A female-specific RdDM-associated element guides de novo DNA methylation during rice gametophyte development

DNA methylation is crucial for regulating gene expression and silencing transposons. While the spatiotemporal specificity of DNA methylation controls organ formation and development, the molecular mechanisms underlying locus- or tissue-specific DNA methylation remain unclear. Previously, locus- or tissue-specific DNA methylation was thought to depend solely on the recognition of histone methylations. Here we show that high expression of 24-nucleotide small interfering RNAs (24-nt siRNAs) leads to incremental CHH methylation at specific loci via the RNA-directed DNA methylation (RdDM) pathway during female gametophyte development in rice. We also identify a DNA motif, the Female-specific RdDM-associated Element (FRE), which guides methylation to these loci. Furthermore, we show that the female-preferentially expressed protein OsCLSY3 directly interacts with FRE to regulate CHH methylation specifically in female reproductive tissues. These findings uncover a novel molecular mechanism necessary for tissue- and locus-specific de novo DNA methylation that might be conserved across diverse organisms including animals.