Mechanism of self/nonself-discrimination in Brassica self-incompatibility

Abstract

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S₉-SRK ectodomain (eSRK) and S₉-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S₈-eSRK-S₈-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Fig. 1. Structure determination of S₈-meSRK–S₈-SP11 complex.

a Eleven amino acid mutations of S₈-eSRK (S₈-meSRK) suppress self-aggregation. The immunoblots show the fractions from size exclusion chromatography of S₈-eSRK and S₈-meSRK ectodomains expressed in insect cells. Arrows show the elution positions of molecular mass markers. C, control supernatants of insect cell culture expressing the recombinant proteins. b S₈-meSRK possesses S₈-SP11 recognition activity. The panel shows immunoblot analysis of pull-down eluates using biotin-S₈-SP11 and insect culture media expressing S₈-meSRK or S₈-meSRK-HLH, which is an artificial fusion with the dimerization domain of a bHLH-ZIP protein. c Chemical shift perturbation analysis of S₈-SP11. Overlay of the spectrum of ¹⁵N-labeled S₈-SP11 (blue) with that of ¹⁵N-labeled S₈-SP11 co-existing with unlabeled S₈-meSRK (red). d ITC analysis of the S₈-meSRK–S₈-SP11 interaction. Upper panel, thermogram; lower panel, integrated titration curve. e Overall structure of S₈-meSRK–S₈-SP11 heterotetramer. Two S₈-meSRK (pink and yellow) and two S₈-SP11 (cyan and green) molecules are shown as cartoon models. Dotted lines indicate disordered regions. Details of the labeled circles are shown in Fig. 2a, d, g.

Fig. 2. Interface between S₈-meSRK and S₈-SP11.

a, d Close-up views of SP11-binding site 1 on S₈-meSRK (pink) with S₈-SP11 (cyan). g Close-up view of SP11-binding site 2 on S₈-meSRK with another molecule of S₈-SP11 (green). b, e, h Comparison of SP11-binding sites of eSRK between the S₈-meSRK–S₈-SP11 and S₉-eSRK–S₉-SP11 complexes. The eSRK molecules were superimposed using C_α atoms and show the same views as in a, d, and g, respectively. S₉-eSRK is shown in silver, and S₉-SP11 in yellow (interacting with SP11-binding site 1) and orange (site 2). c, f, i Close-up views of S₉-eSRK–S₉-SP11 complex in the same orientation as in (b), (e), and (h). a–i, Dotted lines represent hydrogen bonds. Water molecules are shown as small cyan spheres. j Pull-down of S₈-meSRK-HLH mutants with biotin-S₈-SP11 as in Fig. 1b.

Fig. 3. MM–GBSA analysis using our modeled eSRK–SP11 complexes.

a Calculated ΔG values for modeled SP11s against eSRKs. Red rectangles represent tightly bound eSRK–SP11 pairs, and white to blue rectangles represent pairs with weak affinity. These values were calculated by the MM–GBSA method. b, c Sequence alignments of SP11 (b) and the hypervariable regions (HVs) of SRK (c) haplotypes with heat maps indicating energy contributions from each residue. Residues consisting of an α-helix and β-strands are shown in dark and light blue, respectively. These alignments were generated using PROMALS3D.

Fig. 4. Identifying important residues for self/nonself-discrimination between similar eSRK–SP11 pairs.

a Model structure of S₄₆-eSRK–S₄₆-SP11 complex. Green and cyan represent two S₄₆-SP11 molecules in the heterotetramer, and pink and yellow represent two S₄₆-eSRK molecules. Residues mutated in the pull-down assay are shown in orange. Left and right panels show close-up views of interfaces around CR III and around CR I–II, respectively. a, c Subscripts to the right of residue numbers indicate chain ID in the complex (a, b, eSRK; c, d, SP11). b Pull-down of S₈-meSRK-HLH and its derivatives with biotin-S₈-SP11 was performed as in Fig. 1b. S₈-meSRK-HLH^{N271S,E273D,N337I} and S₈-meSRK-HLH^{N271S,E273D,N337I,E80G,S190P,Y198F,R367T} proteins lost the ability to bind S₈-SP11. c Model structure of the S₃₆-eSRK–S₃₆-SP11 complex. An overview of S₃₆-SP11 docked with S₃₆-eSRK dimer is shown in the left panel. Cyan represents S₃₆-SP11 molecule, and pink and yellow represent two S₃₆-eSRK molecules. Residues mutated in the bioassay are shown in orange. A close-up view of the CR III of S₃₆-SP11 is shown in the right panel. The H62R mutation is shown in pink. Distances are shown in angstroms. d Pollination bioassay. S₃₆-SP11- or mutant-treated (50 pmol) S₃₆S₃₆ pistils were pollinated with S₁₂ pollen grains. Pollen tubes were observed by UV fluorescence microscopy after aniline blue staining. Arrow shows pollen tubes. Scale bars, 100 μm.