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 nucleotide-binding 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 strain-specific 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.

Keywords: NLR proteins; effector-triggered immunity; nuclear magnetic resonance spectroscopy; plant innate immunity; resistance protein.

Fig. 1.

Solution structure of Sr33 reveals a four-helix bundle fold. (A) NMR structure of Sr33^6-120 in cartoon representation, with the individual helices and N and C termini labeled. The conserved EDVID motif (EDAVD in Sr33) is shown in stick representation (colored green in B and D). (B) Superposition of the Sr33^6-120 structure (blue) and the crystal structure of MLA10^5-120 (yellow) in cartoon representation. Missing residues in the MLA10^5-120 structure (amino acids 91–95) are shown by a dotted line. The crystallographic dimer observed for MLA10^5-120 is shown as a black and white outline. (C) Superposition, as shown in B, rotated 90° around the y axis. (D) Superposition of the Sr33^6-120 structure (blue) and the crystal structure of Rx^1-122 (red) in cartoon representation. Missing residues in the Rx^1-122 structure (amino acids 40–50) are shown by a dotted line. (E) Superposition, as shown in D, rotated 90° around the y axis.

Fig. 2.

Molecular mass calculations based on SEC-MALS analysis for Sr33^6-120 (A), MLA10^5-120 (B), Rx^1-122 (C), and Sr50^5-123 (D). For all proteins, solid black lines represent the normalized refractive index trace (arbitrary units, y axis) for proteins eluted from an in-line Superdex 200 10/300 column. Colored lines under the peaks correspond to the averaged molecular mass (right-hand y axis) distributions across the peak as determined by MALS (MW_MALS). Dotted lines indicate the predicted molecular masses of a monomer. The average MW_MALS values compared with predicted monomeric molecular mass values are 13.7/13.1 kDa for Sr33^6-120, 13.3/13.4 kDa for MLA10^5-120, 13.3/14.3 kDa for Rx^1-122, and 14.7/14.1 kDa for Sr50^5-123.

Fig. 3.

SAXS data from monomeric fractions of Sr33^6-120, MLA10^5-120, and Rx^1-122 are consistent with compact, globular particles. (A) Datasets from SEC-SAXS are shown as colored lines, with the MLA10^5-120 and Rx^1-122 data scaled to overlay with the Sr33^6-120 data. Arb., Arbitrary. (B) Normalized distance distribution functions, P(r), are shown as colored lines matching the scattering curve from which they were calculated. All distributions have been scaled to the maxima of the highest peak. (C) SEC-SAXS datasets again plotted as colored lines, now arbitrarily offset in y for clarity. Experimental errors are displayed at 1σ in lighter colors. The theoretical scattering predicted from each 3D structural model is shown as a black line against the corresponding dataset. (D) The first member of the Sr33^6-120 NMR ensemble (blue), the Rx^1-122 crystal structure (red), and the dimeric MLA10^5-120 crystal structure (yellow) are shown in cartoon representation, docked into ab initio envelopes calculated from their respective scattering datasets. Ab initio models are shown in transparent surface representation, with the average model from 16 independent runs shown in light gray and the filtered model shown in darker gray.

Fig. 4.

Solution studies of CC domains with extended sequences of Sr33 and MLA10. Molecular mass calculations from SEC-MALS analysis for Sr33^6-120, Sr33^6-144, and Sr33^6-160 (A) and MLA10^5-120 and MLA10^5-144 (B). Solid gray, dark gray, and black lines represent the refractive index for the three proteins, respectively, when eluted from an in-line Superdex 200 5/150 GL column, normalized to the height of the major peak for clarity. Dotted lines indicate the predicted molecular masses of both monomeric and dimeric species, and colored lines show the experimental molecular mass distributions as determined by MALS (values are shown in SI Appendix, Table S5).

Fig. 5.

Minimal autoactive domains of MLA10, Sr33, and Sr50 self-associate in planta. (A) MLA10, Sr33, and Sr50 protein fragments fused to HA or CFP were transiently expressed in N. benthamiana. The autoactive MLA10^1-160:CFP, Sr33^1-160:CFP, and Sr50^1-163:CFP constructs were used as positive controls. Cell death was visualized 5 d after infiltration. Equivalent results were obtained in three independent experiments. (B) Indicated proteins, transiently expressed in N. benthamiana leaves, were extracted 20 h after infiltration and analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Proteins were immunoprecipitated with anti-GFP beads (IP-GFP) and analyzed by immunoblotting with anti-GFP and anti-HA antibodies. RGA4 (CC domain):CFP fusion was used as a control for specificity. Sr50^1-163 was used as a positive control (25). Ponceau staining of the RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit shows equal protein loading.