Control of Rice Tillering by RNA-directed DNA Methylation at Miniature Inverted-repeat Transposable Elements

In rice, tillering is an important agricultural trait that affects plant architecture and grain yield. RNA-directed DNA methylation (RdDM), which establishes DNA methylation in all CG, CHG and CHH sequence contexts and maintains CHH methylation in plants, silences transposable elements (TEs) and regulates gene expression. Here we report that knockdown and knockout of OsNRPD1a and OsNRPD1b, genes encoding two orthologues of the largest subunit of OsPol IV holoenzyme that transcribes 24-nucleotide (nt) siRNAs in RdDM, lead to a high-tillering phenotype, in addition to dwarfism and smaller panicles. In the shoot base of the osnrpd1a/b RNAi line, many genes are dysregulated, due to loss of 24-nt siRNAs and CHH methylation. Among these genes, OsMIR156d and OsMIR156j, which promote rice tillering, is derepressed when CHH methylation at Miniature Inverted-Repeat Transposable Elements (MITEs) in the promoters of OsMIR156d and OsMIR156j is lost. By contrast, D14, which suppresses rice tillering, is repressed when CHH methylation at a MITE in the 3’UTR of D14 is lost. Deletion of the MITE that is targeted by RdDM in D14 is sufficient to cause high-tillering. Our findings reveal control of rice tillering by RdDM at MITEs and provide the potential targets for agronomic trait enhancement by epigenome editing. We conducted small RNA sequencing, mRNA sequencing and whole-genome bisulfite sequencing in three biological replicates using the shoot base of 4-week old seedlings of wild-type (WT, japonica) and the osnrpd1a/b RNAi lines.