A wheat resistosome defines common principles of immune receptor channels

Abstract

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses¹. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity^2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr35⁵ in complex with the effector AvrSr35⁶ of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.

Fig. 1. 3D reconstruction of the Sr35 resistosome.

a, Negative staining of purified Sr35 in complex with AvrSr35 (Sr35 resistosome). Star-shaped particles were enriched by affinity purification and size-exclusion chromatography. Monodisperse Sr35 resistosomes have an average size of approximately 24 nm. Scale bar, 100 nm. b, 2D classifications of the Sr35 resistosome particles from the cryo-EM sample. Particles show preferential orientations for bottom and top views. Fewer, but sufficient, particles are in side view. Scale bar, 20 nm. c, Cryo-EM density map with 3 Å resolution (top) and the finally refined structure model (bottom) of the Sr35 resistosome shown in three different orientations. AvrSr35 is coloured green and Sr35 domains are shown according to the colour codes in the inset panel.

Fig. 2. Assembly of the Sr35 resistosome.

a, Sr35 resistosome showing a lateral dimer. Boxes in green, yellow and pink indicate positions of the zoomed views in c–f. Sr35 domains and AvrSr35 coloured according to the inset panel. b, Structure of one Sr35 protomer in complex with AvrSr35. Colour codes as in a. The blue box indicates position of structural detail in f. c, Structural detail of ATP binding in one protomer. Note the specific hydrogen bond of R311 with the γ-phosphate group of ATP at a 2.8 Å distance. Grey and white residue labels correspond to NBD and WHD residues, respectively. d, Structural detail of the interface between NBDs of a lateral dimer. Dashed lines represent polar interactions. Grey and white residue labels correspond to two neighbouring protomers from the pentamer. e, Structural detail of interface between two coiled-coil (CC) protomers. f, Structural detail of coiled-coil and LRR domain intramolecular packing in one Sr35 protomer. Acidic residues in the CC^EDIVD form salt-bridges with basic Arg (R) residues of the LRR^R-cluster. g, Cotransfection of Sr35 and Sr35 mutants with AvrSr35 in wheat protoplasts. Relative luminescence as readout for cell death. EV treatment defined the relative baseline (mean ± s.e.m.; n = 5). Test statistics derived from analysis of variance (ANOVA) and Tukey post hoc tests (P < 0.05). Exact P values for all protoplast plots are provided in Supplementary Table 3. Bar colours as box colours in c and d. h, Tobacco cell death data of Sr35 and Sr35 mutants with AvrSr35. i, Wheat protoplast data of EDIVD and R-cluster mutants. Experiment and statistics as in g. Bar colours as box colour in f. j, Nicotiana benthamiana cell death data of EDVID and R-cluster mutants. Representative data in h and j shown from seven replicates and scored for leaf cell death.

Fig. 3. The Sr35 resistosome forms a Ca²⁺-permeable non-selective cation channel.

a, Representative measurements from two-electrode (TEVC) recordings from Xenopus oocytes expressing Sr35, AvrSr35 and Sr35/AvrSr35. Effects of CaCCinh-A01 (Ca²⁺-activated chloride channel inhibitor) and LaCl₃ (Ca²⁺ channel blocker) on the Sr35-mediated currents in ND96 solution (96 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.6). Current traces shown at different voltages from −110 mV to +70 mV in 20 mV increments and current amplitudes at −110 mV. b, Quantitative measurements of data as in a. c, Structure-based mutagenesis of Sr35 residues at the interface between the LRR domain of Sr35 and AvrSr35, and Sr35 α1-helix. TEVC recordings in ND96 solution, and current amplitudes at −110 mV. d, Wheat protoplast data of Sr35 mutations at α1-helix. Relative luminescence as readout for cell death. EV treatment defined the relative baseline (mean ± s.e.m.; n = 5). Test statistics derived from ANOVA and Tukey post hoc tests (P < 0.05). Exact P values are provided in Supplementary Table 3. e, Tobacco cell death data of Sr35 and Sr35 channel mutants. Representative data shown from a minimum of three replicates. f, The Sr35 channel is selective for cations. TEVC recordings performed in various solutions, including KCl (96 mM), K-gluconate (96 mM), NaCl (96 mM), Na-gluconate (96 mM) and TBA-Cl (96 mM). g, Cationic currents of CaCl₂, Ca-Glu, MgCl₂ and Mg-Glu in the presence of CaCCinh-A01 and CaCCinh-A01+LaCl₃. Data are mean ± s.e.m., n ≥ 8 (b,c,f,g). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, one-way ANOVA analyses and Tukey post hoc test in b, c and g, and two-sided Student’s t-tests in f.

Fig. 4. Direct AvrSr35 effector recognition is mediated by the Sr35 LRR domain.

a, Interface between Sr35 LRR and AvrSr35. Red colour indicates the critical LRR residues within 5 Å from AvrSr35. b, Structural detail of Sr35 receptor and AvrSr35 effector interface. Dashed lines represent polar interactions. Grey and white residue label boxes correspond to Sr35 and AvrSr35 sidechains, respectively. c, Cotransfection of Sr35 LRR mutants with AvrS35 in wheat protoplasts. Relative luminescence as readout for cell death. EV treatment defined the relative baseline (mean ± s.e.m.; n = 5). Test statistics derived from ANOVA and Tukey post hoc tests (P < 0.05). Exact P values for all protoplast plots are provided in Supplementary Table 3. Bar colours correspond to box colours in b. d, Nicotiana benthamiana cell death data of Sr35 LRR mutations at the receptor–effector interface. Representative data are shown from 11 replicates and scored for leaf cell death. e, Cotransfection of Sr35 with AvrS35 mutants in wheat protoplasts. Experimental layout and statistics as in c (mean ± s.e.m.; n = 5). Bar colours as domain colours in a. f, Nicotiana benthamiana cell death data of AvrSr35 mutants co-expressed with Sr35. Representative data are shown from nine replicates and scored for leaf cell death.

Fig. 5. Structure-guided neofunctionalization of orphan CNLs and MLA receptor hybrids.

a, Illustration of Sr35 domain structure and hybrid receptors made from Sr35 homologues (Sh) in bread wheat (Triticum aestivum; TaSh1) and barley (Hordeum vulgare; HvSh1). Sr35^LRR (red) substitutes TaSH1^LRR and HvSH1^LRR (TaSH1^Sr35LRR and HvSH1^Sr35LRR). GOF receptor variants (TaSh1^GOF and HvSh1^GOF) were derived from sequence polymorphisms between Sr35, TaSH1 and HvSH1. b, Wheat protoplast transfections of TaSh1^Sr35LRR, HvSh1^Sr35LRR and controls co-expressed with AvrSr35. EV treatment defined the relative baseline (mean ± s.e.m.; n = 6). Test statistics derived from ANOVA and Tukey post hoc tests (P < 0.05). Exact P values for all protoplast plots are provided in Supplementary Table 3. c, Tobacco cell death of TaSh1^Sr35LRR and HvSh1^Sr35LRR. Representative data shown from seven replicates and scored for cell death. d, Western blot of hybrid receptors tested in tobacco. Pooled three replicates. Ponceau S staining as loading control. Composite image of same blot. e, Cryo-EM structure of Sr35 and structural predictions of TaSH1^LRR and HvSH1^LRR (ref. ). Polymorphisms between TaSH1 or HvSH1, and Sr35 are shown (orange). Residues mutated are shown red. f, Wheat protoplast transfections of TaSh1^GOF, HvSh1^GOF and controls co-expressed with AvrSr35. Experiment and statistics as in b. g, Tobacco cell death of TaSh1^GOF and HvSh1^GOF. Replicates and scoring as in c. h, Western blot of GOF experiment in tobacco. Replicates and loading control as in d. i, Graphical illustration of MLA hybrid receptors; design as in a (HvMla10^Sr35LRR and HvMla13^Sr35LRR). j, Wheat protoplast transfections of HvMla10^Sr35LRR, HvMla13^Sr35LRR and controls co-expressed with AvrSr35. Experiment and statistics as in b. k, Tobacco cell death of HvMla10^Sr35LRR and HvMla13^Sr35LRR. Replicates and scoring as in c. l, Western blot of MLA hybrid receptors in tobacco. Replicates and loading control as in d. Composite image of two independent blots.

Extended Data Fig. 1. Sr35-AvrSr35 complex reconstitution in Sf21 insect cells.

a, Cell viability data in Sf21 insect cells. Sr35 constructs carrying amino-terminal 6xHis-Sumo-tag and Avr35 constructs carrying N-terminal GST-tag. Cell viability was determined using trypan blue stain (mean ± SEM; n = 3 technical replicates). Six biological replicates were performed with comparable results. b, Chromatogram of Sr35-AvrSr35 resistosome purification using HiLoad S6 column. Red arrows corresponding to 669 kDa (66 mL) thyroglobulin molecular weight marker, 440 kDa (72 mL) to ferritin. c, SDS–PAGE of individual fractions collected in (b). Numbers represent elution volumes. Molecular weight marker (MWM) on left. d, Representative cryo-EM micrograph of Sr35-AvrSr35 complex. e, Representative 2D class averages of Sr35-AvrSr35 complex. f, Flowchart of cryo-EM data processing and Sr35-AvrSr35 3D reconstruction. g, FSC curves at 0.143 of the final model of Sr35-AvrSr35 complex. h, FSC curves at 0.143 of the final model of Sr35LRR-AvrSr35.

Extended Data Fig. 2. AvrSr35 structure from the Sr35 resistosome.

α10-helix (red) is involved in most extensive contacts with Sr35 LRR.

Extended Data Fig. 3. Western blot of N. benthamiana experiments.

Pooled samples from 3 technical replicates. Ponceau S staining as a loading control. a, Sr35 NBD ATP-binding and coiled-coil protomer interface mutants. Myc-tagged protein. Left and right side merged from the same blot. b, Sr35 EDVID and arginine-cluster mutants. Myc-tagged protein. Last lane cropped from the same blot. c, Sr35 channel mutants. Myc-tagged protein. d, Sr35 LRR mutants. Myc-tagged protein. e, AvrSr35 mutants. YFP-tagged protein detected by GFP antibody.

Extended Data Fig. 4. Details of EDIVD and R-cluster.

a, Multiple protein sequence alignment of HvMLA10, HvMLA13, Sr35 and ZAR1. Amino acids highlighted in red and in red text are identical and possess similar properties, respectively. Alignment of the EDIVD motif and arginine cluster are boxed in black (Robert and Gouet 2014). b, Electrostatic surface charge of Sr35 LRR around the EDVID motif. c, Structural alignment of Sr35 inactive structure prediction (cyan) and one protomer (yellow) from Sr35 resistosome. Detailed view of EDVID and arginine cluster interactions. In analogy to ZAR1, the Sr35 coiled-coil (CC) α1-helix might undergo structural rearrangement, which likely requires EDVID with arginine cluster interactions to transiently resolve.

Extended Data Fig. 5. CNL resistosome structural conservation.

The structures (in surface representation) of the ZAR1 resistosome and the Sr35 resistosome are shown. Zar1 is indirectly activated by the host proteins PBL2 and RKS1. Sr35 is directly activated by the fungal effector AvrSr35. The first, second, and third row show the top, side, and bottom views of these structures, respectively. Domains are coloured according to in-figure legend. Sizes are indicated by scale bar.

Extended Data Fig 6. Recognition of AvrSr35 effector by Sr35 LRR domain.

a, Shape and charge complementarity of Sr35 LRR and AvrSr35 at their interface. (Left) AvSr35 shown as cartoon (lime) and Sr35 as electrostatics surface model. (Right) Sr35 LRR shown as cartoon (cyan) and AvrSr35 as electrostatics surface model. b, Wheat protoplast data of AvrSr35 mutants predicted to impair Sr35 recognition. Relative luminescence as readout for cell death. Empty vector treatment defined the relative baseline (mean ± SEM; n = 3). Test statistics derived from ANOVA and Tukey post hoc tests (P <0.05). Exact p values provided in Supplementary Table 3.

Extended Data Fig. 7. Comparison of the Sr35 prediction (AlphaFold2) with the Sr35 protomer from the cryo-EM structure.

a, Structural alignment of WHD and LRR domains from Sr35 AlphaFold2 prediction (cyan) and from Sr35 resistosome Cryo-EM structure (blue). b, Structural comparison of monomeric Sr35 from prediction (left) and from Cryo-EM structure (right). Substantial differences exist highlighting the structural re-organization within the NOD module (NBD-HD1 relative to WHD). Domain color code: coiled-coil (yellow), NBD (light pink), HD1 (cyan), WHD (purple), and LRR (blue).

Extended Data Fig. 8. Steric clash between AvrSr35 and Sr35 NBD mediates Sr35 receptor activation.

Inactive Sr35 inside the cell comes in contact with Pgt effector AvrSr35. In avoidance of a steric clash (red) between AvrSr35 and the Sr35 NBD domain, the Sr35 NBD domain is forced to structurally rearrange and a ‘primed’ receptor-effector complex is formed. Full activation and oligomerization requires subsequent ADP release, ATP binding and, NOD module rearrangement and coiled-coil (CC) domain structural rearrangement. Sr35 domains and AvrSr35 are coloured according to in-figure legend.

Extended Data Fig. 9. Comparison of ZAR1, Sr35, ROQ1, RPP1 ligand binding sites.

Ligand binding to LRR of CNLs (Zar1, Sr35) and LRR-CJID of TNLs (Roq1, RPP1) occurs in equivalent region in the ascending lateral side of the LRR domain (compare concave, convex, ascending and descending lateral sides defined on Zar1).

Extended Data Fig. 10. Rationale for hybrid receptor design.

a, Structural (top) and sequence (bottom) alignment of Sr35, HvMLA10, HvMLA13, TaSH1 and HvSH1. Amino acid 505 in the structurally and sequence conserved α4-helix of the WHD of Sr35 was included in hybrid CNL receptors. Structure of Sr35 is isolated from the cryo-EM Sr35 resistosome structure, while HvMLA10, HvMLA13, TaSH1 and HvSH1 were predicted using AlphaFold2. b, Structural alignment of Sr35 LRR (light blue) with structural prediction of TaSh1 (yellow) and c, HvSh1 (orange). d, Multiple protein sequence alignment of Sr35, TaSH1 and HvSH1. Circled amino acids were substituted to corresponding amino acids in the Sr35 sequence for the generation of TaSh1^GOF and HvSh1^GOF constructs. Amino acids highlighted in red and in red text are identical and possess similar properties, respectively.