The solution structure of Sr33 challenges paradigms for coiled-coil domain dimerization in plant NLR immunity receptors

LW Casey1, A Bentham1,2, P Lavrencic1,4, S Cesari3, AE Mark1, P Anderson2, PN Dodds3, M Mobli4, B Kobe1 and SW Williams1,2,5

  1. School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia
  2. School of Biological Sciences, Flinders University, Adelaide, SA 5001, Australia
  3. CSIRO Agriculture, GPO Box 1600, Canberra ACT 2601, Australia
  4. Centre for Advanced Imaging, University of Queensland, Brisbane, Queensland 4072, Australia
  5. Plant Sciences Division, Research School of Biology, The Australian National University, Canberra 2601, Australia

Plants utilize intracellular immunity receptors, known as NLRs (nucleotide-binding oligomerization domain-like receptors) to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world’s most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein Sr33, which is responsible for strain-specific resistance to the wheat stem-rust pathogen, Puccinia graminis f. sp. tritici. We present the solution NMR structure of a coiled-coil fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate via SEC-MALS and SAXS that these regions are in fact largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta and this self-association correlates with their cell death signaling activity. We show that the minimal region of the CC domain required for both in planta cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but that this is likely to involve a different structural basis than previously suggested.