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CellPlant BiologyPeer-reviewed journal article

An activated wheat CCG10-NLR immune receptor forms an octameric resistosome

Guanghao GuoiD, He ZhaoiD, Kaihong BaiiD, Professor Jian Lu, Qiuhong Wu, Lei Lu, Yue Zhang, Lingli Dong, Guangwei LiiD, Yongxing Chen, Yikun HouiD, Ping LuiD, Miaomiao LiiD, Huaizhi Zhang, Gaojie WangiD, Keyu Zhu, Baoge Huang, Xuejia Cui, Hongkui Fu, Chenchen Hu, Zhiying Chu, Xue Lyu, Sophien KamouniD, Chao WangiD, Zhiyong Liu, , Jonathan D. G. JonesiD

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Abstract

Nucleotide-binding and leucine-rich repeat (LRR) receptors (NLRs) are widespread intracellular immune sensors across kingdoms. In plants, the G10-type coiled-coil (CCG10)-NLRs form a distinct phylogenetic clade that remains poorly characterized. Here, we identified a gain-of-function (GOF) mutant of Wheat Autoimmunity 3 (WAI3), designated WAI3GOF, which encodes a constitutively activated CCG10-NLR protein due to an amino acid substitution. Cryo-EM structural analysis revealed that activated WAI3 assembles into a distinctive octameric resistosome. Arabidopsis RPS2, another CCG10-NLR, also forms an octamer, indicating a conserved structural property across monocot and dicot plants. The WAI3 resistosome mediates a prolonged and sustained increase in cytosolic calcium influx, facilitated by a unique channel architecture arising from its divergent CC domain configuration. Notably, this domain arrangement may be shared by many plant NLRs that lack the conserved EDVID motif in their CC domains. Our findings uncover a previously uncharacterized resistosome structure and provide insights into plant immune receptor plasticity.

In-depth summary

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (27 Aug 2025)

Activated wheat CCG10-NLR immune receptor WAI3 and Arabidopsis RPS2 assemble into octameric calcium-permeable resistosomes with an inverted coiled-coil domain topology associated with lacking the conserved EDVID motif.

Previously solved plant coiled-coil NLR (CC-NLR) resistosomes form pentamers or hexamers stabilized by a conserved EDVID motif, leaving the activation mechanism of EDVID-lacking CCG10-NLRs unknown. Determining the cryo-EM structure of auto-active wheat WAI3 reveals an octameric channel assembly and an inverted domain arrangement shared across monocot and dicot CCG10-NLRs.

Key results

  1. ShownA single amino acid substitution in the leucine-rich repeat (LRR) domain of wheat CCG10-NLR protein WAI3 causes constitutive immune activation, spontaneous cell death lesions, and powdery mildew resistance.

    Map-based cloning of EMS mutant M3405 narrowed the locus to a 600 kb region on chromosome 2D where a C1855T mutation in gene C5 (TraesCS2D01G380500, 919 aa) causes an L619F substitution, validated across 3 independent T1 transgenic Fielder wheat lines and 10 Nicotiana benthamiana hypersensitive response (HR) replicates. · WAI3GOF triggers constitutive activation of…; Figure legends

  2. ShownActivated WAI3 and Arabidopsis RPS2 oligomerize into octameric resistosomes, with WAI3 also forming two distinct dual-octamer assemblies in vitro.

    Cryo-EM of 7,043 micrographs of purified WAI3^(GOF/L12E/L15E) resolved a C8 single octamer at 3.95 A resolution (200 A diameter, 135 A height, ~1,000 kDa on BN-PAGE, burying 4,980 A^2 per protomer interface across 38 and 35 interacting residues), an N-to-N dual-octamer at 4.5 A, and a B-to-B dual-octamer at 3.6 A, while negative stain EM showed octameric rings for purified Arabidopsis RPS2^DeltaN26. · Activated CCG10-NLR WAI3 forms an octameric…; The CCG10-NLR RPS2 also forms an octameric…; Multiple interfaces stabilize the octameric WAI3…

  3. ShownThe WAI3 resistosome forms a wider ion-conducting pore than hexameric NRC4 and mediates a two-phase prolonged cytosolic calcium influx required for cell death.

    In GCaMP3-transgenic N. benthamiana, WAI3^GOF induced an initial transient Ca2+ peak followed by a sustained elevation that was abolished by 2 mM LaCl3 or by membrane-binding (L12E/L15E) and pore-lining (D3K/E17K/D20K) mutations; PoreAnalyzer showed a minimum pore radius of 12.7 A at Lys140 for WAI3 versus 4.3 A at Gln126 for the NRC4^DV hexamer. · The WAI3 resistosome triggers prolonged and…; Figure legends

  4. ShownAbsence of the EDVID motif and arginine cluster rotates the WAI3 coiled-coil domain by approximately 180 degrees, placing it opposite the LRR domain and inverting membrane topology.

    Unlike ZAR1, Sr35, NRC2, and NRC4, WAI3 positions its CC domain adjacent to NB-ARC like TIR-NLRs; AlphaFold3 models of 5 Arabidopsis CC-NLRs per subgroup (A-D) showed EDVID-lacking groups A and B adopt WAI3-like CC orientations while EDVID-containing groups C and D adopt ZAR1-like orientations, and HD1-WHD hinge angles tracked oligomeric state (80 deg in WAI3 octamer vs 88 in NRC2 hexamer and 92 in ZAR1 pentamer). · The WAI3 resistosome reveals a unique molecular…; CC-NLRs without the EDVID motif may exhibit…; Different oligomeric states of plant…

Methods, data and reproducibility

Cloned the causal mutation of autoimmune wheat mutant M3405 and purified full-length membrane-detaching WAI3^(GOF/L12E/L15E) and truncated Arabidopsis RPS2^DeltaN26 oligomers from Nicotiana benthamiana leaves for negative-stain and cryo-EM structure determination. Paired structural analysis and AlphaFold3 modeling with mutagenesis, GCaMP3 live-imaging of cytosolic Ca2+ influx, and hypersensitive response assays.

  • EMS-mutagenized M3405 mutant (in Zhongke331), F2 mapping population (M3405 x Liangxing 99), and transgenic wheat lines (in cultivar Fielder) · wheat (Triticum aestivum) · n = 3 independent T1 transgenic lines; n = 3 biological replicates for PR qPCR · TraesCS2D01G380500
  • Cryo-EM micrographs of tandem-affinity-purified WAI3^(GOF/L12E/L15E) resistosome complexes expressed in Nicotiana benthamiana · wheat protein expressed in Nicotiana benthamiana · n = 7,043 cryo-EM images
  • Negative-stain EM, BN-PAGE, HR cell death, and GCaMP3 calcium reporter assays of WAI3 and Arabidopsis RPS2 variants in Nicotiana benthamiana · wheat and Arabidopsis thaliana proteins expressed in Nicotiana benthamiana · n = 10 biological replicates for HR assays
  • Published plant resistosome structures used for comparative structural and pore analysis (ZAR1, NRC4, RPP1) · Arabidopsis thaliana and Nicotiana benthamiana · n = 3 structures · PDB: 6J5T
Methods & tools
AlphaFold3, PoreAnalyzer, GCaMP3, BSR-seq, BN-PAGE, Cryo-EM
Reproducibility gaps
No Electron Microscopy Data Bank (EMDB) or Protein Data Bank (PDB) accession codes for the resolved WAI3 cryo-EM maps and atomic models are provided in the text.
Limitations (3)
  • The N-terminal alpha1-helix (residues 1-35) and C-terminal residues 811-830 of WAI3 were unresolved in the cryo-EM density map, likely due to flexibility without plasma membrane stabilization, leaving the membrane-inserted channel pore structure uncharacterized. · stated by the authors
  • Purified Arabidopsis RPS2^DeltaN26 exhibited high structural heterogeneity under negative-stain EM, with only a small fraction of particles forming octamers, likely because transiently expressed RPS2 without RIN4 fragments is incompletely activated. · stated by the authors
  • The physiological relevance of the N-to-N and B-to-B dual-octamer configurations of WAI3 is unknown, and the B-to-B assembly may represent an overexpression artifact. · stated by the authors

Read this if you study plant NLR immune receptor structure, oligomerization stoichiometry, or calcium channel signaling mechanisms across divergent coiled-coil NLR clades.

Citations
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