Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula


The legume–Rhizobium symbiosis leads to the formation of a new organ, the root nodule, involving coordinated and massive induction of specific genes. Several genes controlling DNA methylation are spatially regulated within the Medicago truncatula nodule, notably the demethylase gene, DEMETER (DME), which is mostly expressed in the differentiation zone. Here, we show that MtDME is essential for nodule development and regulates the expression of 1,425 genes, some of which are critical for plant and bacterial cell differentiation. Bisulphite sequencing coupled to genomic capture enabled the identification of 474 regions that are differentially methylated during nodule development, including nodule-specific cysteine-rich peptide genes. Decreasing DME expression by RNA interference led to hypermethylation and concomitant downregulation of 400 genes, most of them associated with nodule differentiation. Massive reprogramming of gene expression through DNA demethylation is a new epigenetic mechanism controlling a key stage of indeterminate nodule organogenesis during symbiotic interactions.

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Figure 1: DNA methylation and demethylation genes exhibit differential expression in M. truncatula nodule zones.
Figure 2: A subset of transposable elements (TEs) is transcriptionally upregulated in nodules.
Figure 3: MtDME transcriptional activation precedes the NCR-associated differentiation stage of M. truncatula nodules and is developmentally regulated.
Figure 4: Decreasing MtDME by RNA interference (DMEi) leads to strong defects in nodule differentiation.
Figure 5: Distribution amongst expression clusters of the genes down- and upregulated in pDME–DMEi versus control pDME–GUS nodules.
Figure 6: Differential cytosine methylation during nodule development (CG and CHG contexts).


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We thank S. Fénart (Roche Diagnostics, France) for his advice on genome capture, M. Crespi and C. Lelandais (IPS2, Saclay) for critical reading of the manuscript, M. Marchetti and O. Catrice (LIPM, Toulouse) for their help with the flow cytometer (FR AIB, Toulouse). We are grateful to T. Ott (Munich University) for the LjDME sequence, R. Geurts and E. Limpens (Wageningen University, the Netherlands) for the S. meliloti pnifH–GFP strain, J. Gouzy and S. Carrère (LIPM) for data submissions to SRA, L. Sauviac and C. Rosenberg (LIPM) for providing us with oligonucleotides for S. meliloti genes and cloning vectors respectively, and J.-M. Prosperi (INRA Montpellier) for M. truncatula seeds. Sequencing was performed by the GeT genotoul platform (Toulouse). This work was supported by the INRA SPE (EPINOD project), the ANR (EPISYM project), the Laboratoire d'Excellence (LABEX) TULIP (ANR-10-LABX-41), as well as a doctoral grant from the French Ministry of Education and Research for Carine Satgé.

Author information




C.S. and S.M. did most experiments, with contributions from M.C.A., C.R. and K.G. C.N. and E.S. performed bioinformatic analyses. G.L. and M.F.J. did the statistical analyses. L.C. carried out phylogenetic analyses. C.S., M.F.J. and P.G. analysed data. C.S., S.M., M.F.J. and P.G. conceived the research plans. P.G. conceived the project. P.G., C.S. and M.F.J. wrote the article with contributions from K.G. and C.N.

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Correspondence to Pascal Gamas.

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Satgé, C., Moreau, S., Sallet, E. et al. Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula. Nature Plants 2, 16166 (2016).

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