Abstract
Salicylic acid (SA) is a plant hormone that is critical for resistance to pathogens1,2,3. The NPR proteins have previously been identified as SA receptors4,5,6,7,8,9,10, 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.
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Data availability
Uncropped gels and DNA sequencing results of all constructs are included in Supplementary Data. Structural coordinates and structural factors have been deposited in the Protein Data Bank under accession number 6WPG. All reagents are available from the corresponding authors upon request. Source data are provided with this paper.
Code availability
All software used in this study is publicly available. These include HKL-2000 v.720 package, GraphPad Prism 7.00 and 8, Phenix 1.14-3260, Protein Prospector v.5.23.1, Microsoft Excel 2018, Clustal Omega and R Studio v.1.3.1225 with a script listed in Reporting Summary.
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Acknowledgements
We thank the beamline staff at ALS for help with data collection; T. Sun and Y. Zhang for advice on optimizing the radiolabelled ligand binding assay for NPR1; members of the N.Z., X.D. and W. Xu laboratories for discussion and help; Z. Mou for an NPR1 antibody; Y. Tada for the wheat-germ-based in vitro translation system; M. Mwimba for help with statistical analysis; and C. Sponsel for assisting with nucleic acid preparations for quantitative PCR and screening transgenic lines. This work is supported by the Howard Hughes Medical Institute to N.Z. and X.D., the Gordon and Betty Moore Foundation (GBMF3032) and the National Institutes of Health (2R01-GM069594-09 and 5R35-GM118036) to X.D., Henan University to W.W., an NRSA fellowship from NIGMS (1F32-GM122250-01A1) to P.J.Z., and Duke University Department of Biology Hargitt postdoctoral research fellowships to J.W. and P.J.Z.
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Authors and Affiliations
Contributions
W.W., J.W., H.L., S.Y., X.D. and N.Z. conceived the project. H.L. designed, and H.L. and W.W. performed, the proteolytic mapping experiments. H.L. determined the SBC of NPR4. H.L., D.-V.R. and H.S. tested different constructs for protein purification and crystallization. H.L. and W.W. obtained the diffracting crystals. W.W. and N.Z. determined and analysed the structures. J.W. created NPR4 point mutations, carried out all in planta experiments, performed the majority of the domain swaps, and conducted most of the radiolabelled SA ligand binding assays, with the exception of the NPR1 SA-binding curves, which were conducted by T.R.H. M.G. and W.W. conducted the HDX experiments and analysed the data. P.J.Z. performed the NPR1 degradation assays and phylogenetic analysis of NPR proteins for selected land plants, L.L. contributed constructs for the NPR1–NPR4 domain swaps, S.Y. conducted the initial SA-binding assays for the NPR1–NPR4 domain swaps, W.W., J.W., P.J.Z., X.D. and N.Z. wrote the manuscript with help from all of the co-authors.
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Competing interests
N.Z. is a cofounder of Coho Therapeutics and a Scientific Advisory Board member of Kymera Therapeutics. X.D. is a cofounder of Upstream Biotechnology, Inc., and a Scientific Advisory Board member of Inari Agriculture.
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Extended data figures and tables
Extended Data Fig. 1 Mapping and refolding of the NPR4 SBC.
Related to Figs. 1, 2. a, Domain arrangements of A. 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 SA or 3-OH 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 owing 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 Fig. 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 s.d. (centre value) are shown, but are often too small to be seen. The peptide sequences, amino acid numbers and structural domain to which they belong are indicated on top of the plots. b, The SA-insensitive deuterium uptake plots of three peptides containing residues that belong to the proposed ethylene-responsive element-binding-factor-associated amphipathic repression (EAR) motif (underlined). c, The SA-free HDX profile is mapped on the NPR4 SBC crystal structure for the four time points, with a colour ramp scheme indicative of the percentage of exchange. Regions coloured in grey were outside of the peptide coverage.
Extended Data Fig. 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 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 (Oryza 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 and red sticks) modelled onto SA (yellow and 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 as a 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 NPR3 (WT) and NPR3(R428A) as determined with radiolabelled ligand binding assay with 100 nM 3H-SA. n = 6 independent samples. Error bars indicate s.d. (centre value).
Extended Data Fig. 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 SBC regions. Bootstrap values are noted for the branching of each node. Numbers 1–6 correspond to the six Arabidopsis NPRs. 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, NPR2, NPR3 and NPR4. b, Size-exclusion chromatography elution profile of His–MBP–NPR1, which was first purified by amylose affinity chromatography. The inset shows the final purified His–MBP–NPR1 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 radiolabelled 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 s.e.m. (centre value). cpm, counts per minute. d, Diagrams of NPR1 and NPR4 domain boundaries that are relevant to Fig. 3d.
Extended Data Fig. 5 NPR amino acid sequence homology in angiosperms.
Related to Fig. 3. a, Neighbour-joining tree depicting the divergence of the CT domains of A. thaliana NPRs and O. sativa NH proteins (highlighted), as well as relationship with other NPR-like proteins in angiosperms. Black, out groups; blue, NPR1 and NPR2 clade; and orange, NPR3 and NPR4 clade. b, c, Amino acid sequence alignments of NPR C terminal domains 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 Fig. 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 h after treatment with 0.1 mM SA. An antibody against GFP (anti-GFP) was used. Asterisk denotes a nonspecific 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; 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 (anti-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 pretreated with 0.1 mM SA for 24 h. NPR1 was detected using an anti-NPR1 antibody (c) and the relative band intensities were quantified (d). n = 3 independent biological samples. Error bars indicate s.d. (centre values). e, Western blot analysis of transgenic npr3 npr4 seedlings expressing NPR4–GFP or NPR4(F426L)–GFP after a 24-h treatment with 0.1 mM SA. L1, L2, L6 and L7, independent transgenic lines; TPE, total protein extract; CBB, Coomassie brilliant blue. An antibody against GFP (anti-GFP) was used. Asterisks denotes a non-specific band. f, Fold change of PR1 expression in seedlings from e 24 h after 0.1 mM SA treatments. The data are normalized to UBQ5 expression, error bars indicate s.d. (n = 3). Statistical significance was determined by one-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 npr4 plants expressing NPR4–GFP, NPR4(F426L)–GFP or NPR4(F426L/T459G)–GFP after a 6-h treatment with 0.5 mM SA spray. L8 and L11 denote independent transgenic lines. An antibody against GFP (anti-GFP) was used. Asterisk denotes a non-specific band. h, Fold change of PR1 expression in leaves from g 6 h after mock or 0.5 mM SA spray. The data are normalized to UBQ5 expression. n = 5 biologically independent samples. Error bars indicate s.d. (centre values). Statistical significance was determined by one-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 P. syringae pv. maculicola ES4326 infection. Images of the development of disease symptoms (i) and bacterial growth in infected leaves (j) were recorded 3 d after inoculation at OD600 nm = 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 = 3 experiments each with 8 biological repeats per genotype and treatment. Error bars indicate s.d. (centre value). Statistical significance was determined by two-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 pretreated with 1 mM SA for 24 h as in c, d. n = 5 biologically independent samples. Error bars indicate s.e.m. (centre values).
Supplementary information
Supplementary Information
Supplementary Discussion; Supplementary Method. This file contains a discussion section on the mapping of NPR4-SBC by limited proteolytic digestion of various NPR4 constructs and the characterization of full-length NPR4 by HDX-MS analysis. It also contains addition protein expression and purification methods for non-crystallographic assays.
Supplementary Data
Supplementary Data. This file contains the sequencing data for all constructs used in this study and uncropped gels and photos.
Supplementary Table
Supplementary Table. This file contains the raw data for HDX-MS analysis of SA-free and SA-bound NPR4.
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Wang, W., Withers, J., Li, H. et al. Structural basis of salicylic acid perception by Arabidopsis NPR proteins. Nature 586, 311–316 (2020). https://doi.org/10.1038/s41586-020-2596-y
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DOI: https://doi.org/10.1038/s41586-020-2596-y
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