Dominance modifiers at the Arabidopsis self-incompatibility locus retain proto-miRNA features and act through non-canonical pathways

Self-incompatibility in flowering plants is a common mechanism that prevents self-fertilization and promotes outcrossing. In Brassicaceae, there is high genetic diversity at the locus controlling self-incompatibility, and dozens of distinct alleles are organized in a complex dominance hierarchy: the gene controlling self-incompatibility specificity in pollen shows monoallelic expression in heterozygote individuals. This is achieved through the action of sRNA precursors that resemble miRNAs, although the underlying molecular mechanisms remain elusive. Here, we engineered Arabidopsis thaliana lines expressing components of the Arabidopsis halleri self-incompatibility 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 self-incompatibility 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 of 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 apparently arbitrary features, which are often associated with lack of effects on gene expression, are crucial in the context of the self-incompatibility dominance hierarchy since they allow for one sRNA precursor of a given allele to repress multiple other 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 along the proto-miRNA - miRNA evolutionary continuum.