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 environment1-7. 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 activation9-12, 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.