Plant epigenetic regulations are involved in transposable element silencing, developmental processes and responses to the environment^{1,2,3,4,5,6,7}. They often involve modifications of DNA methylation, particularly through the DEMETER (DME) demethylase family and RNA-dependent DNA methylation (RdDM)⁸. Root nodules host rhizobia that can fix atmospheric nitrogen for the plant's benefit in nitrogen-poor soils. The development of indeterminate nodules, as in Medicago truncatula, involves successive waves of gene activation^9,10,11,12, control of which raises interesting questions. Using laser capture microdissection (LCM) coupled to RNA-sequencing (SYMbiMICS data¹¹), we previously identified 4,309 genes (termed NDD) activated in the nodule differentiation and nitrogen fixation zones, 36% of which belong to co-regulated genomic regions dubbed symbiotic islands¹³. We found MtDME to be upregulated in the differentiation zone and required for nodule development, and we identified 474 differentially methylated regions hypomethylated in the nodule by analysing ~2% of the genome⁴. Here, we coupled LCM and whole-genome bisulfite sequencing for a comprehensive view of DNA methylation, integrated with gene expression at the tissue level. Furthermore, using CRISPR–Cas9 mutagenesis of MtDRM2, we showed the importance of RdDM for CHH hypermethylation and nodule development. We thus proposed a model of DNA methylation dynamics during nodule development.

植物表观遗传调控涉及转座因子沉默、发育过程和对环境的反应^{1,2,3,4,5,6,7}。它们通常涉及 DNA 甲基化的修饰，特别是通过 DEMETER (DME) 去甲基化酶家族和 RNA 依赖性 DNA 甲基化 (RdDM) ⁸。根瘤寄生的根瘤菌可以固定大气中的氮，从而使植物在贫氮土壤中受益。不确定结节的发展，如Medicago truncatula，涉及基因激活的连续波^9,10,11,12，其控制引发了有趣的问题。使用激光捕获显微切割 (LCM) 与 RNA 测序相结合（SYMbiMICS 数据¹¹)，我们之前确定了 4,309 个基因（称为 NDD）在根瘤分化和固氮区被激活，其中 36% 属于被称为共生岛¹³的共同调控的基因组区域。我们发现MtDME在分化区上调，是结节发育所必需的，我们通过分析约 2% 的基因组⁴鉴定了结节中低甲基化的 474 个差异甲基化区域。在这里，我们将 LCM 和全基因组亚硫酸氢盐测序结合起来，以全面了解 DNA 甲基化，并与组织水平的基因表达相结合。此外，使用 CRISPR–Cas9 诱变MtDRM2，我们展示了 RdDM 对 CHH 高甲基化和结节发育的重要性。因此，我们提出了结节发育过程中的 DNA 甲基化动力学模型。