Research ArticleCELL BIOLOGY

Horizontal genome transfer by cell-to-cell travel of whole organelles

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Science Advances  01 Jan 2021:
Vol. 7, no. 1, eabd8215
DOI: 10.1126/sciadv.abd8215
  • Fig. 1 Detection of fluorescent markers by confocal laser scanning microscopy in leaf cells of plants used for grafting experiments (Nuc-kan:YFP and Pt-spec:dsRed) and a plant obtained from horizontal plastid genome transfer (bottom panel).

    Nuc-kan:YFP plants express the YFP reporter protein from their nuclear genome, whereas Pt-spec:dsRed plants express the dsRed reporter from their plastid genome (fig. S1A). YFP fluorescence is represented in blue and occurs in the nucleus and the cytosol. dsRed fluorescence is shown in green and, together with the red chlorophyll fluorescence, occurs exclusively in the plastids. Plants originating from horizontal plastid genome transfer show all three fluorescences (bottom panel).

  • Fig. 2 SEM image of a graft junction.

    Upon formation of the graft union, callus tissue proliferates from both stems, thus sealing the gap between stock and scion and reestablishing intercellular communication. Later, some callus cells differentiate into phloem and xylem, mediating vascular reconnection of scion and stock.

  • Fig. 3 Intercellular exchange of plastid genomes during formation of the graft union.

    (A) Confocal images of callus cells within a (manually dissected) graft junction. Five different cell types can be distinguished. Types I and V represent Nuc-kan:YFP and Pt-spec:dsRed cells, respectively. Cells with a mixed population of plastids (types III and IV) originate from intercellular transfer of plastid genomes. Type II cells can arise through intercellular genome transfer but could alternatively represent YFP (protein or mRNA) exchange between neighboring cells. See text for details. Arrows mark transferred plastids. Note that transferred plastids are smaller than resident plastids (table S1). Nc, nucleus; Pt, plastid. (B) Callus cells forming a graft union. Within a single graft junction, multiple events of plastid genome exchange (▲) are detected, in addition to a number of events that represent either complete plastid genome exchange (i.e., complete replacement of the plastid population of the recipient cell) or macromolecular trafficking of the YFP (∆).

  • Fig. 4 Plastid mixing in grafted callus cells.

    (A) Callus grafting. Callus tissue was obtained by placing stem sections from Nuc-kan:YFP and Pt-spec:dsRed plants on regeneration medium. Tissue proliferating from the cambium ring was grafted by aligning callus explants with a micromanipulator and placing them between agarose blocks. Continued growth results in cell adhesion and tissue fusion. (B) Confocal images (with YFP and dsRed fluorescences merged) of the graft junction before (left) and after (right) cell adhesion. Nuc-kan:YFP cells contain plastid genomes acquired from Pt-spec:dsRed (white dashed box). The red dashed line indicates the border between the grafted tissues. (C) Images of individual callus grafts with cells containing a mixed population of plastids. The top panel shows an event of plastid genome transfer from a Nuc-kan:YFP cell into a Pt-spec:dsRed cell. The two bottom panels show transfer events from Pt-spec:dsRed into Nuc-kan:YFP cells. Note the initially small size of the acquired plastids (arrows) within the adhered callus cells (top and middle panels). They later regain normal size (bottom panel). (I) and (II) indicate two independent transfer events in adjacent cells. Costaining of cell walls reveals protrusions of adhered callus cells (asterisks in top and middle panels) and irregular staining of the cell walls after establishment of the graft union (bottom).

  • Fig. 5 Cell wall pores allow exchange of large cellular structures between callus cells.

    (A) Overview electron microscopy image (S3EM) of a cryo-fixed callus assembly containing cells with transferred plastid genomes. The black box indicates a pair of adhered cells at the graft junction; the red dotted line marks the border between the grafted tissues. (B) BSE images of eight consecutive 70-nm-thick sections (z-stack) spanning the cell wall contact zone of two adhered callus cells [within the boxed area in (A)]. The middle lamella between the cells identifies them as adhered cells (cells A and B) rather than daughter cells. By contrast, no middle lamella is present between cell B and cell C [in (A)]. (C) Tomogram of a cell wall (magenta) between adhered callus cells. The tomogram reveals a direct cytoplasmic connection (arrow) and organelles (green) that are small enough for direct passage. (D) Individual cell wall openings between adhered callus cells (marked by red dotted lines) identified by electron microscopy. M, mitochondrion; V, vacuole; CW, cell wall.

  • Fig. 6 Budding callus cells in graft unions.

    (A) Confocal image of a cell wall at the graft junction. Staining with calcofluor white shows open pores (left). In addition, bud-like protrusions emerge from the cell surface (middle panel), which are also seen by cryo-SEM (right). The structures collapse under high vacuum, but the central pore remains visible (inset). (B) Confocal images of the bud-like protrusions. In Nuc-kan:YFP lines, the cytosolic YFP signal extends into the buds (right two images, blue signal). Chlorophyll fluorescence is detectable within some buds, indicating the presence of plastids (magenta signal). The presence of plastids (white arrows) was confirmed with transplastomic lines expressing GFP (6) or dsRed (left two images). (C) Analysis of the bud-like protrusions by TEM of cryo-fixed cells. The buds emerged from cell wall pores with an approximate width of 1.5 μm. Some buds keep narrow connections to their mother cell (black arrows). (D) Ultrastructural analysis of buds reaching into neighboring cells. The buds are rich in cytosol and organelles. The red dashed lines in (C) and (D) mark the openings in the cell walls of adhered callus cells. (E) Schematic representation of the cellular events described in (A) to (D).

  • Fig. 7 Plastids of callus cells in the graft union dedifferentiate into amoeboid proplastids.

    (A) Confocal microscopy images of cells harboring transferred plastids. Whereas plastids newly acquired from a neighboring donor cell have a diameter of approximately 1 μm, resident plastids in the recipient cells have a diameter of approximately 5 μm. Only the small plastids are likely able to pass through the cell wall pores with an estimated size exclusion limit of 1.5 μm (see Figs. 5, B to D, and 6, C and D). (B) Callus cells contain a mixed population of morphologically distinguishable plastids. Starch granule-containing amyloplast-like plastids represent the main plastid type. The inset in the right image shows a starch granule (white) at high magnification. Immunoelectron microscopy of cryo-fixed samples (using anti-ClpP and anti-dsRed antibodies) additionally identified small rod-like amoeboid organelles and proplastid-like small spherical or bean-shaped organelles (black arrows and inset in the second image from the right) as plastids. Most of these small plastids contain few or no internal membranes and lack starch granules.

  • Fig. 8 Dedifferentiated plastids are highly mobile and move from cell to cell.

    (A) Dedifferentiated small and amoeboid plastids show increased mobility. Although normal plastids remain at their position within the cell for a long time (top series), amoeboid and spherical plastids move rapidly inside the cell (bottom series), often along the plasma membrane (see movies S1 and S2). (B) Transfer of dedifferentiated plastids between living cells in real time. The mobile plastids are marked by white arrows; the cell wall crossed by them is represented as dotted line (top and middle panels). A cell wall pore through which a plastid travels is marked by the dotted circle (bottom). See also movies S3 to S5. (C) Plastid dedifferentiation facilitates intercellular transfer. When plastid dedifferentiation is induced by keeping callus tissue in the dark for 3 days in the absence of an external carbon source, loss of chlorophyll fluorescence occurs, indicating thylakoid degradation. In parallel, plastid size and shape markedly change. Experimental stimulation of dedifferentiation increases the frequency of intercellular organelle transfer across the graft junction more than fivefold. The x axis represents the number of successful grafts analyzed; the y axis represents the number of doubly resistant calli obtained per graft.

  • Fig. 9 Dedifferentiated plastids of callus cells in the graft union contain DNA.

    Staining of DNA in callus cell plastids. (A) Detection of DNA in plastids by colocalization of the fluorescences of (i) the DNA-intercalating dye SYBR Green, (ii) the plastid-expressed dsRed protein, and (iii) the chloroplast-specific pigment chlorophyll in Pt-spec:dsRed callus cells via confocal laser scanning microscopy. (B) Detection of DNA in dedifferentiated plastids. Plastid dedifferentiation in Pt-spec:dsRed callus cells was induced by carbon starvation (growth in the dark and in the absence of sucrose), followed by staining of DNA with SYBR Green. Colocalization of SYBR Green fluorescence (blue) with plastid-specific dsRed fluorescence (green) confirms the presence of DNA in dedifferentiated plastids. (C) Detection of plastid DNA in Nuc-kan:YFP cells. Although SYBR Green and YFP fluorescence strongly overlap (YFP + SYBR), colocalization of SYBR Green fluorescence (green) and chlorophyll fluorescence (red) in dedifferentiated plastids of Nuc-kan:YFP cells can be seen and confirms the presence of DNA. The asterisks denote the plastids that are shown enlarged in the insets.

  • Fig. 10 Transferred plastids contain DNA.

    Confocal images of callus cells within a (manually dissected) Pt-spec:dsRed/Nuc-kan:YFP graft junction. (A) Overview image of the graft union, with the cell walls stained with calcofluor white and the (nuclear and organellar) DNA visualized with SYBR Green. In Nuc-kan:YFP cells, SYBR Green fluorescence is detected in the nucleus and the organelles, together with the YFP fluorescence in the nucleus and the cytosol (YFP + SYBR). Chloroplasts are recognized by their red chlorophyll fluorescence. In Pt-spec:dsRed cells, DNA in the nucleus and the organelles is visualized by SYBR Green staining. In addition, the plastids show chlorophyll fluorescence and dsRed fluorescence. (B) Among the callus cells, cells with mixed populations of plastids represent events of organelle transfer across the graft junction. (C) Enlargement of the region boxed in (B) (white dotted square). Note that transferred plastids are smaller than resident plastids and contain DNA, as evident from colocalization of dsRed and SYBR Green fluorescence (arrows).

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