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 Echevarria 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). (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 (late-anaphase chromosomes indicated by a filled arrowhead) 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. Images are maximum intensity projections except the top-right panel (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 WT roots colonized by Rhizophagus irregularis (15 dpi). Plant and fungal cell walls were stained with Calcofluor white (grayscale). H3.1 was 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. Images are maximum intensity projections. Scale bars: 50 μm. Two independent transformation experiments were performed with 3 to 5 composite plants analyzed per replica.

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). (A) Left panel: the solid-line frame indicates the outer and middle cortical regions of an early nodule primordium shown in the close up. Right panel: dotted-line regions of interest (ROIs) indicate 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 just penetrated by the infection thread. Scale bars: 20 μm. (B) Left panel: dotted-line ROIs indicate nuclei of infected cells (IC) being passed (C4) or recently passed (C3) by a cortical infection thread. Right panel: dotted-line ROIs indicate 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). (E) 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. Nuclei of increasing size and DNA content are coloured according to their merged H3.3 (magenta) and H3.1 (green) content in decondensed chromatin (G1, S, G2). This diagram illustrates cells exiting the cell cycle within proliferating or endoreduplicating populations, which proceed to a massive H3.1 eviction following their last DNA replication round (Otero et al., 2016).

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), daphne-like (14 dpi) and nf-ya1-1 (12 dpi), 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 below each panel 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). Rhizobia inside infection threads are depicted in red. Confocal images are maximum intensity projections and show merged fluorescent channels (eGFP: green; mCherry: magenta; Calcofluor white cell-wall staining: grayscale in the middle and right panels). Scale bars: 20 μm. IC: infected cell. Ep: epidermis. (B-C) Quantification of the nuclear area (B) and the relative corrected total H3.1-eGFP nuclear fluorescence (IC/NC) (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 with magenta or green indicating relative H3.1-eGFP nuclear signals (IC/NC) below 1,5 or above 1,6, respectively. 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.

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

(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 Echevarria 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 (dotted-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 are maximum intensity projections and show merged fluorescent channels (eCFP and GFP: cyan; mCherry: red; YFP: yellow; Calcofluor white cell-wall staining: grayscale). (B) The filled arrowhead points to a cell of the C3 layer just penetrated by an infection thread. Cell-cycle phases of non-infected neighbouring cells are indicated. (C) Left panel: the dotted-line ROI indicates 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. Right panels: fluorescence intensity profiles of CDT1a-, H3.1- and N-CYCB1.1-associated signals along the cyan and green transects shown in (C). Scale bars: 20 μm. (D) 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. (E) 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. (D-E) Images are maximum intensity projections and 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, with blue or yellow indicating low or high fluorescence levels, respectively. The dotted-line ROIs 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 are maximum intensity projections and 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 (pUbi) promoters (n = 55 nuclei). All data points are shown and are from 2 (pUbi) 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 primodium 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. Dotted-line ROIs 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). In the Green Fire Blue colour scheme, blue or yellow indicate low or high fluorescence levels, respectively. 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. 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 dotted-line frame 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 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-line ROIs indicate 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 are maximum intensity projections (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. (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 are maximum intensity projections and 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 7 – 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 ectopically expressing CYCD3;1 and the Medicago pKNOLLE::GUS (pKNOLLE) reporter construct. Fluorescence curves (GUS-mediated hydrolysis of 4-MUG) over time are shown for 3 biological replicates. Error bars indicate standard deviation. Data are from 1 of 4 independent transient transformations. (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.

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 (this study, Torrey and Barrios, 1969) 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 presumably enter a prolonged G2-phase (this study, Yang et al., 1994) 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.