NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid1,2,3,4. Despite the important role of NPR1 in plant immunity5,6,7, 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.
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The data supporting the findings of this study are available within the paper and its Supplementary Information. The cryo-EM structures of the apo NPR1, NPR12–TGA32 and TGA32–NPR12–TGA32, and X-ray crystal structures of the NPR1(ΔSBD) and TGA3 NID have been deposited at the PDB (www.pdb.org) under accession codes 7MK2, 7TAD, 7TAC, 7MK3 and 7TAE, respectively. The cryo-EM density maps of apo NPR1, NPR1–SA, NPR12–TGA32 and TGA32–NPR12–TGA32 have been deposited to the Electron Microscopy Data Bank under accession codes EMD-23884, EMD-23885, EMD-25771 and EMD-25769, respectively. There are no restrictions on data availability. Source data are provided with this paper.
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We thank Y. Tada for discussion on the project and Z. Yu, X. Zhao, S. Yang and R. Yan for support in cryo-EM data collection at the Janelia Research Campus. This work was supported by grants from the National Institutes of Health R01 GM115355 to P.Z., R35 GM118036 to X.D., R01 GM141223 to A.B., R01 AI148366 to Z.G., the Duke University School of Medicine Bridge Fund to P.Z., the Howard Hughes Medical Institute to X.D. and the Intramural Research Program of the National Institute of Environmental Health Sciences (NIEHS) grant ZIC ES103326 to M.J.B. X-ray fluorescence and diffraction data were collected at the Northeastern Collaborative Access Team beamline 24-ID-C, which is funded by the National Institute of General Medical Sciences (grant P30 GM124165). The Pilatus 6M detector on the 24-ID-C beamline is funded by the NIH Office of Research Infrastructure Programs (Hight-End Instrumentation grant S10 RR029205). The SAXS measurements were performed at Beamline 12-ID-B. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by the Argonne National Laboratory under contract DE-AC02-06CH11357. Cryo-EM data were collected using the Thermo Fisher Scientific Titan Krios transmission electron microscopes at the Janelia Research Campus and Duke University Shared Materials Instrumentation Facility (SMIF), and the Thermo Fisher Scientific Talos Arctica transmission electron microscopes at NIEHS and the University of North Carolina at Chapel Hill (UNC).
X.D. is a cofounder of Upstream Biotechnology and a scientific advisory board member of Inari Agriculture. The other authors declare no competing interests.
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Extended data figures and tables
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.
a, Interaction of Myc-tagged CUL3A (Myc-CUL3) with free GST, or GST-fused WT NPR1 (GST–NPR1), or NPR1ΔBHB deletion mutant (deletion of E200-L258; GST–NPR1ΔBHB) 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.
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).
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.
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).
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 genes32. 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.
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.
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Kumar, S., Zavaliev, R., Wu, Q. et al. Structural basis of NPR1 in activating plant immunity. Nature 605, 561–566 (2022). https://doi.org/10.1038/s41586-022-04699-w