s (yellow), 1 min (red), 30 min (blue), and 20 hr (black) for each observable peptide at the midpoint of their primary sequence for the apo (top) and SA-bound (bottom) NPR4 with SBC region highlighted in light blue. d, Net difference in percentage deuterium exchange at each time point plotted for each observable peptide. Regions with slowed exchange upon SA binding fall in the positive y-axis and are highlighted in yellow. Broken lines indicate gaps in the sequence coverage. Blue bars and arrows indicate SBC secondary structure elements. e, f, SA binding by NPR3, NPR4, and NPR3-NPR4 chimeric proteins in the presence of 200 nM (e) and 100 nM (f) ³H-SA. Horizontal blue lines above the bar graph in (f) indicate the NPR4 regions swapped into NPR3 in comparison to NPR4-SBC. NPR4 regions: 4CT (345–574); 4CT1 (345–394), 4CT2 (369–428), 4CT3 (395–444), 4CT4 (445–494), 4CT5 (469–518) and 4CT6 (495–574). n=3 independent samples.

Figure 2.. Crystal structure of the SA-bound NPR4 SBC.

a, Overall views of the SA-bound NPR4-SBC structure with NPR4 in green, SA in space-filling model, and secondary structural elements labelled. Dashed lines represent disordered regions. b, Close-up views of NPR4-SBC bound to SA (yellow-red stick) with its positive Fo-Fc electron density contoured at 4σ (purple mesh). SA-contacting residues are shown in stick model with polar interactions represented by yellow dashes. c, A close-up view of the NPR4-SBC internal cavity (orange) with SA completely buried inside. SA-contacting NPR4 residues are shown in stick (green) with their atoms forming the internal cavity surface coloured in orange. d, Structural mapping of SA-induced HDX differences of NPR4-SBC sequence regions at the 30 min time point. Regions in blue are protected upon SA (yellow-red sticks) binding. e, SA-binding activities of wild type and mutant NPR4 at 500 nM ³H-SA. GST; glutathione S-transferase; cpm, counts per minute. n=3 independent samples. f, In vitro pull-down of MBP-NPR1-StrepII by GST-NPR4 under two different SA concentrations. Protein interactions were assessed by SDS-PAGE followed by Coomassie staining. g, h, AlphaScreen competition and titration assays assessing the NPR1-NPR4 binding affinity with IC₅₀ value indicated (g) and the potency of SA in disrupting NPR1-NPR4 binding with BA as a negative control (h). n=3 independent samples. i, In vitro pull-down of MBP-NPR1-StrepII with GST-NPR4 point mutants under 0.1 mM SA. Protein interactions were assessed by western blot using GST (α-GST) and StrepII (α-StrepII) antibodies. Numbers below each sample are ratios indicating NPR1 levels (+SA) compared to the controls (-SA).

Figure 3.. Differential SA binding by NPR1 and NPR4 proteins.

a, A comparison of SA binding site occupancy of His-MBP-NPR1 and GST-NPR4 at 800 nM ³H-SA. b, Co-purification of His-MBP-NIMIN-2 with His-NPR1-SBC or His-NPR1-SBC-R432Q co-expressed in E. coli, with His-MBP-NIMIN-2 alone as a control. Protein interactions were examined by SDS-PAGE followed by Coomassie staining. c, A comparison of SA-binding activities of MBP-NIMIN-2 alone, MBP-NIMIN-2-bound NPR1-SBC, and MBP-NIMIN-2-bound NPR1-SBC-R432Q, with a SA-only sample indicating the background signal. Inset, a dose-response curve for SA binding to MBP-NIMIN-2-bound NPR1-SBC. d, Design of NPR1-NPR4 chimeric proteins (A to G). The residues involved in the swaps are shown in Extended Data Fig. 4d. e, SA binding by NPR1, NPR4, and NPR1-NPR4 chimeric proteins illustrated in (d). f, Structural positions of NPR4 SBC surface residues (orange sticks) selected for mutational analysis. g, SA-binding activities of NPR4-SBC surface residue mutants at 100 nM ³H-SA. h, Co-immunoprecipitation (Co-IP) of NPR1-FLAG with NPR4-HA or npr4^F426L-HA with ratios indicating NPR1 levels (+SA, 0.1 mM) compared to the controls (-SA). i, In vitro pull-down of NPR1 with NPR4 or npr4^{F426L T459G} with the ratios of NPR1 compared to 0 mM SA listed below. j, Saturation binding analysis of GST-NPR4 (Kd: 49.9 ± 9.2 nM; h: 0.9; R²: 0.96) and GST-npr4^{F426L T459G} (Kd: 17.2 ± 2.5 nM; h: 1.3; R²: 0.98). n=3 independent samples for all statistical data.

Figure 4.. Functional impacts of two NPR4-SBC surface residue mutants.

a, Cell-free protein degradation assays comparing the degradation rates of endogenous NPR1 in npr3 npr4 (npr3/4) lines expressing similar amounts of NPR4, npr4^F426L, or npr4^{F426L T459G}. The samples were pretreated with 0.1 mM SA for 24 hr prior to extraction. The data represent the mean ± SEM (n = 3) of the relative band intensities quantified from western blots (Extended Data Fig. 6b). b, PR1 expression 24 hr after mock (−) or 0.1 mM SA (+) treatments. The data were normalized to UBQ5 expression. n=6 biologically independent samples. Statistical significance was determined by 1-way ANOVA on log-transformed data followed by Tukey’s multiple comparison correction, letters indicate statistical significance (p < 0.05). c, SA protection against Psm ES4326 infection. Bacterial growth in infected leaves was recorded 3 days after inoculation of mock (−)- or 0.1 mM SA (+)-treated plants. n=8 biologically independent samples. Statistical significance was determined by 2-way ANOVA on log-transformed data. ns p > 0.9971, **** p < 0.0001. d, e, Psm/AvrRpt2 infection assays. Ion leakage data were normalized to a total ion count recorded 28 hr post inoculation (hpi) (d), and colony forming units (cfu) in infected leaves were determined 24 hpi (e). n=3 (d) and n=8 (e) biologically independent samples. Statistical significance was determined by 1-way ANOVA on log-transformed data followed by Tukey’s multiple comparison correction, letters indicate statistical significance, p < 0.05. f, The NPR4^{F426L T459G} double mutation enhances SA perception and SA-induced resistance without compromising effector-triggered immunity (ETI). At higher SA concentrations, a second SA-binding site might exist to promote NPR4-NPR1 re-association.