Intrinsic DNA motif-binding capability of OsCLSY3 guides female-preferential RNA-directed DNA methylation

Abstract

DNA methylation is essential for regulating gene expression and transposon silencing. In plants, RNA-directed DNA methylation (RdDM) mediates de novo DNA methylation. In Arabidopsis, RdDM is targeted to specific loci through either histone methylation or DNA motifs, the latter involving the chromatin remodelers CLASSY3 and CLASSY4 (CLSY3/4) in reproductive tissues mediated through the interaction of REPRODUCTIVE MERISTEM (REM) transcription factors and the GENETICS DETERMINES EPIGENETICS1 (GDE1) linker protein with an AT-rich DNA motif. Here, we identify a transcription factor-independent mechanism for female-preferential RdDM in rice, in which OsCLSY3 directly recognizes a GC-rich DNA motif, termed the Female-preferential RdDM-associated Element (FRE). OsCLSY3's intrinsic motif-binding capability is distinct from CLSY proteins in Arabidopsis. Interaction between OsCLSY3 and FRE-associated loci produces abundant 24-nucleotide small interfering RNAs in female reproductive tissues, driving progressive CHH methylation during development. These findings uncover a rice-specific mode of RdDM and provide insight into conserved and divergent regulation of DNA methylation across plant lineages.