Modifiers of genetic dominance at the Arabidopsis self-incompatibility locus retain proto-miRNA features and act through non-canonical gene silencing pathways - BSAS data

Self-incompatibility (SI) in flowering plants is a common mechanism that prevents self-fertilization and promotes outcrossing. In Brassicaceae, the locus controlling SI is highly diverse and dozens of distinct alleles are organized in a complex dominance hierarchy: the gene controlling SI specificity in pollen always shows monoallelic expression in heterozygote individuals, and this is achieved through the action of sRNA precursors that resemble miRNAs, although the mechanism behind this remains elusive. Here, we engineered Arabidopsis thaliana lines expressing components of the Arabidopsis halleri SI system and used a reverse genetics approach to pinpoint the pathways underlying the function of these sRNA precursors. We showed that they trigger a robust decrease in transcript abundance of the recessive pollen SI genes, but not through the canonical transcriptional or post transcriptional gene silencing pathways. Furthermore, we observed that single sRNA precursors are typically processed into hundreds of sRNA molecules, with distinct sizes, abundance levels and ARGONAUTE loading preferences. This heterogeneity closely resembles that of proto-miRNAs, the evolutionary ancestors of miRNAs. Our results suggest that these apparent arbitrary features, which are often associated with lack of effects in gene expression, are crucial in the context of the SI dominance hierarchy since they allow one sRNA precursor of a given allele to repress multiple other (more recessive) alleles. This study not only provides an in-depth characterization of the molecular features underlying complex dominance interactions, but also constitutes a unique example of how specific evolutionary constraints shape the progression of sRNA precursors within the proto-miRNA – miRNA evolutionary continuum. Overall design: To measure DNA methylation in SCR01 target sites we used the BiSulfite Amplicon Sequencing Technique - BSAS (Masser et al., 2013; Sigman et al., 2021) which relies on PCR enrichment of regions of interest using bisulfite converted DNA as a template. To assess if the presence of S-locus sRNA precursors is associated with DNA methylation changes in the respective SCR01 target regions, we isolated immature buds of SCR01, SCR01*mir1887, SCR01 Ah04mir1887 and SCR01*mir1887 Ah20mirS3 plants.