Whole-genome landscape of Medicago truncatula symbiotic genes

Abstract

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Overview of the M. truncatula A17 genome.
Fig. 2: Symbiotic gene expression patterns and organization in M. truncatula.
Fig. 3: Analysis of potential regulatory elements in nodule development islands.

Data availability

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PSQE00000000. The version described in this paper is version PSQE01000000. Raw reads from PacBio, ChIP-seq and small RNAseq experiments have been deposited at the Sequence Read Archive (SRA) (project accession number: SRP131849). Data related to gene annotation, transposable element annotation and ChIP-seq analyses, as well as Supplementary Table 6, are available at the web portal: https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/; downloads section.

References

  1. 1.

    Martin, F.M., Uroz, S. & Barker, D.G. Ancestral alliances: plant mutualistic symbioses with fungi and bacteria. Science 356, eaad4501 (2017).

    Article  Google Scholar 

  2. 2.

    Young, N. D. & Udvardi, M. Translating Medicago truncatula genomics to crop legumes. Curr. Opin. Plant Biol. 12, 193–201 (2009).

    CAS  Article  Google Scholar 

  3. 3.

    Young, N. D. et al. The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480, 520–524 (2011).

    CAS  Article  Google Scholar 

  4. 4.

    Tang, H. et al. An improved genome release (version Mt4.0) for the model legume Medicago truncatula. BMC Genomics 15, 312 (2014).

    Article  Google Scholar 

  5. 5.

    Moll, K. M. et al. Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula. BMC Genomics 18, 578 (2017).

    Article  Google Scholar 

  6. 6.

    Kamphuis, L. G. et al. The Medicago truncatula reference accession A17 has an aberrant chromosomal configuration. New Phytol. 174, 299–303 (2007).

    CAS  Article  Google Scholar 

  7. 7.

    de Bang, T. et al. Genome-wide identification of Medicago peptides involved in macronutrient responses and nodulation. Plant Physiol. 175, 1669–1689 (2017).

    CAS  Article  Google Scholar 

  8. 8.

    Miller, J. R. et al. Hybrid assembly with long and short reads improves discovery of gene family expansions. BMC Genomics 18, 541 (2017).

    Article  Google Scholar 

  9. 9.

    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 

  10. 10.

    Jardinaud, M. F. et al. A laser dissection-RNAseq analysis highlights the activation of cytokinin pathways by nod factors in the Medicago truncatula root epidermis. Plant Physiol. 171, 2256–2276 (2016).

    CAS  Article  Google Scholar 

  11. 11.

    Stanton-Geddes, J. et al. Candidate genes and genetic architecture of symbiotic and agronomic traits revealed by whole-genome, sequence-based association genetics in Medicago truncatula. PLoS ONE 8, e65688 (2013).

    CAS  Article  Google Scholar 

  12. 12.

    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 

  13. 13.

    Krzyczmonik, K., Wroblewska-Swiniarska, A. & Swiezewski, S. Developmental transitions in Arabidopsis are regulated by antisense RNAs resulting from bidirectionally transcribed genes. RNA Biol. 14, 838–842 (2017).

    Article  Google Scholar 

  14. 14.

    Swiezewski, S., Liu, F., Magusin, A. & Dean, C. Cold-induced silencing by long antisense transcripts of an Arabidopsis polycomb target. Nature 462, 799–802 (2009).

    CAS  Article  Google Scholar 

  15. 15.

    Fedak, H. et al. Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript. Proc. Natl Acad. Sci. USA 113, E7846–E7855 (2016).

    CAS  Article  Google Scholar 

  16. 16.

    Henriques, R. et al. The antiphasic regulatory module comprising CDF5 and its antisense RNA FLORE links the circadian clock to photoperiodic flowering. New Phytol. 216, 854–867 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Vernié, 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).

    Article  Google Scholar 

  18. 18.

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

    Article  Google Scholar 

  19. 19.

    Kalo, P. et al. Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 308, 1786–1789 (2005).

    CAS  Article  Google Scholar 

  20. 20.

    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 

  21. 21.

    Marsh, J. F. et al. Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase. Plant Physiol. 144, 324–335 (2007).

    CAS  Article  Google Scholar 

  22. 22.

    Ovchinnikova, E. et al. IPD3 controls the formation of nitrogen-fixing symbiosomes in pea and Medicago Spp. Mol. Plant Microbe Interact. 24, 1333–1344 (2011).

    CAS  Article  Google Scholar 

  23. 23.

    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 

  24. 24.

    Berrabah, F. et al. A nonRD receptor-like kinase prevents nodule early senescence and defense-like reactions during symbiosis. New Phytol. 203, 1305–1314 (2014).

    CAS  Article  Google Scholar 

  25. 25.

    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 

  26. 26.

    Graham, M. A., Silverstein, K. A., Cannon, S. B. & VandenBosch, K. A. Computational identification and characterization of novel genes from legumes. Plant Physiol. 135, 1179–1197 (2004).

    CAS  Article  Google Scholar 

  27. 27.

    Pan, H. & Wang, D. Nodule cysteine-rich peptides maintain a working balance during nitrogen-fixing symbiosis. Nat. Plants 3, 17048 (2017).

    CAS  Article  Google Scholar 

  28. 28.

    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 

  29. 29.

    Alunni, B. & Gourion, B. Terminal bacteroid differentiation in the legume-rhizobium symbiosis: nodule-specific cysteine-rich peptides and beyond. New Phytol. 211, 411–417 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394–408 (2014).

    CAS  Article  Google Scholar 

  31. 31.

    Hurst, L. D., Pal, C. & Lercher, M. J. The evolutionary dynamics of eukaryotic gene order. Nat. Rev. Genet. 5, 299–310 (2004).

    CAS  Article  Google Scholar 

  32. 32.

    Nutzmann, H. W., Huang, A. & Osbourn, A. Plant metabolic clusters – from genetics to genomics. New Phytol. 211, 771–789 (2016).

    Article  Google Scholar 

  33. 33.

    Reimegard, J. et al. Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana. Nucleic Acids Res. 45, 3253–3265 (2017).

    Article  Google Scholar 

  34. 34.

    Plaza, S., Menschaert, G. & Payre, F. In search of lost small peptides. Annu. Rev. Cell Dev. Biol. 33, 391–416 (2017).

    CAS  Article  Google Scholar 

  35. 35.

    Hnisz, D. & Young, R. A. New insights into genome structure: genes of a feather stick together. Mol. Cell 67, 730–731 (2017).

    CAS  Article  Google Scholar 

  36. 36.

    Rowley, M. J. et al. Evolutionarily conserved principles predict 3D chromatin organization. Mol. Cell 67, 837–852 e7 (2017).

    CAS  Article  Google Scholar 

  37. 37.

    Mele, M. & Rinn, J. L. ‘Cat’s cradling’ the 3D genome by the act of LncRNA transcription. Mol. Cell 62, 657–664 (2016).

    CAS  Article  Google Scholar 

  38. 38.

    Mercer, T. R. & Mattick, J. S. Structure and function of long noncoding RNAs in epigenetic regulation. Nat. Struct. Mol. Biol. 20, 300–307 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Heo, J. B. & Sung, S. Vernalization-mediated epigenetic silencing by a long intronic noncoding RNA. Science 331, 76–79 (2011).

    CAS  Article  Google Scholar 

  40. 40.

    Mayjonade, B. et al. Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules. Biotechniques 61, 203–205 (2016).

    CAS  Article  Google Scholar 

  41. 41.

    Berlin, K. et al. Corrigendum: assembling large genomes with single-molecule sequencing and locality-sensitive hashing. Nat. Biotechnol. 33, 1109 (2015).

    CAS  Article  Google Scholar 

  42. 42.

    Berlin, K. et al. Assembling large genomes with single-molecule sequencing and locality-sensitive hashing. Nat. Biotechnol. 33, 623–630 (2015).

    CAS  Article  Google Scholar 

  43. 43.

    Badouin, H. et al. The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution. Nature 546, 148–152 (2017).

    CAS  Article  Google Scholar 

  44. 44.

    Koren, S. et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722–736 (2017).

    CAS  Article  Google Scholar 

  45. 45.

    Chin, C. S. et al. Phased diploid genome assembly with single-molecule real-time sequencing. Nat. Methods 13, 1050–1054 (2016).

    CAS  Article  Google Scholar 

  46. 46.

    Raymond, O. et al. The Rosa genome provides new insights into the domestication of modern roses. Nat. Genet. 50, 772–777 (2018).

    CAS  Article  Google Scholar 

  47. 47.

    Tayeh, N. et al. A tandem array of CBF/DREB1 genes is located in a major freezing tolerance QTL region on Medicago truncatula chromosome 6. BMC Genomics 14, 814 (2013).

    CAS  Article  Google Scholar 

  48. 48.

    Kulikova, O. et al. Satellite repeats in the functional centromere and pericentromeric heterochromatin of Medicago truncatula. Chromosoma 113, 276–283 (2004).

    CAS  Article  Google Scholar 

  49. 49.

    Chin, C. S. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10, 563–569 (2013).

    CAS  Article  Google Scholar 

  50. 50.

    Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014).

    Article  Google Scholar 

  51. 51.

    Foissac, S. et al. Genome annotation in plants and fungi: EuGene as a model platform. Current Bioinformatics 3, 87–97 (2008).

    CAS  Article  Google Scholar 

  52. 52.

    Bao, W., Kojima, K. K. & Kohany, O. Repbase Update, a database of repetitive elements in eukaryotic genomes. Mob. DNA 6, 11 (2015).

    Article  Google Scholar 

  53. 53.

    Zerbino, D. R. Using the Velvet de novo assembler for short-read sequencing technologies. Curr. Protoc. Bioinformatics 11, Unit11 5 (2010).

    Google Scholar 

  54. 54.

    Wu, T. D. & Watanabe, C. K. GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21, 1859–1875 (2005).

    CAS  Article  Google Scholar 

  55. 55.

    Tephra: A Tool for Discovering Transposable Elements and Describing Patterns of Genome Evolution v.0.12.2 (Staton, S., 2017); https://github.com/sestaton/tephra

  56. 56.

    Generic Feature Format Version 3 (GFF3) v.1.23 (Stein, L., 2013); https://github.com/The-Sequence-Ontology/Specifications/blob/master/gff3.md

  57. 57.

    Staton, S. E. & Burke, J. M. Transposome: a toolkit for annotation of transposable element families from unassembled sequence reads. Bioinformatics 31, 1827–1829 (2015).

    CAS  Article  Google Scholar 

  58. 58.

    Kurtz, S., Narechania, A., Stein, J. C. & Ware, D. A new method to compute K-mer frequencies and its application to annotate large repetitive plant genomes. BMC Genomics 9, 517 (2008).

    Article  Google Scholar 

  59. 59.

    Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).

    CAS  Article  Google Scholar 

  60. 60.

    Guizard, S., Piegu, B. & Bigot, Y. DensityMap: a genome viewer for illustrating the densities of features. BMC Bioinformatics 17, 204 (2016).

    Article  Google Scholar 

  61. 61.

    Veluchamy, A. et al. LHP1 regulates H3K27me3 spreading and shapes the three-dimensional conformation of the Arabidopsis genome. PLoS ONE 11, e0158936 (2016).

    Article  Google Scholar 

Download references

Acknowledgements

We thank C. Ben and L. Gentzbittel (EcoLab, Université de Toulouse, CNRS, Toulouse INP, UPS, France), G. Aubert, R. Thompson and K. Gallardo (INRA, UMR 1347, Agroécologie, Dijon, France) and B. Gronenborn (I2BC, CNRS, Paris Sud, CEA, University of Paris Saclay, Gif sur Yvette, France) for providing small RNA data on disease responses, seeds and viroid-infected plants, respectively, as well as N. Peeters (LIPM, Toulouse) for mRNA data used for genome annotation. We thank M.C. Le Paslier for her help in Illumina sequencing. This work was supported by the ANR grants EPISYM (grant no. ANR-15-CE20-0002), NODCCAAT (no. ANR-15-CE20-0012), REGULEG (no. ANR-15-CE20-0001), the ‘Laboratoire d’Excellence (LABEX)’ TULIP (no. ANR-10-LABX-41), the LABEX Saclay Plant Sciences (SPS; no. ANR-10-LABX-40) and the European Research Council (no. ERC-SEXYPARTH), and we made use of data previously generated in the ANR SYMbiMICS (ANR-08-GENO-106) and the INRA SPE EPINOD projects. The sequencing platform was supported by France Génomique National infrastructure (grant no. ANR-10-INBS-09) and by the GET-PACBIO programme (Programme opérationnel FEDER-FSE MIDI-PYRENEES ET GARONNE 2014-2020). We are grateful to the Genotoul bioinformatics platform Toulouse Midi-Pyrenees (Bioinfo Genotoul) for providing computing and storage resources. C. Satgé was supported by a doctoral grant from the French Ministry of Education and Research.

Author information

Affiliations

Authors

Contributions

S.Mo., B.M., C.L-R. and O.B. prepared DNA samples and performed PacBio sequencing. S.Cau., C.C-D., W.M. and H.B. built the Bionano optical maps. B.M., J.G., W.M., S.Mu. and A.Ber. designed and performed Illumina seq of BAC end sequencing (EcoR1 library). F.D. prepared DNA samples and managed Illumina sequencing. J.G. assembled the genome. E.S., S.Car. and J.G. annotated protein-coding genes and miRNAs. S.E.S. annotated and analysed repeats and transposable elements. S.Car. developed the Medicago bioinformatics portal. J.K. positioned HapMap data on the new reference genome. C.S. and C.L.-B. prepared samples for the sRNA analyses. C.L.-B. conducted the miRNA analyses. T.B., C.L.-B. and Y.P. conducted the siRNA analyses. S.Mo. and M.P. prepared the histone mark samples. D.L. and M.P. performed the ChIP experiments. M.B., D.L. and A.Ben. performed ChIP-seq. M.Za., M.Zo., M.B., S.Car., Y.P. and P.G. performed the analysis of the ChIP-seq data. Y.P. and P.G. conducted the lncRNA analyses. Y.P., S.Car. and P.G. performed the gene family analyses. J.G. and T.B. performed the sRNA and mRNA expression analyses. M.-F.J. performed the gene and siRNA differential expression analyses. Y.P. and P.G. performed the integrated analyses of the symbiotic islands. P.G., J.G., M.C., A.N. and J.B. contributed to the project set-up. P.G., J.G., S.E.S. and C.L.-B. wrote the manuscript, with contributions from M.C., F.F., J.B., B.M., Y.P., F.D., A.N., M.Zo., E.S. and S.Mu. P.G., J.G. and M.C. coordinated the project.

Corresponding authors

Correspondence to Jérôme Gouzy or Pascal Gamas.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–6, Supplementary Tables 1 and 2, Supplementary Notes on genome sequencing and assembly; genome annotation; transposable elements and repeats; transcriptome analysis; analysis of symbiosis-related islands, and Supplementary References. Supplementary Table 6 (M. truncatula gene annotation, RNAseq data, MtV4 ID and affymetrix probe correspondence) can be found at https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/; downloads section.

Reporting Summary

Supplementary Table 3

Transduplicate analyses

Supplementary Table 4

miRNA analyses

Supplementary Table 5

siRNA analyses

Supplemental Table 7

Expression correlation analyses

Supplementary Table 8

Genes expressed in symbiosis-related islands

Supplementary Table 9

Conservation of symbiosis-related island genes in M. truncatula R108 genome

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pecrix, Y., Staton, S.E., Sallet, E. et al. Whole-genome landscape of Medicago truncatula symbiotic genes. Nature Plants 4, 1017–1025 (2018). https://doi.org/10.1038/s41477-018-0286-7

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing