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

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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).

References

  1. 1

    Vasse, J., de Billy, F., Camut, S. & Truchet, G. Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. J. Bacteriol. 172, 4295–4306 (1990).

    CAS  Article  Google Scholar 

  2. 2

    Kondorosi, E., Mergaert, P. & Kereszt, A. A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors. Annu. Rev. Microbiol. 67, 611–628 (2013).

    CAS  Article  Google Scholar 

  3. 3

    Maunoury, N. et al. Differentiation of symbiotic cells and endosymbionts in Medicago truncatula nodulation are coupled to two transcriptome-switches. PLoS ONE 5, e9519 (2010).

    Article  Google Scholar 

  4. 4

    Limpens, E. et al. Cell- and tissue-specific transcriptome analyses of Medicago truncatula root nodules. PLoS ONE 8, e64377 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Roux, B. et al. An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing. Plant J. 77, 817–837 (2014).

    CAS  Article  Google Scholar 

  6. 6

    Mergaert, P. et al. A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs. Plant Physiol. 132, 161–173 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Alunni, B. et al. Genomic organization and evolutionary insights on GRP and NCR genes, two large nodule-specific gene families in Medicago truncatula. Mol. Plant Microbe. Interact. 20, 1138–1148 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Durgo, H. et al. Identification of nodule-specific cysteine-rich plant peptides in endosymbiotic bacteria. Proteomics 15, 2291–2295 (2015).

    CAS  Article  Google Scholar 

  9. 9

    Wang, D. et al. A nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis. Science 327, 1126–1129 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Van de Velde, W. et al. Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 327, 1122–1126 (2010).

    CAS  Article  Google Scholar 

  11. 11

    Farkas, A. et al. Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms. Proc. Natl Acad. Sci. USA 111, 5183–5188 (2014).

    CAS  Article  Google Scholar 

  12. 12

    Horváth, B. et al. Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant. Proc. Natl Acad. Sci. USA 112, 15232–15237 (2015).

    Article  Google Scholar 

  13. 13

    Vernie, T. et al. EFD is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula. Plant Cell 20, 2696–2713 (2008).

    CAS  Article  Google Scholar 

  14. 14

    Sinharoy, S. et al. The C2H2 transcription factor REGULATOR OF SYMBIOSOME DIFFERENTIATION represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula. Plant Cell 25, 3584–3601 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Frugier, F., Poirier, S., Satiat-Jeunemaitre, B., Kondorosi, A. & Crespi, M. A Kruppel-like zinc finger protein is involved in nitrogen-fixing root nodule organogenesis. Genes. Dev. 14, 475–482 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Godiard, L. et al. MtbHLH1, a bHLH transcription factor involved in Medicago truncatula nodule vascular patterning and nodule to plant metabolic exchanges. New Phytol. 191, 391–404 (2011).

    CAS  Article  Google Scholar 

  17. 17

    Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).

    CAS  Article  Google Scholar 

  18. 18

    Zemach, A. et al. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153, 193–205 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Zhu, J. K. Active DNA demethylation mediated by DNA glycosylases. Annu. Rev. Genet. 43, 143–166 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Choi, Y. et al. DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110, 33–42 (2002).

    CAS  Article  Google Scholar 

  21. 21

    Le, B. H. et al. Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors. Proc. Natl Acad. Sci. USA 107, 8063–8070 (2010).

    CAS  Article  Google Scholar 

  22. 22

    Ibarra, C. A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360–1364 (2012).

    CAS  Article  Google Scholar 

  23. 23

    Mok, Y. G. et al. Domain structure of the DEMETER 5-methylcytosine DNA glycosylase. Proc. Natl Acad. Sci. USA 107, 19225–19230 (2010).

    CAS  Article  Google Scholar 

  24. 24

    Ardourel, M. et al. Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses. Plant Cell 6, 1357–1374 (1994).

    CAS  Article  Google Scholar 

  25. 25

    Yang, C., Signer, E. R. & Hirsch, A. M. Nodules initiated by Rhizobium meliloti exopolysaccharide mutants lack a discrete, persistent nodule meristem. Plant Physiol. 98, 143–151 (1992).

    CAS  Article  Google Scholar 

  26. 26

    Glazebrook, J., Ichige, A. & Walker, G. C. A Rhizobium meliloti homolog of the Escherichia coli peptide-antibiotic transport protein SbmA is essential for bacteroid development. Genes. Dev. 7, 1485–1497 (1993).

    CAS  Article  Google Scholar 

  27. 27

    Bobik, C., Meilhoc, E. & Batut, J. Fixj a major regulator of the oxygen limitation response and late symbiotic functions of Sinorhizobium meliloti. J. Bacteriol. 188, 4890–4902 (2006).

    CAS  Article  Google Scholar 

  28. 28

    Alves-Carvalho, S. et al. Full-length de novo assembly of RNA-seq data in pea (Pisum sativum L.) provides a gene expression atlas and gives insights into root nodulation in this species. Plant J. 84, 1–19 (2015).

    CAS  Article  Google Scholar 

  29. 29

    Truchet, G. et al. Alfalfa nodulation in the absence of Rhizobium. Mol. Gen. Genet. 219, 65–68 (1989).

    CAS  Article  Google Scholar 

  30. 30

    Liu, J. et al. Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula. Plant Physiol. 141, 167–177 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Hsieh, T. F. et al. Regulation of imprinted gene expression in Arabidopsis endosperm. Proc. Natl Acad. Sci. USA 108, 1755–1762 (2011).

    CAS  Article  Google Scholar 

  32. 32

    Van de Velde, W. et al. Aging in legume symbiosis. A molecular view on nodule senescence in Medicago truncatula. Plant Physiol. 141, 711–720 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Plongthongkum, N., Diep, D. H. & Zhang, K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat. Rev. Genet. 15, 647–661 (2014).

    CAS  Article  Google Scholar 

  34. 34

    Mascher, M. et al. Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond. Plant J. 76, 494–505 (2013).

    CAS  Article  Google Scholar 

  35. 35

    Akalin, A. et al. Methylkit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol. 13, R87 (2012).

    Article  Google Scholar 

  36. 36

    Fedorova, M. et al. Genome-wide identification of nodule-specific transcripts in the model legume Medicago truncatula. Plant Physiol. 130, 519–537 (2002).

    CAS  Article  Google Scholar 

  37. 37

    Ott, T. et al. Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development. Curr. Biol. 15, 531–535 (2005).

    CAS  Article  Google Scholar 

  38. 38

    Lefebvre, B. et al. A remorin protein interacts with symbiotic receptors and regulates bacterial infection. Proc. Natl Acad. Sci. USA 107, 2343–2348 (2010)

    CAS  Article  Google Scholar 

  39. 39

    Gamas, P., Niebel, F. D. C., Lescure, N. & Cullimore, J. V. Use of a subtractive hybridization approach to identify new Medicago truncatula genes induced during root nodule development. Mol. Plant Microbe. Interact. 9, 233–242 (1996).

    CAS  Article  Google Scholar 

  40. 40

    Mortier, V., De Wever, E., Vuylsteke, M., Holsters, M. & Goormachtig, S. Nodule numbers are governed by interaction between CLE peptides and cytokinin signaling. Plant J. 70, 367–376 (2012).

    CAS  Article  Google Scholar 

  41. 41

    Pislariu, C. I. & Dickstein, R. An IRE-like AGC kinase gene, MtIRE, has unique expression in the invasion zone of developing root nodules in Medicago truncatula. Plant Physiol. 144, 682–694 (2007).

    CAS  Article  Google Scholar 

  42. 42

    Combier, J. P. et al. MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula. Genes Dev. 20, 3084–3088 (2006).

    CAS  Article  Google Scholar 

  43. 43

    Ariel, F. et al. Noncoding transcription by alternative RNA polymerases dynamically regulates an auxin-driven chromatin loop. Mol. Cell 55, 383–396 (2014).

    CAS  Article  Google Scholar 

  44. 44

    Kawakatsu, T. et al. Unique cell-type-specific patterns of DNA methylation in the root meristem. Nat. Plants 2, 16058 (2016).

    CAS  Article  Google Scholar 

  45. 45

    Yu, A. et al. Dynamics and biological relevance of DNA demethylation in Arabidopsis antibacterial defense. Proc. Natl Acad. Sci. USA 110, 2389–2394 (2013).

    CAS  Article  Google Scholar 

  46. 46

    Chabaud, M., Larsonneau, C., Marmouget, C. & Huguet, T. Transformation of barrel medic (Medicago truncatula Gaertn.) by Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12 nodulin promoter fused to the gus reporter gene. Plant Cell Rep. 15, 305–310 (1996).

    CAS  Article  Google Scholar 

  47. 47

    Boisson-Dernier, A. et al. Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol. Plant Microbe. Interact. 14, 695–700 (2001).

    CAS  Article  Google Scholar 

  48. 48

    Verdier, J. et al. Gene expression profiling of M. truncatula transcription factors identifies putative regulators of grain legume seed filling. Plant Mol. Biol. 67, 567–580 (2008).

    CAS  Article  Google Scholar 

  49. 49

    Sun, Z., Cunningham, J., Slager, S. & Kocher, J. P. Base resolution methylome profiling: considerations in platform selection, data preprocessing and analysis. Epigenomics 7, 813–828 (2015).

    CAS  Article  Google Scholar 

  50. 50

    Feng, H., Conneely, K. N. & Wu, H. A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data. Nucleic Acids Res. 42, e69 (2014).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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

Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Pascal Gamas.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-10, Supplementary Methods, Supplementary References, Supplementary Tables 2, 9 and 10. (PDF 2214 kb)

Supplementary Table 1

Supplementary Table 1. (XLSX 117 kb)

Supplementary Table 3

Supplementary Table 3. (XLSX 884 kb)

Supplementary Table 4

Supplementary Table 4. (XLSX 15072 kb)

Supplementary Table 5

Supplementary Table 5. (XLSX 147 kb)

Supplementary Table 6

Supplementary Table 6. (XLSX 113 kb)

Supplementary Table 7

Supplementary Table 7. (XLSX 6712 kb)

Supplementary Table 8

Supplementary Table 8. (XLSX 14 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/nplants.2016.166

Download citation

Further reading