Go Suzuki

Abstract Key message S²⁹ haplotype does not require the MLPK function for self-incompatibility in Brassica rapa. Abstract Self-incompatibility (SI) in Brassicaceae is regulated by the self-recognition mechanism, which is based on the S-haplotype-specific direct interaction of the pollen-derived ligand, SP11/SCR, and the stigma-side receptor, SRK. M locus protein kinase (MLPK) is known to be one of the positive effectors of the SI response. MLPK directly interacts with SRK, and is phosphorylated by SRK in Brassica rapa. In Brassicaceae, MLPK was demonstrated to be essential for SI in B. rapa and Brassica napus, whereas it is not essential for SI in Arabidopsis thaliana (with introduced SRK and SP11/SCR from related SI species). Little is known about what determines the need for MLPK in SI of Brassicaceae. In this study, we investigated the relationship between S-haplotype diversity and MLPK function by analyzing the SI phenotypes of different S haplotypes in a mlpk/mlpk mutant background. The results have clarified that in B. rapa, all the S haplotypes except the S²⁹ we tested need the MLPK function, but the S²⁹ haplotype does not require MLPK for the SI. Comparative analysis of MLPK-dependent and MLPK-independent S haplotype might provide new insight into the evolution of S-haplotype diversity and the molecular mechanism of SI in Brassicaceae.

<title>Abstract</title> Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In <italic>Brassica</italic>, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (<italic>S</italic> receptor kinase) and pollen determinant SP11 (<italic>S</italic>-locus Protein 11, also named SCR) from the <italic>S</italic>-locus. Although the structure of the <italic>B. rapa S</italic>₉-SRK ectodomain (eSRK) and <italic>S</italic>₉-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 <italic>Brassica</italic> SI by determining the <italic>S</italic>₈-eSRK–<italic>S</italic>₈-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 <italic>Brassica</italic> SI.

Self-compatibility in Arabidopsis thaliana represents the relatively recent disruption of ancestral obligate cross pollination, recognized as one of the prevalent evolutionary pathways in flowering plants, as noted by Darwin. Our previous study found that inversion of the male specificity gene (SP11/SCR) disrupted self-incompatibility, which was restored by overexpressing the SCR with the reversed inversion. However, SCR in A. thaliana has other mutations aside from the pivotal inversion, in both promoter and coding regions, with probable effects on transcriptional regulation. To examine the functional consequences of these mutations, we conducted reciprocal introductions of native promoters and downstream sequences from orthologous loci of self-compatible A. thaliana and self-incompatible A. halleri. Use of this inter-species pair enabled us to expand the scope of the analysis to transcriptional regulation and deletion in the intron, in addition to inversion in the native genomic background. Initial analysis revealed that A. thaliana has a significantly lower basal expression level of SCR transcripts in the critical reproductive stage compared to that of A. halleri, suggesting that the promoter was attenuated in inducing transcription in A. thaliana. However, in reciprocal transgenic experiments, this A. thaliana promoter was able to restore partial function if coupled with the functional A. halleri coding sequence, despite extensive alterations due to the self-compatible mode of reproduction in A. thaliana. This represents a synergistic effect of the promoter and the inversion resulting in fixation of self-compatibility, primarily enforced by disruption of SCR. Our findings elucidate the functional and evolutionary context of the historical transition in A. thaliana thus contributing to the understanding of the molecular events leading to development of self-compatibility.