The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins

Abstract

Plants use intracellular immunity receptors, known as nucleotidebinding oligomerization domain-like receptors (NLRs), 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 strainspecific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici. We present the solution structure of a coiled-coil (CC) 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 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. The minimal region of the CC domain required for both 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 is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.plant innate immunity | resistance protein | NLR proteins | effectortriggered immunity | nuclear magnetic resonance spectroscopy

The Coiled-Coil and Nucleotide Binding Domains of BROWN PLANTHOPPER RESISTANCE14 Function in Signaling and Resistance against Planthopper in Rice

“…Like MLA10, the NMR structure of the wheat Sr33 protein, an ortholog of MLA, was also solved and found to form dimers in solution. These examples suggest that self-association is critical for signaling involving CNLs (Maekawa et al, 2011;Casey et al, 2016). A similar mechanism has been demonstrated in more extensive studies of animal NLRs.…”

Signaling from the plasma-membrane localized plant immune receptor RPM1 requires self-association of the full-length protein

“…It was recently demonstrated that the solution structure of the wheat CNL Sr33 CC fragment differs significantly from that of the published crystal structure of a barley paralog, MLA10, and, rather surprisingly, resembles the structure of the CC fragment from the distantly related potato CNL Rx (52) solved in complex with its interacting protein RanGAP2 (53).…”

“…The hydrophobicity of these residues is conserved in RPM1, Sr33, and Rx (Fig. 3A), suggesting that these residues can be involved either in dimer formation or in holding together the monomeric four-helix bundle (52,54). We wanted to know whether these conserved hydrophobic residues also play a role in RPM1 function.…”

“…CC domain dimerization has been shown to be important for function of the barley CNL MLA10 (18). Recently, it was shown that only relatively longer, functional fragments of the MLA10, Sr33, and Sr50 CC domains dimerize in planta, whereas shorter, nonfunctional CC domain fragments that are disrupted in one alphahelix presumed to be important for CC function do not (44,52). Thus, we wanted to know whether the RPM1 CC domain also self-associates in planta.…”

“…However, the RPM1 CC-1 fragment (amino acids 1-135) includes the full predicted alpha-helical region possibly important for CC function (SI Appendix, Fig. S7A), as is the case for the larger and functional Sr33 CC fragment (amino acids 1-142) (44,52). Therefore, all of our CC fragments should include the last alpha-helix, a structural characteristic important for MLA10 and Sr33 CC function.…”