Structural basis of NPR1 in activating plant immunity

Abstract

NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid^1-4. Despite the important role of NPR1 in plant immunity^5-7, understanding of its regulatory mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding domain. Crystal structure analysis reveals a unique zinc-finger motif in BTB for interacting with ankyrin repeats and mediating NPR1 oligomerization. We found that, after stimulation, salicylic-acid-induced folding and docking of the salicylic-acid-binding domain onto ankyrin repeats is required for the transcriptional cofactor activity of NPR1, providing a structural explanation for a direct role of salicylic acid in regulating NPR1-dependent gene expression. Moreover, our structure of the TGA3₂-NPR1₂-TGA3₂ complex, DNA-binding assay and genetic data show that dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form an enhanceosome. The stepwise assembly of the NPR1-TGA complex suggests possible hetero-oligomeric complex formation with other transcription factors, revealing how NPR1 reprograms the defence transcriptome.

Extended Data Fig. 1 |. Biochemical characterization and cryo-EM reconstruction of apo NPR1.

a, SDS–PAGE gel of purified NPR1. b, Size-exclusion chromatography shows that NPR1 elutes between molecular weight markers of 440 kDa and 158 kDa on a Superose 6 Increase 10/300 GL column. c, Crosslinking of NPR1 by BS3 at varying concentrations reveals a dominant dimer band. d, Flowchart of the reconstruction. Details are described in Methods. Green circles highlight representative NPR1 particles on the micrograph with the enlarged view in the inset. e, The global Fourier Shell Correlation (FSC) curve. f, Local resolution map. g, Euler angle distribution of the particles. h, Representative regions of the EM density map. Images in a and c are representative of 3 and 2 biological replicates, respectively.

Extended Data Fig. 2 |. Biochemical characterization of the NPR1 BHB and SBD domains.

a, Interaction of Myc-tagged CUL3A (Myc-CUL3) with free GST, or GST-fused WT NPR1 (GST–NPR1), or NPR1^ΔBHB deletion mutant (deletion of E200-L258; GST–NPR1^ΔBHB) in E. coli. The proteins were co-expressed in E. coli and total lysate was used for pull-down with glutathione affinity resin. Images are representative of 2 biological replicates. b, Solution small angle X-ray scattering (SAXS) data for the Arabidopsis apo NPR1 SBD purified from insect cells at a concentration of 1 mg ml⁻¹. The SAXS scattering curve, the Kratky plot, and the Guinier analysis are shown in the top, middle, and bottom panels, respectively. The non-bell shape of the Kratky plot indicates the protein is unfolded and exhibits a random-coil behaviour. The Guinier analysis yields a large radius of gyration (Rg) value of 47 Å for the NPR1 SBD (~20 kDa) in comparison with folded lysozyme (14.3 kDa; Rg = ~16 Å) and Bovine serum albumin (66 kDa; Rg = ~30 Å), indicating a disordered conformation.

Extended Data Fig. 3 |. NPR1 harbours a unique zinc finger.

a, X-ray fluorescence scanning data revealed the presence of Zn²⁺ in NPR1(ΔSBD) crystals. Scanning results for the NPR1 protein crystal and buffers are shown in the left and right panels, respectively. b, Sequence alignment of BTB domains. Conserved cysteine and histidine residues in a unique cysteine cluster preserved in NPR proteins are highlighted in pink. Dots indicate residues participating in zinc coordination, and triangles denote residues mutated in npr1(dim). Listed plant species include: Arabidopsis thaliana (At), Brassica rapa (Br), Brassica juncea (Bj), Brassica napus (Bn),Raphanus sativus (Rs), Oryza sativa (Os), Nicotiana tabacum (Nt), Populus trichocarpa (Pt), Zea mays (Zm), Solanum lycopersicum (Sl), Vitis vinifera (Vv), Hordeum vulgare (Hv), Medicago truncatula (Mt), and Glycine max (Gm).

Extended Data Fig. 4 |. Cryo-EM reconstruction of the NPR1-SA complex.

Details of the flowchart are described in Methods. a, Flowchart of the reconstruction. b, Global Fourier Shell Correlation (FSC) curve. c, Euler angle distribution of the particles.

Extended Data Fig. 5 |. Cryo-EM reconstruction of the NPR1–TGA3 complex.

a, Flowchart of the reconstruction. b, Local resolution, global Fourier Shell Correlation (FSC) curve, Euler angle distribution of the particles, and representative regions of the EM density map of the TGA3₂–NPR1₂–TGA3₂ complex. c, Local resolution, global FSC curve, Euler angle distribution of the particles, and representative regions of the EM density map of the NPR1₂–TGA3₂ complex.

Extended Data Fig. 6 |. Crystallographic and mass spectrometry characterization of the TGA3 NID–palmitate complex.

a, Crystal structure of the TGA3 NID dimer. The two TGA3-NID molecules are shown in the rainbow colour, with N-terminus coloured in blue and C-terminus coloured in red. b, A zoomed-in view of the location of the palmitate. Polar interactions with the carboxylate group of the palmitates are indicated with dashed lines. For clarity, only one TGA3 molecule is coloured in rainbow, and the other molecule is coloured in grey. Purple meshes in panels a, b represent 2mFo-DFc omit map of the palmitate plotted at the 1.0 σ level. c, Mass spectrometry analysis of the fatty acid extracted from the protein sample, verifying the fatty acid as the palmitic acid (C16:0).

Extended Data Fig. 7 |. The as-1 elements in SA-induced gene promoters.

a, Distribution of as-1 element in the promoters (3 kb) of SA-uninduced genes compared to the promoters (3 kb) of top 100 SA-induced genes. A statistically significant difference of as-1 element distribution was seen in the promoters of the top 100 SA-induced genes compared to the promoters of SA-uninduced genes (p-value < 0.001). b, Promoter analysis for as-1 elements of the top 100 SA-induced genes after 8 h treatment. c, Frequency plot of distances between as-1 elements from the promoters of the top 100 SA-induced genes. d, Electrophoresis mobility shift assay of NPR1 and TGA3 using 6-fluorescein-labelled DNA spanning the LS5-to-LS7 region of the PR1 promoter containing two as-1 elements (LS5/LS7) or a single as-1 element in the LS5 region (LS5/LS7⁻) or in the LS7 region (LS5⁻/LS7). d is a representative image of 3 biological replicates.

Extended Data Fig. 8 |. Characterization of the npr1(dim) mutant.

a, Elution profiles of the WT NPR1, npr1(C82A), and npr1(dim) samples on a Superose 6 Increase 10/300 GL column. b, A low resolution cryo-EM map of npr1(dim) reveals the expected shape of a monomeric NPR1. The monomeric model of NPR1 has been fitted into the EM density and displayed in the cartoon representation.

Fig. 1 |. Cryo-EM analysis of apo NPR1 and NPR1–SA.

a, The NPR1 homodimer (residues 40–402). b, Topology of NPR1 BTB, BHB and ANKs. Cys, cysteine cluster. c, Alignment of NPR1 BHB with the 3-box motif of SPOP (Protein Data Bank (PDB): 3HTM). d, Stabilization of NPR1 ANKs through a His300/His334-mediated hydrogen-bond network. bb, backbone amides. e, Representative 2D classes of NPR1 cryo-EM images without SA (left) and with SA (right). The arrows indicate the folded SBD. Scale bar, 5 nm. f, 3D reconstruction of full-length NPR1 showing SBD–ANK docking in the presence of SA. g, Hydrophobic interactions between SBD and ANK3/4. Interfacial residues (stick model), Cα atoms of Gln400 and Arg506 (yellow spheres), and SA (pink sphere model) are shown. h, i, Induction of the PR1 promoter 24 h after SA treatment (h) and interaction with TGA3–mRFP (i) tested for HA-tagged NPR1 (NPR1–HA), npr1(L346D) (L346D–HA), npr1(L393D) (L393D–HA) and npr1(I397D) (I397D–HA). j, k, Induction of the PR1 promoter 30 h after SA treatment (j) and the interactions between TGA3–mRFP and NPR1 variants (k), tested for NPR1–HA or npr1(Q400C/E401L/R506C) (Q400C/E401L/R506C–HA). For h and j, data are mean ± s.d. n = 3 independent biological replicates. P values were calculated using one-way analysis of variance (ANOVA) with Tukey’s post test. The images in i and k are representatives of two biological replicates each.

Fig. 2 |. NPR1 contains a unique zinc finger and a redox sensor.

a, NPR1 homodimer (residues 39–405) with the zinc finger detailed. The coordination of Zn²⁺ (coral sphere) by side chains of Zn²⁺-chelating residues (shown in the stick model and labelled in blue) is indicated by black dashed lines. The hydrogen-bond network between the zinc finger and ANK4 is indicated by blue dashed lines, with the pertinent side chain and backbone groups shown in the stick model. The side chain of Cys156 is shown in the stick model. b, Consensus (top) and alignment showing conserved zinc finger residues (bottom) in NPR1s from different species generated by Clustal Omega. Conserved residues in the consensus sequence are coloured, whereas variable residues are indicated by Xs. Proteins with and without Cys156 are separated with a dashed line. Listed plant species include: Arabidopsis thaliana (At), Brassica rapa (Br), Brassica juncea (Bj), Brassica napus (Bn),Raphanus sativus (Rs), Nicotiana tabacum (Nt), Populus trichocarpa (Pt), Solanum lycopersicum (Sl), Vitis vinifera (Vv), Medicago truncatula (Mt),Hordeum vulgare (Hv), Oryza sativa (Os), Zea mays (Zm), and Glycine max (Gm). c, d, Interaction between TGA3–mRFP and NPR1 variants (c) and induction of PR1 promoter 24 h after SA treatment (d), tested for HA-tagged NPR1 (NPR1–HA), npr1(C150A) (C150A–HA), npr1(C150Y) (C150Y–HA), npr1(C155A) (C155A–HA), npr1(C155Y) (C155Y–HA), npr1(H157A) (H157A–HA), npr1(C160A) (C160A–HA) and npr1(A151P/D152R) (A151P/D152R–HA). For d, data are mean ± s.d. n = 3 independent biological replicates. P values were calculated using one-way ANOVA and Tukey’s post test. e, The NPR1(ΔSBD) (Thr39–Asp410) tetramer in the protein crystal. f, In vitro oligomerization of NPR1 (residues Thr39–Lys262), WT or the C156A, C82A and C212V/C216A/C223L mutants, induced by hydrogen peroxide. The hash symbol (#) indicates impurity. The images in c and f are representative of two and three biological replicates, respectively.

Fig. 3 |. The structure and function of the NPR1–TGA3 complexes.

a, Cryo-EM density map and cartoon representation of the TGA3₂–NPR1₂–TGA3₂ complex. b, Cryo-EM density map of the NPR1₂–TGA3₂ assembly intermediate. The ANK region (grey) with weak density is indicated. c, The palmitate-containing TGA3 NID dimer (coloured rainbow and grey). d, Palmitic acid recognition by the TGA3 NID in the 1.5-Å-resolution crystal structure of the TGA3 NID–palmitate complex. Polar interactions are denoted (dashed lines), and van der Waals contacts are shown (curved lines). e, The molecular interactions between the NPR1 ANK1 (green) and the TGA3 NID (purple). Polar interactions are indicated (dashed lines). f, g, Interactions between TGA3–mRFP and the indicated NPR1 variants (f) and the induction of the PR1 promoter 24 h after SA treatment (g), tested for HA-tagged NPR1 (NPR1–HA), npr1(L281D) (L281D–HA), npr1(L284D) (L284D–HA) and npr1(L281D/L284D) (L281D/L284D–HA). For g, data are mean ± s.d. n = 3 independent biological replicates. P values shown were calculated using one-way ANOVA and Tukey’s post test. The image in f is representative of two biological replicates.

Fig. 4 |. The NPR1 dimer is required for SA-mediated immunity activation.

a, Electrophoresis mobility shift assay for TGA3 in complex with NPR1 or npr1(dim) (dim). The PR1 promoter region with (LS5/LS7) or without (LS5⁻/LS7⁻) two as-1 elements was used as the probe. b, The NPR1 dimerization interface. c, Self-interaction of NPR1 or npr1(dim) tested between GFP- and HA-fused proteins, with and without SA. d, Cell-free oligomerization of NPR1–GFP, npr1(dim)–GFP or npr1(C82A)–GFP (C82A–GFP). M, monomerized; O, oligomerized; T, total reduced proteins. e, Localization (left) and nucleocytoplasmic partitioning (right) of NPR1–GFP and npr1(dim)–GFP. Scale bar, 20 μm. f, The induction of the PR1 promoter by NPR1–HA or npr1(dim)–HA 24 h after SA treatment. g, h, Defence gene expression (g) and growth of Pseudomonas syringae pv. maculicola (Psm) ES4326 (h) in transgenic npr1 plants expressing NPR1–GFP, npr1(dim)–GFP lines 3 and 5, or C82A–GFP treated with 1 mM SA for 24 h. The box plot in e shows the median (centre line), and 25th and 75th percentiles (box limits), with the whiskers marking the minimum and maximum values. n = 15 (NPR1–GFP) and n = 16 (npr1(dim)–GFP) micrographs examined over 3 independent biological replicates. c.f.u., colony-forming units. Data are mean ± s.d. of n = 3 independent biological replicates (f and g); and mean ± 95% confidence intervals of n = 8 independent biological replicates (h). P values shown were calculated using either two-tailed Student’s t-tests (e), or one-way ANOVA (f and g) and two-way ANOVA (h) followed by Tukey’s post test. The experiments in c–h were repeated at least twice with similar results. i, The enhanceosome model of dimeric NPR1 bridging two dimeric TGA transcription factors. SA-induced SBD–ANK docking creates a new interface to facilitate post-translational modifications and/or to facilitate the recruitment of transcriptional regulators (X) for the activation of defence genes. The images are representative of three (a, c and e) and four (d) biological replicates.