Reinforcement of DNA methylation in CHH context through RNA-directed DNA methylation ensures sexual reproduction in rice [BS-Seq]. Reinforcement of DNA methylation in CHH context through RNA-directed DNA methylation ensures sexual reproduction in rice [BS-Seq]

DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. Although the mechanism of RdDM has been well understood in Arabidopsis, mutations in RdDM genes cause no visible developmental defects in Arabidopsis. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes RNA-dependent RNA polymerase 2 in rice. Mutation in OsRDR2 abolished the accumulation of 24-nt small interfering RNAs, and consequently substantially decreased genome-wide CHH methylation levels. Moreover, male and female reproductive development was disturbed, which led to the sterility of osrdr2 mutants. We discovered that OsRDR2-dependent DNA methylation likely regulates the expression of multiple key genes involved in stamen development and meiosis. In wild-type (WT) plants but not in osrdr2 mutants, genome-wide CHH DNA methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of methylation in reproductive organs of the WT was mainly explained by the enhancement of RdDM activity including OsRDR2 activity. Our results, which revealed a global increase in DNA methylation through enhancement of RdDM activity in reproductive organs, demonstrate the crucial role of OsRDR2 in the sexual reproduction of rice. Overall design: whole genome wide DNA methylation data by bisulphite sequencing

DNA甲基化是一类重要的表观遗传标记，可调控基因与转座子（transposons）的表达。RNA指导的DNA甲基化（RNA-directed DNA methylation, RdDM）是植物中负责从头DNA甲基化（de novo DNA methylation）的核心分子通路。尽管拟南芥中RdDM的作用机制已得到充分解析，但RdDM基因突变并未使拟南芥产生可见的发育缺陷。本研究分离并克隆了水稻中的五元素山1（Five Elements Mountain 1, FEM1），该基因编码RNA依赖的RNA聚合酶2（RNA-dependent RNA polymerase 2）。OsRDR2基因突变会阻断24-nt小干扰RNA（small interfering RNAs, siRNAs）的积累，进而大幅降低全基因组CHH甲基化水平。此外，水稻雌雄生殖发育过程受到干扰，最终导致osrdr2突变体完全不育。研究发现，OsRDR2依赖的DNA甲基化或许可调控多个参与雄蕊发育与减数分裂（meiosis）的关键基因的表达。相较于幼苗，野生型（wild-type, WT）植株的穗部、雄蕊与雌蕊的全基因组CHH DNA甲基化水平显著更高，而osrdr2突变体中未观察到此现象。野生型生殖器官的甲基化水平全局升高，主要源于包括OsRDR2活性在内的RdDM活性增强。本研究结果揭示了通过增强生殖器官中的RdDM活性可实现DNA甲基化的全局升高，证实了OsRDR2在水稻有性生殖过程中的关键作用。实验整体设计：采用亚硫酸氢盐测序（bisulphite sequencing）获取全基因组DNA甲基化数据。