Plant epigenetic regulations are involved in transposable element silencing, developmental processes and responses to the environment1,2,3,4,5,6,7. 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 activation9,10,11,12, 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.
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Raw reads from BsSeq and RNA-seq experiments have been deposited at the Sequence Read Archive (SRA) (project accession numbers: SRP355902 and SRP349933). Data related to gene annotation, TE annotation (Tephra-based, TASR10_round2_RepeatMasker–based and EDTA-based), methylome and DMR analyses are available at the M. truncatula genome portal and browser https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/. Source data are provided with this paper.
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We thank the Engineering Nitrogen Symbiosis for Africa (ENSA) project for providing plasmid backbones for Golden Gate cloning and M. truncatula root transformation. This work was supported by the Agence Nationale de la Recherche (ANR) grants EPISYM (ANR-15-CE20-0002) (P.G.) and Laboratoire d’Excellence (LABEX) TULIP (ANR-10-LABX-41) (P.G., J.G.), and made use of data previously generated in the ANR SYMbiMICS (ANR-08-GENO-106) (P.G.) and the INRA SPE ‘EPINOD’ projects (P.G.). The sequencing platform was supported by France Génomique National infrastructure (ANR-10-INBS-09) (O.B.). We are grateful to the Genotoul bioinformatics platform Toulouse Midi-Pyrenees (Bioinfo Genotoul) for providing computing and storage resources.
The authors declare no competing interests.
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Screenshot from the M. truncatula 5.0 genome browser (https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/), showing M. truncatula 5.1.8 annotation, root and nodule RNAseq, 24nt-siRNA mapping, BsSeq data, CG-CHG and CHH DMRs. DMRs were detected next to a gene specifically expressed in the nodule (MtNCR060).Transposable elements are represented as red horizontal bars in M. truncatula 5.1.8 annotations. 24nt-siRNA are from root, 4, 6 and 10 dpi nodule small RNA libraires13, depicted as brown, orange, pink and red bars, respectively. Methylation levels are shown for one biological replicate of root tips and nodule fixation zone, as purple, blue and orange vertical lines for the CG, CHG and CHH contexts, respectively. Hypo CG-CHG DMRs and hyper CHH DMRs (nodule fixation zone vs root tips) are depicted as green and orange horizontal bars, respectively.
The number of genes extended by 1 kb on each side and intersecting with CG-CHG DMRs (nodule fixation zone vs root tips) are represented by blue bars. The total number of nuclear genes per pattern are represented by grey bars.
Extended Data Fig. 3 Average mC profile of NDD genes with (DMR-plus; 1,723 genes) and without (DMR-minus; 2,572 genes) CG-CHG DMRs (Fix vs root tips), in whole root tips and 6 dpi nodules.
Two replicates of each sample. The metaplots were generated using Cytosines shared by all samples with a minimum coverage of 5 (9,870,327 and 12,178,901 Cs in the CG and CHG context, respectively). Gene sizes normalized to 3 kb. Note that the mC profiles of DMR-minus genes are indistinguishable in the root tip and nodule samples and exhibit a lower mC density than DMR-plus genes.
a, phylogenetic tree with Arabidopsis thaliana DRM1, DRM2 and DRM3 proteins and the four related M. truncatula proteins (Mt5.0 annotation). b, multiple alignment analysis. Conserved sites in DRM2-like proteins22 are boxed in blue and the ubiquitin-associated domains (UBA) found in DRM2 proteins (but not DRM3) are boxed in red (two in AtDRM2, one in MtDRM2 proteins). DRM3 proteins also lack invariant residues in sites IV, IX and X.
a, Position of the six guides used simultaneously for editing MtDRM2 and MtDRM2L2. b, Table showing the editing efficiency of each of the six guides. c, Representative example (out of 16 gels, each with 20 lanes) of a genotyping experiment with products of nested genomic PCR analyzed by agarose gel electrophoresis. Lane 1 is a control non-edited sample. Mono-guide edited amplicons run at the same position as the control while multi-guide editions can generate large deletions. The four non-labeled lanes correspond to the 1 kb Plus DNA ladder (Thermo Fisher). d, Representative example of the sequencing product of a mono-guide-edited (guide c) amplicon.
Extended Data Fig. 6 Analyses of CRISPR-Cas9-induced MtDRM2/MtDRM2L2 (drm) mutants and control samples.
a, number of nodules produced in drm mutants vs control transformed roots (drm / pAtUBI:DRM2-R, CRISPR-GUS, pAtUBI:DRM2-R), at 16 days post inoculation with S.meliloti 2011 nifH:GFP. Kruskal-Wallis non parametric test (chi-squared = 2.9866, df = 3, p-value = 0.3937). Dunn’s test of multiple comparisons using rank sums (α = 0.05) (two-sided tests). Boxes indicate the second and third quartiles, with median values indicated by a line, and whiskers represent the first and fourth quartiles. b, examples of 16 dpi root nodules induced by S.meliloti 2011 nifH:GFP, produced in roots transformed with CRISPR-GUS, CRISPR-DRM2/DRM2L2 (drm mutant), pAtUBI:DRM2-R, and CRISPR-DRM2/DRM2L2 pAtUBI:DRM2-R (complementation experiment). Nodulated root fragments analyzed using a stereomicroscope, with the left and right panels corresponding to bright field and fluorescence observations, respectively. The GFP scores were determined by counting the number of GFP + and GFP- nodules (score 1 and 4: ≤25% and 75-100% GFP + nodules, respectively). Nitrogen fixation efficiency of nodulated roots determined by an acetylene reduction assay (ARA), with calculation of ethylene production per nodule. pA*s = amplitude of detected ethylene per second (as provided by Agilent 7820 A gas chromatograph). Of note, nodule chains were occasionally observed in both drm and control samples. A total of 268 nodulated root fragments were analyzed both by stereomicroscopy and ARA, in three independent experiments.
Extended Data Fig. 7 Acetylene reduction assay for the complementation experiment of drm mutants (violin plots).
The following root fragments nodulated by S. meliloti nifH:GFP were tested, from left to right: roots transformed with CRISPR-DRM2/DRM2L2, with a GFP score <3 (<50% GFP + nodules, as determined using a stereomicroscope: drm2/drm2L2 biallelic mutations), with a GFP score >3 (no biallelic mutations); control CRISPR-GUS and pAtUBI:DRM2-R roots; drm2/drm2L2 mutants complemented by pAtUBI:DRM2-R. Boxes indicate the second and third quartiles, with median and mean values indicated as a line and a red dot, respectively. Whiskers represent the first and fourth quartiles. Non-parametric Games-Howell test (two-sided).
Extended Data Fig. 8 Screenshot of the Mt5.0 genome browser showing the expression of a TE located downstream of the MtNCR774 gene.
The predicted readthrough transcription from the NCR gene is visible with the log scale only (RNAseq data). Are also shown the alignment of 24nt-siRNAs siRNAs from root, 4, 6 and 10 dpi dpi nodule small RNA libraries13, depicted as brown, orange, pink and red bars, respectively, as well as corresponding siRNAs clusters (defined using Shortstack3.5.8, this study). CHH DMRs (Fix zone vs. root tips) are depicted as horizontal orange bars. BsSeq methylation data are shown for one replicate of root tip, nodule fixation zone, CRISPR-GUS and CRISPR-DRM2 nodule samples, with CG, CHG and CHH methylation levels represented as violet, blue and orange bars, respectively. Of note MtNCR774 shows a 4-fold down-regulation in drm mutant nodules vs. control CRISPR-GUS nodules.
Extended Data Fig. 9 DAPI-stained nodule sections show nucleus enlargement in the late nodule development zones but no genome-wide chromatin relaxation.
Representative images from confocal microscopy of 15 dpi nodules (n = 16). The right panels are a close up of the left panel (red boxes). Red arrows indicate ongoing cell divisions in zone I. Note the presence of well fluorescent chromocenters in nuclei of zone IIp, interzone II-III and zone III. Scale bars = 100 µm (left panels) or 40 µm (right panels).
Supplementary Tables 1, 8 and 9, and Note 1.
Supplementary Table 2. List of differentially methylated regions (DMRs) detected on the eight M. truncatula chromosomes when comparing the laser-dissected nitrogen-fixation zone (Fix) and root tip (RT) genomic DNA. Tab 1: CG context, RT versus Fix. Tab 2: CHG context, RT versus Fix. Tab 3: CG-CHG context, RT versus Fix. Tab 4: CHH context, Fix versus RT. Supplementary Table 3. Genes associated with CG-CHG DMRs (nodule fixation zone versus root tips). The genes shown are within 1 kb of a CG-CHG DMR. Nodule expression patterns were previously defined13. Those were defined by clustering analysis of the mean relative expression in five nodule zones [RNA-seq analysis of laser-dissected nodule ZI (zone I), meristematic region; ZIId: distal zone II, (pre)infection; ZIIp: proximal zone II, early differentiation; IZ: interzone II-III, late differentiation; ZIII: zone III, nitrogen-fixation]; a value of 0 indicates genes without statistically significant difference between gene expression levels in the five nodule zones. Supplementary Table 4. M. truncatula gene expression analyzed by RNA sequencing. Tab 1 and Tab 2: NDD genes with and without CG-CHG DMRs, respectively. Tab 3: genes putatively involved in DNA methylation and demethylation: RdDM machinery8, CMT2 and CMT3 chromomethylases with associated genes, and DNA demethylases (DME family4); Tab 4: histone H2A.W genes. RNA-seq data and expression patterns were previously described11,13. These patterns were defined from the mean relative expression in five nodule zones [RNA-seq analysis of laser-dissected nodule ZI (zone I), meristematic region; ZIId: distal zone II, (pre)infection; ZIIp: proximal zone II, early differentiation; IZ: interzone II-III, late differentiation; ZIII: zone III, nitrogen-fixation]. In Tab 3, except for the DME family, poorly expressed members of gene families were not indicated. CPM = EdgeR-normalized counts per million reads. LFC = log2(fold change). Supplementary Table 5. Differentially methylated regions (DMRs) detected on the eight M. truncatula chromosomes when comparing CRISPR-GUS and CRISPR-MtDRM2/MtDRM2L2 nodules. Hairy root transformation. Two biological replicates per sample. Tab 1: CHH context. Tab 2: CHG context. Tab 3: CG context. Supplementary Table 6. Differentially expressed genes (DEGs) in CRISPR-DRM2/DRM2L2 versus CRISPR-GUS nodules. Three biological replicates per sample. LFC: log(fold change); CPM: counts per million read pairs. Tab 1: RNA-seq data, following Edge R normalization. Tab 2: GO enrichment analysis for genes downregulated in CRISPR-DRM2/DRM2L2 versus CRISPR-GUS nodules. Tab 3: GO enrichment analysis for genes upregulated in CRISPR-DRM2/DRM2L2 versus CRISPR-GUS nodules. Nodule expression patterns were previously described;13 a value of 0 indicates genes without statistically significant difference between gene expression levels in the five nodule zones. In column B, negative distances indicate upstream DMRs. Gene names are from release 20210906. Supplementary Table 7. Transposable and repeat elements (TEs) expressed in M. truncatula nodules 10 dpi. Only the TEs mapped on the eight M. truncatula chromosomes and with a spatial expression pattern (RG Pattern: repeat and gene pattern) are shown. Tab 1: RNA-seq data from laser-dissection nodule zone I (ZI), zone II distal (ZIId), zone II proximal (ZIIp), interzone II-III (IZ) and zone III (ZIII) (mean of three biological replicates11 remapped on Mt5.1.8 genome release, and quantified using Salmon) and differential expression analysis as compared to zone I; columns S-W: percentage of total expression in the five nodule zones. Tab 2: definition of RG patterns, with the average percentage of total expression in the five nodule zones for each pattern. Tab 3: TE families expressed in nodules versus all M. truncatula TE families.
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Pecrix, Y., Sallet, E., Moreau, S. et al. DNA demethylation and hypermethylation are both required for late nodule development in Medicago. Nat. Plants 8, 741–749 (2022). https://doi.org/10.1038/s41477-022-01188-w