Medicago histone H3.1 patterns reveal sustained mitotic and endocycling activities in a symbiotic context.

(A) Schematic representation of histone H3.3-mCherry and H3.1-eGFP distribution (horizontal bars) and fluorescence intensity (grey saturation) throughout the different cell-cycle phases (adapted from Echevarría et al., 2021). H3.1 is predominantly expressed during S-phase and incorporated during DNA replication in proliferating (G1, S, G2, M) and endocycling (G1, S, G2) cells. H3.3 is constitutively produced. Nuclei of increasing size and DNA content are coloured according to their H3.3 (pink) and H3.1 (green) content in decondensed chromatin (G1, S, G2) or condensed chromosomes (M). This diagram also illustrates cells exiting the cell cycle within proliferating or endoreduplicating populations, which leads to a significant H3.1 eviction following their last DNA replication round (Otero et al., 2016). (B) Confocal images of nodule sections isolated from WT transgenic roots inoculated with mCherry-producing S. meliloti (35 to 40 dpi), showing H3.1-eGFP (green) and H3.3-mCherry (magenta) localization across characteristic developmental zones. H3.1 accumulates in the meristematic zone (ZI) enriched in proliferating cells and in regions with high endoreduplication activity (ZIId and ZIIp, distal and proximal parts of the infection zone) where rhizobia are released into membrane-bound compartments called symbiosomes. H3.1 is extensively replaced by H3.3 in the fixation zone (ZIII) where host cells and rhizobia complete their differentiation process. IT: infection thread. Ana: late-anaphase chromosomes. Filled or empty arrowheads point to nuclei with a high (ZIIp) or a low (ZIII) H3-1-eGFP content, respectively. Dashed lines demarcate infected nodule cells containing symbiosomes. Images are maximum intensity projections except the top-right panels (single focal plane). Scale bars: left panel = 50 μm; right panels = 10 μm. Transformation experiments were repeated 3 times with a total of 9 nodules from 6 composite plants showing similar results. (C) Confocal images of whole-mount transgenic roots colonized by Rhizophagus irregularis (15 dpi). Plant and fungal cell walls were stained with Calcofluor white (grayscale). H3.1 is enriched in chromocenters (heterochromatin foci in nuclei indicated by stars) and kept at high levels in the euchromatin (diffuse labeling in nuclei pointed by filled arrowheads) from neighbouring (upper panel) and early-arbusculated cells (lower panel) of the inner cortical tissue. The empty arrowhead points to a nucleus with a low H3.1 content in a fully-arbusculated, differentiated cell. Scale bars: 50 μm. Two independent transformation experiments were performed with 3 to 5 composite plants analyzed per replica. (B-C) The eGFP and mCherry channels are shown in Green Fire Blue when isolated, with blue or yellow indicating low or high fluorescence levels, respectively. CD: cell division. En: endoreduplication. TD: terminal differentiation.

Individual reprogramming for infection includes large-scale chromatin rearrangements.

(A-B) Confocal images of whole-mount WT roots expressing the pH3.1::H3.1-eGFP / pH3.3::H3.3-mCherry reporter and inoculated with mCherry-producing S. meliloti (8 dpi). Images are maximum intensity projections (eGFP: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The eGFP and mCherry channels are shown in Green Fire Blue when isolated. (A) Upper panels: view of inner and middle cortical regions of the early nodule primordium shown in lower panels. Lower panels: view of the outer cortical layers crossed by an infection thread (IT). Dashed lines demarcate nuclei of infected cells (IC) fully passed by a cortical infection thread in the first (C1) and second (C2) cortical layers. NC: neighbouring cell. The filled arrowhead points to the nucleus of a host cell from the middle cortical layer (C3) which is penetrated by the IT. The upper and lower panels are from the same image. Scale bars: 20 μm. (B) Upper panels: dashed lines indicate nuclei of infected cells (IC) being passed (C4) or recently passed (C3) by a cortical infection thread. Lower panels: dashed lines demarcate nuclei of infected cells in the inner cortex (C4/5) of a nodule primordium with several cell layers. Scale bars: 20 μm. (C-D) Quantification of the nuclear area (C) and corrected total H3.1-eGFP nuclear fluorescence (D) at the equatorial plane in couples of neighbouring (NC) and infected cells (IC) from the same cortical layer (C2 to C4, n = 27; see Materials and methods for more details). Roots from 2 to 7 composite plants with visible signs of inner cortical cell division (i.e., showing high H3.1-eGFP signal) were analyzed from 3 independent transformation experiments. All data points are shown and crosses indicate sample means. Differences were statistically significant (p-values < 0.0001) using an unpaired t-test with Welch’s correction (C) or a Mann-Whitney test (D).

H3.1 eviction coincides with cortical infection thread progression.

(A) Confocal images of whole-mount transgenic roots co-expressing H3.1-eGFP and H3.3-mCherry in three different genotypes: WT (11 dpi; upper panels), daphne-like (14 dpi; middle panels) and nf-ya1-1 (12 dpi; lower panels), inoculated with mCherry-producing S. meliloti. WT and nf-ya1-1 plants initiate nodule formation (NOD+) whereas the daphne-like mutant is non-nodulating (NOD-). Numbers indicate the frequencies of observation of a low (WT) or high (daphne-like, nf-ya1-1) H3.1-eGFP content in the last infected cortical cell. Corresponding schematic representations depict cellular proliferation or endocycling activities indicated by nuclear H3.1 levels (low: magenta; high: green) on the cortical infection thread trajectory (C1 to C3; diffuse magenta or green colouring) and in inner cortical layers (C4 to C5; see also Figure 3-figure supplements 1-3). Rhizobia inside infection threads are depicted in red. Confocal images show merged fluorescent channels (eGFP: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale in the middle and lower panels). The eGFP and mCherry channels are shown in Green Fire Blue when isolated. Scale bars: 20 μm. IC: infected cell. Ep: epidermis. (B-C) Quantification of the nuclear area (B) and the relative H3.1-eGFP content (IC/NC; corrected total nuclear fluorescence in arbitrary units) (C) at the equatorial plane in couples of neighbouring (NC) and infected cells (IC) from the same cortical layer in WT (C1 to C4), daphne-like (C1 to C2) and nf-ya1-1 (C1 to C4) genetic backgrounds (n = 43, 19 and 39 nuclei, respectively). Different letters indicate statistically significant differences according to a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (B) All data points are shown and crosses indicate sample means. (C) All data points are shown and horizontal bars indicate sample means with 95% confidence interval. Ten to 21 composite plants (8-14 dpi) from 2 (daphne-like, nf-ya1-1) to 7 (WT) independent transformation experiments were analyzed.

The reduction in the proliferative potential of infected cortical cells is supported by the Arabidopsis PlaCCI reporter.

(A) Schematic representation of Arabidopsis CDT1a-eCFP, H3.1-mCherry and N-CYCB1;1-YFP distribution (horizontal bars) and fluorescence intensity (grey saturation) throughout the different cell-cycle phases (adapted from Echevarría et al., 2021; see also Desvoyes et al., 2020). CDT1a-CFP accumulates in G1 and is rapidly degraded during the G1/S transition (blunt end bar). H3.1 is predominantly expressed during S-phase and incorporated during DNA replication. N-CYCB1;1-YFP is present in late G2 and mitotic cells and is completely degraded in anaphase (blunt end bar). Nuclei of increasing size and DNA content are coloured according to their CDT1a (cyan), H3.1 (red) and N-CYCB1;1 (yellow) content in/around decondensed chromatin (G1, S, G2) or condensed chromosomes (M). H3.1 levels decrease in differentiating cells (dashed line box). (B-C) Confocal images of whole-mount Medicago WT roots expressing the Arabidopsis PlaCCI reporter in nodule primordia at 7 dpi with mCherry-(B) or GFP-producing S. meliloti (C). Images show merged fluorescent channels (eCFP / GFP: cyan; mCherry: red; YFP: yellow; Calcofluor white cell-wall staining: grayscale). Individual channels (eCFP / GFP, mCherry, YFP) are shown in Green Fire Blue when isolated. (B) Upper panels: the thin dashed line demarcates the nucleus of an infected cell (IC) from the C3 layer penetrated by an infection thread. Cell-cycle phases of non-infected neighbouring cells are indicated. The diffusion of the N-CYCB1;1-YFP marker outside the nucleus allows visualization of the phragmosome transvacuolar bridge formed in preparation for mitosis (stars). A cytoplasmic accumulation of the same marker (YFP channel) is visible after nuclear envelope breakdown in a prophase (Pro) cell showing condensing chromosomes (mCherry channel). Scale bar: 20 μm. Lower panels: schematic representations illustrating the different phases of the cell cycle visible above the thick dashed line, revealed by the PlaCCI reporter in the upper panels. Cells engaged in recurrent cell division cycles (G1 late, S, G2 late, prophase) maintain a high level of H3.1 (red). The infected cell (IC) is distinguished by the absence of visible CDT1a-CFP (cyan), a reduced level of H3.1 and a very low level of N-CYCB1;1-YFP (yellow) compared to neighbouring cells. (C) The dashed line demarcates the nucleus of an infected cell (IC) from the C3 layer being passed by a cortical infection thread. Numbers indicate the frequencies of observation of the absence of CDT1a- or N-CYCB1;1-associated signals in the nucleus of the last infected cortical cell. Scale bar: 20 μm. (D) Fluorescence intensity profiles of CDT1a-, H3.1- and N-CYCB1.1-associated signals along the cyan and green transects shown in (C).

A tight control over host cells’ mitotic commitment enables passage of the future nodule meristem.

(A) Left panels: confocal images of a whole-mount WT root co-expressing a destabilized triple-Venus nuclear reporter driven by the Arabidopsis CYCB1;2 promoter (pCYCB1;2::N-CYCB1;2-NLS-3xVenus) and a nuclear-localized tandem-mCherry as a transformation marker, 12 dpi with mCherry-producing S. meliloti. Numbers indicate the frequencies of observation of the absence of the triple-Venus reporter signal in the nucleus of the last infected cortical cell. Two independent transformation experiments were performed with 3 to 4 composite plants analyzed per replica. (B) Left panels: confocal images of a whole-mount WT root co-expressing a transcriptional reporter of Medicago KNOLLE driving a nuclear-localized triple-Venus (pKNOLLE::NLS-3xVenus) together with a nuclear-localized tandem-mCherry as a transformation marker, 12 dpi with mCherry-producing S. meliloti. Numbers indicate the frequencies of observation of nodule primordia where the triple-Venus reporter signal is kept comparably low on the cortical infection thread trajectory. A total of 12 composite plants from 2 independent transformation experiments were analyzed. (A-B) Images show merged fluorescent channels (Venus: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale in the upper panels). The Venus channel is shown in Green Fire Blue when isolated in lower panels. The dashed lines indicate nuclei of infected cells (IC) from the C3 layer being passed by a cortical infection thread. The fluorescence intensity profiles of pCYCB1;2 and pKNOLLE reporter-associated signals along the green transects are shown in the corresponding right panels. NC: neighbouring cell. NOD: nodule primordium. Scale bars: 20 μm.

Dividing cortical cells initiating Medicago nodule primordium formation are tetraploid.

(A) Confocal images of whole-mount WT transgenic roots expressing mCitrine-CENH3 under the control of native (M.t. pCENH3; left and middle panels) or constitutive promoters (pLjUbi; right panel). Simultaneous expression of H3.3-mCherry enables the recognition of condensed (star) and segregating chromosomes (middle panel). mCitrine-CENH3 labels the centromeric region of individual chromosomes (filled arrowheads). The number of CENH3-labeled foci determined across image stacks is indicated in yellow. Images show merged fluorescent channels (mCitrine: Green Fire Blue; mCherry and Calcofluor white cell-wall staining: grayscale). In the Green Fire Blue colour scheme, blue or yellow indicate low or high fluorescence levels, respectively (min. to max. = 1-140 in the middle panel). Scale bars: left and right panels = 10 μm; middle panel = 2 μm. (B) Quantification of the number of centromeric signals in transgenic root tips expressing mCitrine-CENH3 under native (pCENH3) or constitutive (pLjUbi) promoters (n = 55 nuclei). All data points are shown and are from 2 (pLjUbi) to 3 (pCENH3) independent experiments with 7 composite plants analyzed per construct. Horizontal bars indicate sample means with 95% confidence interval. Differences were statistically significant (p-value = 0.0006) using a Mann-Whitney test. (C) Schematic representation of an early nodule primordium where anticlinal cell divisions occurred in the pericycle (P; dark violet) and cortical cells (C2 to C5; light violet). Rhizobia inside the infection thread are depicted in red. Ep: epidermis. En: endodermis. (D) Maximum intensity projections of an early nodule primordium at the developmental stage represented in (C), formed in a WT root expressing the pLjUbi::mCitrine-CENH3 / pH3.3::H3.3-mCherry construct at 7 dpi with mCherry-producing S. meliloti. Dashed lines indicate the contours of divided cells in the inner (C4) and middle (C3) cortical layers. The number of CENH3 - labeled foci, determined across image stacks and indicated in yellow are: 11 and 16 in undivided, infected cells (IC) from the outer cortical layers (C1; left panel and C2, right panel) and 25 to 28 in divided, uninfected cells from the C3 (filled arrowhead; left panel) and C4 layers (right panel). Images show merged fluorescent channels (mCitrine: Green Fire Blue; mCherry and Calcofluor white cell-wall staining: grayscale). Scale bars: 20 μm. (E) Quantification of the number of centromeric signals in early nodule primordia at the developmental stage depicted in (C), formed in inoculated WT roots (7 to 12 dpi) constitutively expressing mCitrine-CENH3. The number (n) of analyzed nuclei in each cell layer is indicated on the top. E: endodermis. P: pericycle. All data points are shown with black or violet symbols indicating undivided or divided cells, respectively. NI: non-infected cell (discs). I: infected cell (triangles). Horizontal dotted lines are positioned at y = 16 and y = 32, corresponding to diploid (2n = 16) or tetraploid (4n = 32) cellular states in Medicago truncatula. Data are from 2 independent experiments with 7 nodule primordia from 5 composite plants analyzed.

Primary infected nodule primordium cells do not reach full competence for chromosome segregation.

(A) Schematic representation of CENH3 deposition at centromeres (blue dots) throughout the different pre-mitotic (G1, S, G2 early and G2 late) cell-cycle phases in plants. Nuclei of increasing size and DNA content are depicted as ovals. The dashed line box indicates the timing of CENH3 loading at replicated centromeres, occurring during G2 before sister kinetochore (blue doublets) split in preparation to mitosis (Lermontova et al., 2007). Right panel: confocal image of a late G2-phase cell observed in a nodule (NOD) in an inoculated WT transgenic root (12 dpi) constitutively expressing mCitrine-CENH3. The majority of fluorescent signals appear as doublets (encircled by dotted lines) corresponding to sister kinetochores. Scale bar: 5 μm. (B) Confocal images of whole-mount WT roots expressing the pLjUbi::mCitrine-CENH3 / pH3.3::H3.3-mCherry construct and inoculated with mCherry-producing S. meliloti (7 to 12 dpi). Filled arrowheads point to nuclei of infected nodule primordium cells. Dotted lines encircle mCitrine-CENH3 doublets appearing as twin spots on the same focal plane. Neighbouring cells in late G2 or undergoing mitosis (M) are indicated. The presence of CENH3-labeled twin spots was assessed across image stacks in the nuclei of infected cells from the middle (C3) and inner (C4/5) cortical layers and colour-coded as follows: grey = no doublet (upper panels); light blue = 1 to 3 doublets (lower left panel); dark violet = 11 or more doublets (lower right panel). Scale bars: 5 μm. (A-B) Images show merged fluorescent channels (mCitrine: Green Fire Blue; mCherry and Calcofluor white cell-wall staining: grayscale). (C) Quantification of the nuclear area at the equatorial plane in cells being passed by a cortical infection thread (infected cells) in nodule primordia formed by WT transgenic roots inoculated with S. meliloti (7 to 12 dpi). The number (n) of analyzed nuclei in each cell layer (C3 to C4/5) is indicated on the top. The 8C chromatin-value is given for uninfected nodule primordium cells in late G2 showing close to 32 mCitrine-CENH3 doublets as identified in (A) and (B). All data points are shown and crosses indicate sample means. Differences were not statistically significant according to a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. At least 9 composite plants from 2 independent experiments were analyzed. (D) Quantification of infected cells in C3 (n = 13) and C4/5 (n = 53) cortical layers in nodule primordia, showing no doublet (grey), 1 to 3 doublets (light blue) or more than 11 doublets (dark violet) labelled by mCitrine-CENH3. Nine composite plants from 2 independent experiments were analyzed. (E) Quantification of the length of mCitrine-CENH3 doublets appearing as twin spots on the same focal plane in early G2 (n = 12), late G2-phase cells (n = 16) and infected cells (IC) from the C4/5 cortical layer (n = 13) in nodule primordia. n = 28 (G2early), 94 (G2late) and 21 (IC C4/5) doublets. Horizontal bars indicate sample means with 95% confidence interval. Differences were not statistically significant according to a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. All data points are shown and are from 5 to 7 composite plants from 2 independent transformation experiments.

NF-YA1 specifically reduces the expression of a G2/M gene in a cell-division enabled system.

(A) Live-cell confocal images of N. benthamiana epidermal cells ectopically expressing eGFP-NF-YA1 variants: WT (left panel) or impaired in DNA recognition (mutDBD; right panel). Both fusion proteins accumulate in the nucleus and were used in fluorometric β-glucuronidase (GUS) assays. Images show merged fluorescent (eGFP: green) and bright field (BF) channels. Scale bars: 10 μm. (B) Schematic representation of the cell division-enabled leaf system (CDELS) on the scale of epidermal pavement cells in N. benthamiana. Differentiated cells in mature leaves (left panel) re-enter the canonical cell cycle upon ectopic expression of the Arabidopsis D-type cyclin CYCD3;1 (right panel). Nearly all re-activated pavement cells (diffuse yellow colouring) have completed cytokinesis after 3 days (filled arrowheads). See also Figure 8 – figure supplement 1B. Scale bars: 20 μm. (C) Left panel: schematic representation of the CDEL system on the scale of the canonical cell cycle. Ectopically-expressed CYCD3;1 targets and activates cyclin-dependent kinases (CDK, not shown), fostering the G1/S transition. Progression throughout the different cell-cycle phases is accompanied by the sequential activation of DNA synthesis (S) and mitotic genes (M). Histone H4 and KNOLLE promoter-reporters were selected as readouts for G1/S and late G2/M transcriptional waves, respectively. Right panel: fluorometric GUS assay in tobacco leaf cells expressing the Medicago pKNOLLE::GUS (pKNOLLE) reporter construct in the absence or presence of ectopically-expressed CYCD3;1. Fluorescence curves (GUS-mediated hydrolysis of 4-MUG) over time are shown for 4 biological replicates. Error bars indicate standard deviation. (D) Activity of GUS driven by the histone H4 promoter (pH4) in the absence or presence of ectopically-expressed eGFP-NF-YA1 (NF-YA1) in CDEL samples (+ CYCD3;1). All data points are shown and crosses indicate sample means. Differences were not statistically significant (p-value = 0.3323) according to an unpaired t test with Welch’s correction. Data are from 3 independent transformation experiments. n = 12 ([-] NF-YA1) and 12 ([+] NF-YA1) biological samples. (E) Activity of GUS driven by the KNOLLE promoter (pKNOLLE) in CDEL samples (+ CYCD3;1) in the absence or presence of ectopically-expressed eGFP-NF-YA1 variants with WT (NF-YA1) or mutated DNA-binding domain (NF-YA1mutDBD). All data points are shown and crosses indicate sample means. Statistically significant differences ([-] NF-YA1 versus [+] NF-YA1; p-value < 0.0001) or non-significant differences ([-] NF-YA1 versus [+] NF-YA1mutDBD; p-value = 0.7861) are based on a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Data are from 4 to 5 independent transformation experiments. n = 18 ([-] NF-YA1), 16 ([+] NF-YA1), 16 ([+] NF-YA1mutDBD) biological samples.

Ectopic expression of NF-YA1 influences both cortical cell division on the trajectory of rhizobial infection and infection thread structure in WT transgenic roots.

(A-E) Data were generated in Medicago WT roots expressing the KNOLLE G2/M transcriptional readout (pKNOLLE::D-box-3xVenus-NLS), without (Control) or with additional production of NF-YA1 on the cortical infection thread trajectory (pENOD11::NF-YA1), 3 to 6 dpi with S. meliloti. Two independent transformation experiments were performed. (A) Schematic representation illustrating the anticlinal division of an outer cortical cell (C2) positioned on the infection thread trajectory (diffuse magenta colouring). Rhizobia inside the infection thread are depicted in magenta. (B) Left panel: quantification of infected cells in outer cortical layers (C1 to C2) of early nodule primordia (stage I to III), divided anticlinally (grey) or undivided (white) in control transgenic roots or roots ectopically expressing Medicago NF-YA1 (pENOD11::NF-YA1). The number (n) of cells analyzed in each transgenic background is indicated on the top. Right panel: table recapitulating the number and the cortical location of divided (split) infected cells in each transgenic background. Data are from 5 to 11 composite plants, with 9 (Control) to 21 (pENOD11::NF-YA1) nodule primordia analyzed. (C) Schematic representation illustrating the general structure of a cortical infection thread, with 2 (C1), 1 (C2) or no additional branch (0; C3) outside the main infection thread. Rhizobia are depicted in magenta. (D) Quantification of the number of infection thread (IT) tips outside the main IT, within individual cortical cells being passed or already fully crossed. All data points are shown and horizontal bars indicate sample means with 95% confidence interval. Differences were not statistically significant (p-value = 0.2814) according to a Mann-Whitney test. (E) Quantification of infected cortical cells in control transgenic roots or roots ectopically expressing NF-YA1, showing 2 or more (grey) or only 1 (white) additional IT tip outside the main IT. (D-E) n = 50 (Control) or 96 (pENOD11::NF-YA1) infected cortical cells (C1 to C4). Data are from 8 to 14 composite plants, with 14 (Control) to 28 (pENOD11::NF-YA1) nodule primordia analyzed.

Model illustrating characteristic cellular traits on the cortical infection thread trajectory.

(A) Cortical cells competent for sustained transcellular infection thread progression are characterized by a reduced proliferation or endoreduplication activity (pink to green two-colour gradient). By the time they are crossed and ultimately internalize bacteria, such tetraploid cells reach a post-replicative, 8C ploidy stage. But, contrary to direct neighbour cells, they do not commit to a subsequent cell division (red to white two-colour gradient). (B) Rather, cells supporting transcellular infection may enter a prolonged G2-phase during which they remodel their histone H3 composition as part of their cellular reprogramming for infection. This differentiation process and concurrent transcellular passage are accomplished within a last cell-cycle round. We propose that cellular factors controlling the G2/M transition and eventually the exit from the canonical cell cycle (dashed arrow) are prominent candidates to support intracellular infection competence.

Labelling patterns of selected Medicago histone H3 variants in transgenic roots.

(A) List of genes encoding for identical H3.1 and H3.3 proteins in Medicago truncatula (Medicago). Accession numbers are given for the Mt4.0v1 and A17 r5.1.9 genome versions. The size of promoters used to express H3.1, H3.1 (2) and H3.3 are indicated in base pairs (bp). (B) Alignment of H3 primary sequences from Arabidopsis thaliana (A.t.H3.1; P59226), Oryza sativa (O.s.H3.1; Q2RAD9), Hordeum vulgare (H.v.H3.1; F2CQJ8), Lotus japonicus (L.j.H3.1; AFK42592.1) and Medicago (M.t.H3.1; B7FN22 and M.t.H3.3; G7I5Z5). Protein accessions are given as GenBank (L. japonicus) or UniProt identifiers (all other sequences). Multiple sequence alignment (MUSCLE with default parameters) and conservation calculations were performed using Jalview (Procter et al., 2021). Amino acids are coloured according to the Percentage Identity scheme. Conservation scores from 4 to 9 highlight the four residues that differ between H3.1 and H3.3 (A31T, F41Y, S87H, and A90L|S). Positions of the N-terminal α-helix (αN) and of the three α-helices (α1, α2, and α3) forming the histone fold domain are indicated and were obtained from HistoneDB 2.0 (Draizen et al., 2016). (C-D) Confocal images of whole-mount WT transgenic roots stained with Calcofluor white (grayscale) and producing the selected H3.3 variant fused to mCherry (magenta) together with either H3.1 (C) or H3.1 (2) (D) proteins fused to eGFP (green). H3.1-eGFP fusions accumulate in a patchy pattern in cells from the meristematic (MZ) and elongation zones (EZ). H3.3-mCherry is constitutively present in cells from all tissues including the differentiated root cap (RC). Scale bars: 50 μm. (E) Confocal images of a WT transgenic root tip producing H3.1-eGFP and H3.3-mCherry. The box indicates the region shown in the close up. H3.1-eGFP marks subnuclear foci corresponding to chromocenters (nuclei indicated by stars). The dashed line demarcates the putative quiescent center (QC) region, with low proliferative potential and almost exclusively labelled with H3.3-mCherry. The full- and close-up views were acquired at two different magnifications. Scale bars: 50 μm. (C-E) Images show merged fluorescent channels (eGFP: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The eGFP and mCherry channels are shown in Green Fire Blue when isolated. Twenty composite plants from 7 independent transformation experiments showed a similar H3.1 / H3.3 pattern. (F) Expression profile of selected H3.1 and H3.3 genes in mock or S. meliloti (S.m.) spot-inoculated roots, the latter forming nodule primordia of several cell layers at 24 hpi (Schiessl et al., 2019). Stars indicate statistically significant differences (p < 0.05) using a Mann-Whitney test. (G) Expression profile of selected H3.1 and H3.3 genes in mature nodule regions isolated by laser-capture microdissection (Roux et al., 2014). ZI: apical zone with meristematic activity. ZIId and ZIIp: distal and proximal parts of the infection zone. IZ: interzone preceding full nitrogen fixation. ZIII: central tissue with differentiated host cells and nitrogen-fixing bacteroids. nd: not detected. Different letters indicate statistically significant differences according to a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (F-G) All data points are shown and were retrieved from MtExpress V3 (Carrere et al., 2021).

A similar localization pattern and reduced H3.1 levels in infected cells are observed using a different H3 gene.

(A-C’’) Confocal images of whole-mount WT roots expressing the pH3.1(2)::H3.1(2)-eGFP / pH3.3::H3.3-mCherry construct and inoculated with mCherry-producing S. meliloti (7 dpi). Images show merged fluorescent channels (eGFP: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The eGFP and mCherry channels are shown in Green Fire Blue when isolated. (A-A’’) Cell division activity within an emerging lateral root (filled arrowhead) or inner cortical layers of an early nodule primordium (star) is associated with the incorporation of H3.1(2)-eGFP. Scale bar: 100 μm. (B-B’’) View of inner cortical cells (C4/5) in a nodule primordium with several cell layers. Nuclei show different levels of H3.1(2)-eGFP fluorescence, indicative of cells at different phases of the canonical cell cycle (see also Figure 1A). The H3.1(2) eGFP-fusion protein is visible in chromocenters (heterochromatin foci in the nucleus indicated by a star), in decondensed chromatin (diffuse labeling in the nucleus pointed by a filled arrowhead) and in condensed chromosomes (empty arrowhead). Note that H3.3-mCherry appears enriched in chromosome extremities (including telomeres) as reported in Arabidopsis (reviewed in Probst et al., 2020). Scale bar: 10 μm. (C-C’’) Dashed lines indicate nuclei of infected cells (IC) passed (C2) or recently passed (C3) by a cortical infection thread, showing lower levels of H3.1-eGFP fluorescence compared to directly adjacent cells. Scale bar: 20 μm. (D-E) Fluorescence intensity profiles of eGFP signal along the cyan (D) or yellow (E) transects shown in (C). Four to 5 composite plants from 2 independent transformation experiments were analyzed.

Infected outer cortical cells do not evict H3.1 when infection and organogenesis processes are uncoupled.

(A-C’’) Confocal images of whole-mount roots of WT composite plants expressing the pH3.1::H3.1-eGFP / pH3.3::H3.3-mCherry construct and inoculated with mCherry-producing S. meliloti. Images show merged fluorescent channels (eGFP: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The eGFP and mCherry channels are shown in Green Fire Blue when isolated. (A-A’’) the box indicates the outer and middle cortical regions of a WT nodule primordium (8 dpi; NOD+) shown in the close up (B-B’’). The full- and close-up views were acquired at two different magnifications. Scale bar: 50 μm. (B-B’’): dashed lines demarcate nuclei of infected cells (IC, H3.1 low) fully passed by a cortical infection thread in the second (C2) and third (C3) cortical layers. Stars indicate periclinal cell divisions in the middle (C3) layer that will give rise to the nodule meristem. Nuclei show different levels of H3.1-eGFP fluorescence, indicative of cells at different phases of the canonical cell cycle. Scale bar: 20 μm. (C-C’’) Confocal image of a WT inoculated root (14 dpi) showing an infection thread aborted in the C2 layer (filled arrowhead), with the nucleus in the contacted cell (CC) positioned closer to the basal membrane. Very few anticlinal divisions (arrows) occurred in the inner layers (NOD-). Numbers indicate the frequencies of observation of a high H3.1-eGFP signal in the nucleus of the last infected cortical cell. Scale bar: 50 μm.

Sustained cell-cycle activity in the outer cortical layers is a characteristic of the non-nodulating daphne-like mutant.

(A-C’’) Confocal images of whole-mount roots of daphne-like composite plants expressing the pH3.1::H3.1-eGFP / pH3.3::H3.3-mCherry construct and inoculated with mCherry-producing S. meliloti. Images show merged fluorescent channels (eGFP: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The eGFP and mCherry channels are shown in Green Fire Blue when isolated. (A-A’’) Maximum intensity projection of an inoculated root (14 dpi) of the non-nodulating daphne-like mutant (NOD-). The presence of H3.1 in the outer layers (filled arrowheads) is indicative of a region with sustained mitotic or endocycling activity. Scale bar = 100 μm. (B-B’’) The box indicates the epidermal and outer cortical regions of an inoculated root (14 dpi) shown in the close up. The full- and close-up views were acquired at two different magnifications. (C-C’’) The filled arrowhead points to an arrested infection thread with a thin, bacterial-free cell-wall extension in the first cortical layer (C1; IC with high H3.1). Scale bars: 20 μm.

Maintenance of H3.1 in infected cells in the nf-ya1-1 mutant coincides with a suboptimal state of cell division and progression of the cortical infection thread.

(A-B) Confocal images of whole-mount roots of nf-ya1-1 composite plants expressing the pH3.1::H3.1-eGFP / pH3.3::H3.3-mCherry construct and inoculated with mCherry-producing S. meliloti. Images show merged fluorescent channels (eGFP: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The eGFP and mCherry channels are shown in Green Fire Blue when isolated. (A) Upper panels: full-view of the confocal image presented in Figure 3A (nf-ya1-1, 12 dpi). The filled arrowhead points to an abnormally bulbous infection thread in the second cortical layer (C2; IC with high H3.1) on top of a nodule primordium (NOD+) with low cell division activity maintained in inner layers (empty arrowhead). Scale bar: 50 μm. Middle panels: the box indicates the middle and inner cortical regions of an early nodule primordium (8 dpi) shown in the close up. The highest frequency of cell divisions (anticlinal) occurred in the C5 layer (empty arrowhead). The full- and close-up views were acquired at two different magnifications. Lower panels: the dashed line demarcates the nucleus of an infected cell being passed by an infection thread in the middle (C3) layer. Numbers indicate the frequencies of observation of a low H3.1-eGFP signal in the nucleus of the last infected cortical cell. Scale bars: 20 μm. (B) Upper and middle panels: maximum intensity projections of an early nodule primordium (NOD+) formed in an inoculated nf-ya1-1 transgenic root (8 dpi). Anticlinal cell divisions occurred in the C5, C4 and occasionally in the C3 layer (empty arrowheads in the upper panels). Middle panels: the box indicates the outer cortical regions (C1, C2) shown in the close up. The full- and close-up views were acquired at two different magnifications. Lower panels: the filled arrowhead indicates an infection thread aborted in the C1 layer, with the nucleus in the contacted cell (CC) positioned closer to the basal membrane. Numbers indicate the frequencies of observation of a high H3.1-eGFP signal in the nucleus of the last infected cortical cell. Scale bars: 20 μm.

KNOLLE and CYCB1;2 transcriptional reporters highlight individual cells preparing for mitosis in Medicago.

(A) Confocal images of a whole-mount Medicago WT root expressing an Arabidopsis CYCB1;2-based fluorescent reporter. The CYCB1;2 promoter (pCYCB1;2) contains several mitosis-specific activator (MSA) cis-elements and is active in late G2 and early M phase. The destruction box (D-box) motif in the N-terminal domain (N-CYCB1;2) ensures the proteolytic degradation of the triple-Venus reporter during anaphase. The pCYCB1;2::N-CYCB1;2-NLS-3xVenus reporter shows a characteristic patchy pattern, reflecting mitotic activity in the root apical meristem (left panel) and labels individual cells that transit through G2/M (right panel) with only occasional, incomplete post-mitotic degradation of the triple-Venus reporter (star). Pro: prophase. Meta: metaphase. Scale bars: 100 μm (left panel); 20 μm (right panel). Images show merged fluorescent channels (Venus: green; Calcofluor white cell-wall staining: grayscale). (B) Upper panel: schematic representation of the Medicago KNOLLE promoter (M.t. pKNOLLE). The three closely-spaced mitosis-specific activator (MSA) cis-elements present in the proximal promoter region are indicated by yellow boxes. Locations of the core MSA motifs (AACGG/CCGTT) are as follows, relative to the ATG start codon: MSA1 = −303 to −307; MSA2 = −335 to −339; MSA3 = −358 to −362. Lower panel: transactivation assay in N. benthamiana leaf cells co-expressing an activator-type MYB3R transcription factor (NtmybA2Δ630) and one of the following target promoter::GUS fusions: Medicago pKNOLLE (native or MSA1-3 point-mutated) or Arabidopsis pCYCB1;2 (native) acting as a positive control. All data points shown correspond to 6 biological replicates. Crosses indicate sample means. Statistically significant differences (pKNOLLE native versus point-mutated; p-value = 0.0003) and non-significant differences (pKNOLLE native versus pCYCB1;2 native; p-value = 0.5390) are based on a Brown-Forsythe and Welch ANOVA test followed by Dunnett’s multiple T3 comparisons test. (C) Confocal images of whole-mount WT roots expressing a destabilized triple-Venus nuclear reporter (fused in-frame to the CYCB1;1 D-box domain) under the control of the native Medicago KNOLLE promoter. A nuclear-localized tandem-mCherry was used as a transformation marker. The pKNOLLE::D-box-3xVenus-NLS construct shows a characteristic patchy pattern, reflecting its G2/M activation in the root apical meristem (left panel) and labels individual late G2-phase cells (right panel) in the root cortex infected by mCherry-producing S. meliloti. By the time it is passed, the infected cell (IC) in the middle cortical layer does not express the KNOLLE G2/M transcriptional readout, by contrast to the directly adjacent cell. Dashed lines indicate cell’s contours visible in the bright field (BF) channel. Images show merged fluorescent channels (Venus: green; mCherry: magenta). The Venus channel is shown in Green Fire Blue when isolated. Scale bars: 100 μm (left panel); 20 μm (right panel).

A PlaCCI sensor optimized for identifying cells competent for infection highlights their pre-mitotic arrest during transcellular passage of the middle cortex.

(A-B) Confocal images of whole-mount Medicago WT roots expressing an alternative version of the PlaCCI sensor (named PlaCCI v2) in nodule primordia at 5 dpi with mCherry-producing S. meliloti. Images show merged fluorescent channels (mTurquoise2: cyan; mCherry: red; mCitrine: yellow; Calcofluor white cell-wall staining: grayscale). Individual channels (mTurquoise2, mCherry, mCitrine) are shown in Green Fire Blue when isolated. Scale bars: 20 μm. (A) Upper panels: the box indicates the middle and inner cortical regions of an early nodule primordium with few cell layers shown in the lower panels. Lower panels: dashed lines demarcate nuclei of an infected cell (IC) from the middle cortex (C3) recently passed by a cortical infection thread and of neighbouring cells in S- or late G2-phase. The star indicates the nucleus of a G1-phase cell located in the vascular cylinder, showing a high CDT1a-associated signal. The full- and zoomed views are from the same image. (B) Dashed lines indicate nuclei of an infected cell (IC) of the middle cortex (C3) being passed by an infection thread and of neighbouring cells (NC) in G1- or late G2-phase. Numbers indicate the frequencies of observation of reduced CDT1a- or N-CYCB1;2-associated signals in the nucleus of infected cortical cells (being crossed or recently crossed) compared to neighbouring cells. (C) Fluorescence intensity profiles of CDT1a-, H3.1- and N-CYCB1.2-associated signals along the magenta (IC), cyan and yellow transects (NC) shown in (B). The dashed black line indicates a baseline of fluorescence intensity (4 arbitrary units) for CDT1a- and N-CYCB1;2-associated signals in late G2- and G1-phase cells, respectively. The infected cell (IC; left panel) is distinguished by reduced levels of CDT1a-mTurquoise2, H3.1-mCherry and N-CYCB1;2-mCitrine compared to neighbouring cells (NC) in G1 (middle panel) and late G2 (right panel). Two independent transformation experiments were performed with 5 to 7 composite plants analyzed per replica.

Proliferating cells in the nodule primordium exhibit significantly larger nuclei compared to meristematic root tip cells.

(A) Confocal images of whole-mount transgenic roots expressing the Arabidopsis PlaCCI reporter in WT Medicago root tips (RT). Images show merged fluorescent channels (CDT1a-eCFP: cyan; H3.1-mCherry: red; N-CYCB1;1-YFP: yellow; Calcofluor white cell-wall staining: grayscale). Right panel: maximum intensity projection. Left panel: single focal plane. Dashed lines indicate meristematic cells at identified stages of the canonical cell cycle. Scale bars: left panel = 100 μm; right panel = 20 μm. (B) Quantification of the nuclear area at the equatorial plane in cells of the root tip (RT) and nodule primordia (NOD) showing either CDT1a-CFP (blue) or N-CYCB1;1-YFP (yellow) associated signals. The number (n) of nuclei measured is indicated above the respective box-whisker plots. All data points are shown and crosses indicate sample means. Multiplication factors of root tip and nodule sample means, indicative of the nuclear enlargement between CDT1a (G1)- and N-CYCB1;1 (late G2)-positive nuclei are given. Different letters indicate statistically significant differences according to a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. At least 2 independent transformation experiments were performed with 3 to 6 composite plants analyzed per replica.

Multiple sequence alignment of plant CENH3 proteins.

Alignment of CENH3 primary sequences from M. truncatula (Medicago, M.t.), L. japonicus (L.j.), A. thaliana (A.t.), H. vulgare (H.v.) and O. sativa (O.s.). The canonical histone H3.1 from Medicago (M.t. H3.1) is included for comparison. Protein accessions are given as GenBank or UniProt identifiers in the upper panel. Multiple sequence alignment (MUSCLE with default parameters) and conservation calculations were performed using Jalview (Procter et al., 2021). Amino acids are coloured according to the Percentage Identity scheme. Positions of the N-terminal α-helix (αN) and of the three α-helices (α1, α2, and α3) forming the histone fold domain are indicated and were obtained from HistoneDB 2.0 (Draizen et al., 2016). Position of the centromere targeting domain (CATD) is indicated below the alignment. CENH3 is the most divergent H3 variant, showing few sequence similarities in the variable N-terminal tail (reviewed in Probst et al., 2020).

Somatic centromere labelling identifies a doubling of the chromosome number as a typical feature of nodule cortical cells.

(A) Schematic representation of two different paths by which somatic polyploid cells can be generated. Endocycling cells (1) successively duplicate genomic DNA without chromosome condensation and segregation, alternating only S- and G-phases. Endomitotic cells (2) abort mitosis (M) during metaphase or anaphase (red arrow) and re-form a single nucleus. Because of complete chromosome replication and partial mitotic progression, endomitotic cells can better retain the ability to re-enter cell division (CD) than highly endoreduplicated cells. After endomitosis, generated polyploid cells hold a doubled number of entire chromosomes individually labelled by CENH3 (blue dots). Chromatin-values (C) are given for a diploid cell (2n) in G1 (2C), G2 (4C), for a 2C cell that has undergone 2 rounds of endocycle (8C) and for a tetraploid cell (4n) in G1 (4C). The number n refers to the number of separate chromosomes in a cell. For simplicity, only 3 segregating chromosomes are represented in M. (B) Confocal images of whole-mount WT roots constitutively producing mCitrine-CENH3 and H3.3-mCherry. Left panels: maximum intensity projections of transgenic root sections colonized by R. irregularis (10 dpi). Plant and fungal cell walls were stained with Calcofluor white (grayscale). The number of CENH3-labeled foci determined across image stacks in early-stage arbusculated cells (filled arrowheads) is indicated in yellow. Scale bars: 20 μm. Right upper panel: maximum intensity projection of the infection zone in a nodule colonized by mCherry-producing S. meliloti (12 dpi). The number of CENH3-labeled foci determined across image stacks in an intracellularly infected cell (star) is indicated in yellow. Scale bar: 20 μm. Right lower panels: maximum intensity projections of early-(left) and late-anaphases (right) observed in transgenic roots, in the root tip and the meristematic zone of a nodule (12 dpi), respectively. The number of CENH3-labeled foci determined across image stacks is indicated in yellow. Scale bars: 2 μm. Images show merged fluorescent channels (mCitrine: Green Fire Blue; mCherry and Calcofluor white cell-wall staining: grayscale). (C) Quantification of the number of centromeric signals in nuclei from root tips (RT; cortical cells), lateral root primordia (LRP; pericycle cells) and the infection zone (IZ) of nodules (NOD; C4/5 cortical cells) from stage IV to VI (Xiao et al., 2014), formed in inoculated WT roots (7 to 12 dpi) constitutively expressing mCitrine-CENH3. All data points are shown with small black symbols indicating infected cells and large blue symbols indicating neighbouring cells in NOD samples. Horizontal dotted lines are positioned at y = 16 and y = 32, corresponding to diploid (2n = 16) or tetraploid (4n = 32) cellular states in Medicago. Data are from 2 independent experiments with 11 (RT), 8 (LRP) and 20 (NOD) samples from 11 composite plants analyzed. n = 55 (RT), 55 (LRP), 66 (infected cell), 40 (neighbouring cell) nuclei.

CENH3 distribution supports the identification of G2-phase cells.

(A-B) Confocal images of whole-mount WT transgenic roots constitutively expressing mCitrine-CENH3 in the meristematic zone of the root tip. Simultaneous expression of H3.3-mCherry enables the recognition of condensed and segregating chromosomes. Images show merged fluorescent channels (mCitrine: Green Fire Blue; mCherry and Calcofluor white cell-wall staining: grayscale). (A) Upper panel: an early G2-phase cell is indicated, exhibiting a large nucleus where not all sister kinetochores have split. Dotted lines encircle mCitrine-CENH3 fluorescent doublets appearing on the same focal plane. The number of CENH3-labeled doublets determined across image stacks is indicated in yellow. Ana: anaphase. Lower panel: an early prophase cell (Pro early) is indicated where all sister kinetochores have split and chromosomes start to condense. The number of CENH3-labeled twin foci determined across image stacks is indicated in yellow. Meta: metaphase. Telo: telophase (one daughter cell visible). Scale bars: 5 μm. (B) Upper left panel: maximum intensity projection of an early G2-phase cell where not all sister kinetochores have split. The dotted line in the corresponding right panel demarcates one mCitrine-CENH3 doublet appearing on a single focal plane. Lower left panel: maximum intensity projection of a late G2-phase cell where the majority of sister kinetochores have split and appear as twin spots on the same focal plane (dotted-line ovals in the corresponding right panel). Scale bars: 5 μm. (C) Quantification of the nuclear area at the equatorial plane in early (n = 25) and late (n = 17) G2-phase cells as identified in (B). All data points are shown and crosses indicate sample means. Differences were not statistically significant (p-value = 0.9190) according to an unpaired t test with Welch’s correction. (D) Quantification of the length of mCitrine-CENH3 doublets as identified in (B) in early (n = 25) and late (n = 10) G2-phase cells. n = 38 (G2 early) and 60 (G2 late) doublets. All data points are shown and horizontal bars indicate sample means with 95% confidence interval. Differences were not statistically significant (p-value = 0.7679) using an unpaired t test with Welch’s correction. (C-D) Data are from 2 independent experiments with 11 root tips from 10 composite plants analyzed.

NF-YA1 modulates the entry into mitosis in a de-differentiating leaf cell population.

(A) Immunoblot analyses of total protein extracts from N. benthamiana leaves co-transformed with the following constructs: pKNOLLE::eGFP-KNOLLE / pH3.3::H3.3-mCherry and p35S::CYCD3;1-HA, pLjUbi::FLAG-NFYA1 or p35S::CYCD3;1-HA / pLjUbi::FLAG-NFYA1. The Coomassie blue-stained membrane (CBS) is shown below and serves as a loading control. The cytokinesis-specific syntaxin KNOLLE was only detected in the samples co-expressing CYCD3;1. Immunoblots were performed 3 times from 3 independent transformation experiments with similar results. (B) Live-cell confocal images of N. benthamiana epidermal cells ectopically expressing CYCD3;1 (p35S::CYCD3;1-HA) and co-transformed with the pKNOLLE::eGFP-KNOLLE construct, 64 hours post-infiltration (hpi). Simultaneous expression of H3.3-mCherry under its native promoter serves as a transformation marker and enables the recognition of segregating chromosomes. FLAG-NF-YA1 was constitutively produced (pLjUbi::FLAG-NF-YA1) from the T-DNA carrying also the CYCD3;1 expression cassette. Left and middle panels: filled arrowheads indicate newly formed cell-division planes in CYCD3;1-expressing samples, in the absence (left) or presence (middle) of NF-YA1. Dashed line boxes in the middle panel indicate cells engaged in mitosis (M). Right panels: close-up images of dividing cells in metaphase (upper panel) or cytokinesis (lower panel) taken from samples co-expressing CYCD3;1 and NF-YA1. Images show merged fluorescent channels (eGFP: Green Fire Blue; mCherry: magenta). Scale bars: left and middle panels = 100 μm; right panel = 20 μm. (C) Quantification of transformed pavement cells showing the eGFP-KNOLLE signal, 64 hpi. Samples were co-infiltrated with p35S::CYCD3;1-HA ([-] NF-YA1) or with p35S::CYCD3;1-HA / pLjUbi::FLAG-NF-YA1 ([+] NF-YA1). All data points are shown and bars indicate sample means with 95% confidence interval. Differences were statistically significant (p-value = 0.0043) according to an unpaired t test with Welch’s correction. N = 13 biological samples per condition with at least 116 cells quantified per sample. (D) Quantification of fully divided (split) cells at 64 hpi, as indicated by the eGFP-KNOLLE signal and filled arrowheads in (B), in samples constitutively expressing CYCD3;1 without [-] or with [+] NF-YA1. Bars indicate sample means with 95% confidence interval. Differences were statistically significant (p-value = 0.0078) according to an unpaired t test with Welch’s correction. n = 12 ([-] NF-YA1) or 10 ([+] NF-YA1) biological samples with at least 78 cells quantified per sample. (C-D) Data are from 3 independent co-infiltration experiments with at least 2 transformed leaves analyzed per replica.

Constitutive expression of Medicago NF-YA1, but not of a MYB3R repressor markedly competes with KNOLLE promoter activation in a CDEL system.

(A) Schematic representation of the Medicago KNOLLE promoter (M.t. pKNOLLE) with the three mitosis-specific activator (MSA) cis-elements indicated by yellow boxes. The repressor-type MYB3R protein (MYB3RR; grey box) binds to MSA motifs and maintains KNOLLE and other late G2/M-specific genes repressed in proliferating cells (outside of M-phase) and in post-mitotic cells. (B) Activity of GUS driven by the KNOLLE promoter (pKNOLLE) in CDEL samples (+ CYCD3;1) in the absence of co-infiltrated transcription factors (TF) or in the presence of ectopically-expressed Medicago HA-MYB3RR (MYB3RR) or eGFP-NF-YA1 (NF-YA1). All data points are shown and crosses indicate sample means. Differences were statistically significant ([-] TF versus [+] MYB3RR samples: p-value = 0.0254; [-] TF versus [+] NF-YA1 samples: p-value < 0.0001) using a Brown-Forsythe and Welch ANOVA test followed by Dunnett’s multiple T3 comparisons test. Data are from 4 to 5 independent transformation experiments. n = 18 ([-] TF), 15 ([+] MYB3RR), 16 ([+] NF-YA1) biological samples.

Infection-induced ENOD11 and NPL transcriptional reporters are activated in the direct vicinity of cortical infection threads.

(A-B) Confocal images of whole-mount WT roots co-expressing a transcriptional reporter of Medicago NPL driving a nuclear-localized triple-Venus (pNPL::NLS-3xVenus), together with a nuclear-localized tandem mCherry under the control of the Medicago ENOD11 promoter (pENOD11::NLS-2xmCherry). Images show merged fluorescent channels (Venus: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The Venus and mCherry channels are shown in Green Fire Blue when isolated, with blue or yellow indicating low or high fluorescence levels, respectively. One transformation experiment was performed with 8 composite plants analyzed. (A) Stars indicate low triple-Venus and tandem mCherry reporter signals located in the pericycle and the vascular cylinder, in the zones of susceptibility (upper panels) and of development of a lateral root primordium (LRP; lower panels) in the absence of rhizobia. Scale bars: 100 μm. (B) Left panels: confocal images of a whole-mount WT root co-expressing ENOD11 and NPL promoter-reporters, 6 dpi with mCherry-producing S. meliloti. The dashed line indicates the nucleus of an infected cell (IC) of the C1 layer showing high fluorescence levels for both reporters. Medium to low fluorescence levels are visible in some directly underlying cells (UC) in C2 to further down in C3 (stars), respectively. IT: infection thread. Scale bars: 20 μm. Right panel: corresponding schematic representation illustrating the activation profile of ENOD11 and NPL promoter-reporters, restricted to the direct vicinity of a cortical infection thread. Promoter activity is sustained in the infected cell (IC; yellow), reaches median levels (green) in direct neighbouring (NC) and underlying cells (UC) and diminishes with distance from the infection site (blue). (C) Confocal images of a whole-mount WT root co-expressing a transcriptional reporter of Medicago ENOD11 driving a nuclear-localized triple-Venus (pENOD11::NLS-3xVenus) and a nuclear-localized tandem mCherry as a transformation marker, 6 dpi with mCherry-producing S. meliloti. Images show merged fluorescent channels (Venus: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The Venus and mCherry channels are shown in Green Fire Blue when isolated. The triple-Venus reporter signal is restricted to the infected cells (IC) in the epidermis (Ep), C1 and C2 layers and to a couple of direct neighbouring cells (NC). IT: infection thread. Scale bars: 50 μm. Two to 5 composite plants from 2 independent transformation experiments were analyzed.

Overexpression of NF-YA1 on the cortical infection thread trajectory has no major impact on nodule primordium colonization.

(A-C) Data are from Medicago WT roots without (Control [EV]; n = 67) or with additional expression of NF-YA1 on the cortical infection thread trajectory (pENOD11::NF-YA1; n = 62), 6 dpi with S. meliloti expressing the lacZ reporter. EV: empty vector. Transformation experiments were performed 2 times with 33 composite plants analyzed for each transgenic background. (A) Quantification of the number of nodule primordia formed per control transgenic root ([EV]) or per root ectopically expressing Medicago NF-YA1 (pENOD11::NF-YA1). The number (n) of roots analyzed for each transgenic background is indicated on the top. All data points are shown and crosses indicate sample means. Differences were statistically significant (p-value = 0.0009) using a Mann-Whitney test. (B) Bright field images of whole-mount, inoculated WT roots (6 dpi) without (Control [EV]) or with additional expression of NF-YA1 (pENOD11::NF-YA1). Rhizobia producing the β-galactosidase enzyme encoded by the lacZ gene (S. meliloti lacZ) appear blue. Numbers indicate the frequencies of observation of uninfected (left panels) or infected (right panels) nodule primordia. Scale bars: 100 μm. (C) Quantification of uninfected (grey) and infected (white) nodule primordia formed in each transgenic background. n = 334 (Control [EV]) or 216 (pENOD11::NF-YA1) nodule primordia.

Overview of transgenic Medicago roots expressing the KNOLLE G2/M transcriptional readout in the absence or presence of ectopically-expressed NF-YA1.

(A) Confocal images of whole-mount WT roots co-expressing the pKNOLLE::D-box-3xVenus-NLS construct and a nuclear-localized tandem mCherry as a transformation marker, without (Control; upper panels) or with additional expression of NF-YA1 on the cortical infection thread trajectory (pENOD11::NF-YA1; lower panels). The root cortex is infected by mCherry-producing S. meliloti (5 to 6 dpi). Nodule primordia were imaged in front view (upper panels) or top view (lower panels). Dashed lines indicate the nuclei of infected cells (IC) from the outer (C2), middle (C3) and inner cortical layers (C4) where the triple-Venus reporter signal is kept low, a characteristic of cells outside the G2/M transition (see also Figure 5-figure supplement 1). The filled arrowheads indicate the nuclei of neighbouring cells (NC) expressing higher levels of the KNOLLE transcriptional reporter. IT: infection thread (indicated by a dotted line in the upper panels). Scale bars: 20 μm. (B) Confocal images of a whole-mount WT root co-expressing the pKNOLLE::D-box-3xVenus-NLS and the pENOD11::NF-YA1 constructs, together with a nuclear-localized tandem mCherry as a transformation marker, 6 dpi with mCherry-producing S. meliloti. Upper panels: view of the middle and inner cortical regions of the early nodule primordium shown in the middle panels. Cell division activity within inner cortical layers is associated with the patchy expression pattern of the pKNOLLE::D-box-3xVenus-NLS construct, reflecting its activation in late G2-phase cells (stars). Middle panels: view of the outer cortical layer (C2) crossed by infection threads. The box indicates the region shown in the close up. The full- and close-up views are from the same image. Lower panels: close-up view of infected cells (IC) in C2, whose nuclei are indicated by dashed lines. An anticlinal division occurred on the infection trajectory (empty arrowhead). Filled arrowheads indicate the tip of two additional infection branches outside the main infection thread (IT) in one of the daughter cells. Scale bars: 20 μm. (A-C) Images show merged fluorescent channels (Venus: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale). The Venus and mCherry channels are shown in Green Fire Blue when isolated. Two independent transformation experiments were performed with 7 to 13 composite plants (3 to 6 dpi) analyzed per replica.

A slightly larger proportion of cells are competent for infection upon local increase in NF-YA1 expression.

(A-G) Data are from Medicago WT roots expressing the pKNOLLE::D-box-3xVenus-NLS construct, without (Control) or with additional production of NF-YA1 on the cortical infection thread trajectory (pENOD11::NF-YA1), 3 to 6 dpi with S. meliloti. Two independent transformation experiments were performed with at least 8 nodule primordia and 5 to 15 composite plants analyzed per transgenic background. (A) Schematic representation emphasizing the respective size of nuclei in neighbouring (NC; dark grey) and infected cells (IC; light grey). Rhizobia inside the infection thread are depicted in magenta. (B) Quantification of the nuclear area at the equatorial plane in couples of neighbouring (NC) and infected cells (IC) from the same cortical layer (C2 to C4; n = 22 [Control] or 38 [pENOD11::NF-YA1]). All data points are shown and crosses indicate sample means. Different letters indicate statistically significant differences according to a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (C) Schematic representation illustrating the typical expression profile of the KNOLLE G2/M transcriptional readout in nodule primordia. This destabilized nuclear reporter indicates the competence of cells to be crossed by an infection thread (low expression signal; pale green) or to divide (medium to high expression signal; light to dark green). Rhizobia are depicted in magenta. IC: infected cell. NC: neighbouring cell. NP: nodule primordium cell. (D) Quantification of the corrected total nuclear fluorescence associated to the pKNOLLE reporter at the equatorial plane, in cortical cells (C2 to C4/5): being passed or already fully crossed by an infection thread (infected cells, IC), in direct contact or from the same layer (neighbouring cells, NCs) and from the nodule primordium (NP). The number (n) of nuclei analyzed in each transgenic background is indicated on the top. All data points are shown and crosses indicate sample means. Different letters indicate statistically significant differences according to a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (E) Schematic representation emphasizing the number of infected cells (diffuse magenta colouring) per cortical cell layer: 1 (C1), 1 (C2) or 2 (C3; split cell). Rhizobia are depicted in magenta. (F) Quantification of the number of infected cells per cortical layer in outer (C1, C2) and middle regions (C3) of nodule primordia. All data points are shown and horizontal bars indicate sample means with 95% confidence interval. Differences were not statistically significant (p-value = 0.4883) according to a Mann-Whitney test. (G) Quantification of instances where 3 or more (grey) or a maximum of 2 infected cells (white) were seen per cortical layer. (F-G) n = 32 (Control) or 70 (pENOD11::NF-YA1) cortical cell layers (C1 to C3) analyzed.

The division of cells adjacent to the infection thread is not affected by ectopic expression of NF-YA1.

(A-B) Data are from Medicago WT roots expressing the pKNOLLE::D-box-3xVenus-NLS construct, without (Control) or with additional production of NF-YA1 on the cortical infection thread trajectory (pENOD11::NF-YA1), 3 to 6 dpi with S. meliloti. Two independent transformation experiments were performed. (A) Schematic representation illustrating the anticlinal division of cells (C1, C2) adjoining a cortical infection thread (diffuse magenta colouring). Rhizobia inside the infection thread are depicted in magenta. (B) Quantification of neighbouring cells in outer cortical layers (C1 to C2) of early nodule primordia (stage I to III), divided anticlinally (grey) or undivided (white) in control transgenic roots or roots ectopically expressing Medicago NF-YA1 (pENOD11::NF-YA1). The number (n) of cells analyzed in each transgenic background is indicated on the top. The considered neighbourhood of a cortical infection thread is constituted by the direct neighbouring cells and the immediate neighbours of the latter, on the plane of the infected cell. Data are from 5 to 11 composite plants, with 9 (Control) to 21 (pENOD11::NF-YA1) nodule primordia analyzed.