DNA demethylation and hypermethylation are both required for late nodule development in Medicago.

Plant epigenetic regulations are involved in transposable element silencing, developmental processes and responses to the environment 1-7 . 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 9-12 , control of which raises interesting questions. Using laser capture microdissection (LCM) coupled to RNA-sequencing (SYMbiMICS data 11 ), 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 islands 13 . 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 genome 4 . 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.