Medicago N2-fixing symbiosomes acquire the endocytic identity marker Rab7 but delay the acquisition of vacuolar identity

Abstract

Rhizobium bacteria form N(2)-fixing organelles, called symbiosomes, inside the cells of legume root nodules. The bacteria are generally thought to enter the cells via an endocytosis-like process. To examine this, we studied the identity of symbiosomes in relation to the endocytic pathway. We show that in Medicago truncatula, the small GTPases Rab5 and Rab7 are endosomal membrane identity markers, marking different (partly overlapping) endosome populations. Although symbiosome formation is considered to be an endocytosis-like process, symbiosomes do not acquire Rab5 at any stage during their development, nor do they accept the trans-Golgi network identity marker SYP4, presumed to mark early endosomes in plants. By contrast, the endosomal marker Rab7 does occur on symbiosomes from an early stage of development when they have stopped dividing up to the senescence stage. However, the symbiosomes do not acquire vacuolar SNAREs (SYP22 and VTI11) until the onset of their senescence. By contrast, symbiosomes acquire the plasma membrane SNARE SYP132 from the start of symbiosome formation throughout their development. Therefore, symbiosomes appear to be locked in a unique SYP132- and Rab7-positive endosome stage and the delay in acquiring (lytic) vacuolar identity (e.g., vacuolar SNAREs) most likely ensures their survival and maintenance as individual units.

Figure 1.

Rab5 and Rab7 Occur on Endosomes. (A) to (D) Confocal image of p35S:GFP-Rab5A2 ([A] and [B]) and p35S:GFP-MtRab7A2 ([C] and [D]) expressing Medicago roots. Bars = 10 μm. (A) GFP-MtRab5A2 marks dot-like structures. Similar localization patterns were observed for Rab5A1 and Rab5B (see Supplemental Figure 3 online). (C) GFP-Rab7A2 (as well as GFP-Rab7A1; see Supplemental Figure 3 online) marks dot-like structures as well as the tonoplast of small and large vacuoles (arrows). (B) and (D) Pulse chase (45 min) with the fluorescent endosomal tracer FM4-64. Yellow dots represent the colocalization of GFP and red fluorescent FM4-64 signal, showing that the structures are endosomes. At this time point, FM4-64 does not yet label the tonoplast. N, nucleus; arrow, vacuole; arrowhead, colocalization of red and green signals.

Figure 2.

Rab5 Marks Prevacuolar Compartments, While Rab7 Marks Different, Partly Overlapping Endosome Populations. (A) Immunolocalization of anti-Rab5 (anti-Ara7) (secondary antibody CY3-tagged; red) on p35S:GFP-Rab5A2–expressing root hairs, showing a high level of colocalization (yellow signal; arrow). (B) Immunolocalization of anti-BP-80 (secondary antibody CY3-tagged; red) on p35S:GFP-Rab5B–expressing root hairs. (C) Immunolocalization of anti-BP-80 (red) on p35S:GFP-Rab5A2–expressing root hairs. The high level of colocalization (yellow signal; arrow) in (B) and (C) indicates that the Rab5-labeled endosomes represent PVC compartments. (D) Immunolocalization of anti-Rab5 (anti-Ara7) (red) on p35S:GFP-Rab7A1–expressing root hairs. Endosomes show partial colocalization of anti-Rab5 and GFP-Rab7 (yellow signal; arrow). (E) Double immunolocalization of anti-Rab7, detected with CY3-tagged secondary antibody (red), and anti-Rab5 (anti-Ara7) detected with Alexa488-tagged secondary antibody (green), on dot-like structures in root hairs of wild-type plants. Rab7 and Rab5 show only partial colocalization (yellow signal). (F) Immunolocalization of BP-80 (secondary Ab CY3-tagged; red) on 35S:GFP-Rab7A2–expressing root; note the low level of colocalization. Arrow indicates colocalization. Bars = 10 μm.

Figure 3.

Rab5 and Rab7 Mark Differently Sized MVBs. (A) and (B) EM immunogold detection (immunogold signal appears as black dots; indicated by white arrow) of anti-GFP in p35S:GFP-Rab5A2–expressing Medicago roots, showing 100- to 300-nm multivesicular endosomes, single (A) or in clusters (B). (C) Anti-Rab5 (anti-Ara7) EM-immunogold detection on p35S:GFP-Rab5A2 roots shows similarly sized multivesicular endosomes. (D) and (E) EM-immunogold labeling of anti-GFP in p35S:GFP-Rab7A2–expressing Medicago roots. Immunogold signal is present over 300- to 500-nm MVBs fusing together (D) and with the tonoplast (E). (F) Double immunolocalization on p35S:GFP-Rab7A2 roots using anti-GFP detected by 15-nm gold particles (white arrowhead) and anti-Rab7 detected by 10-nm gold (black arrowhead). The signal for both GFP and Rab7 is present on the tonoplast. v, vacuole. Bars = 100 nm in (A), 200 nm in (B) to (D), 500 nm in (E), and 200 nm in (F).

Figure 4.

Rab7 Occurs on Symbiosomes, but Rab5 Does Not. (A) Confocal image of pUBQ3:GFP-Rab5A2–expressing nodules. GFP-Rab5A2–labeled endosomes (green dots) are present in the infected cells, but no GFP signal is present on symbiomes after release from the infection thread (It). The rhizobia (R) are expressing monomeric red fluorescent protein (mRFP; red). (B) Immunolocalization of anti-Rab5 (anti-Ara7) on wild-type nodule-infected cells during symbiosome formation in the infection zone. The signal for Rab5 is revealed as red dots (secondary antibody tagged with CY3; arrow). No colocalization of immunosignal with symbiosomes was observed. The rhizobia are counterstained by Sytox Green. (C) Immunolocalization of anti-BP80 (red dots [arrow]; CY3-tagged secondary antibody) in the distal infection zone of wild-type nodules. Rhizobia (R) are counterstained by Sytox Green. No colocalization of immunosignal with symbiosomes was observed. (D) Confocal image of pE12:GFP-Rab7A2 expression in the distal part of the nodule. GFP-Rab7A2 marks both endosomes and the tonoplast (v, vacuole). No labeling of freshly released symbiomes is seen at this stage. The rhizobia are expressing mRFP (red). R, rhizobia. (E) Confocal image of pLB:GFP-Rab7A2 expression in the infection zone of the nodule. The GFP signal is present over the symbiosome membranes of mature symbiosomes (arrow) and the tonoplast. Wild-type bacteria are not counterstained. (F) Immunolocalization of Rab7 in the fixation zone of wild-type nodules using anti-Rab7. The signal (Alexa488-tagged secondary antibody) is present on symbiosome membranes, tonoplast (arrows), and endosomes (arrowheads). Bars = 10 μm.

Figure 5.

Symbiosome Membranes Contain Rab7 and Acquire SYP22 during Natural Senescence. (A) Immunogold detection of anti-GFP in pLB:GFP-Rab7A2–expressing nodules. The 10-nm gold particles are present over the symbiosome membranes. (B) Double immunolabeling using anti-GFP (detected by 15-nm gold; black arrowhead) and anti-MtRab7 (10-nm gold; white arrowhead) confirm the presence of Rab7 on the symbiosome membranes. (C) Immunogold detection of anti-GFP in 5-week-old pLB:GFP-Syp22–expressing nodule. The 15-nm gold signal (arrow) is found over the symbiosome membrane in several cells at the base of the nodule. (D) Close-up of boxed area in (C). Bars = 500 nm in (A) and 200 nm in (B) to (D).

Figure 6.

Rab7 Is Required for Symbiosome Development and Maintenance. (A) Confocal image of dominant-negative p35S:GFP-Rab7A2[T22N]–expressing root, showing GFP-Rab7A2[T22N] fluorescence in the cytoplasm. (B) pLB:GFP-Rab7A2[T22N] expression in the nodule also shows fluorescence in the cytoplasm. (C) Confocal image of constitutively active pLB:GFP-Rab7A2[Q67L] –expressing infected cells in a 14-d-old nodule. Stronger labeling of the tonoplast compared to labeling of the symbiosomes can be observed (cf. Figures 6C and 4E). The always-active construct does not induce fusion symbiosomes or their transformation into lytic compartments. (D) Longitudinal section of a control (empty vector) 14-d-old nodule. Zones: m, meristem; z2, infection zone; z3, fixation zone. (E) Magnification of z3 in (D) showing developed (stage 4) nitrogen-fixing symbiosomes. UIC, uninfected cell; IC, infected cell. (F) Longitudinal section of a 14-d-old Rab7A1-RNAi nodule. (G) Magnification of z3 in (F), showing long rod-type (stage 3) symbiosomes present in z3; note intense accumulation of starch grains in noninfected cells (star). (H) Longitudinal section of a 21-d-old Rab7A1-RNAi nodule showing signs of early senescence; note most of the tissue is degraded. (I) Magnification of (H) showing degraded symbiosomes and dead cells recolonized by saprophytic bacteria (arrow). Bars = 10 μm in (A) to (C), 100 μm in (D), and 50 μm in (E).

Figure 7.

Symbiosomes Do Not Have Vacuolar Identity until the Onset of Senescence. (A) Confocal image of pUBQ3:GFP-MtSYP22–expressing root, showing mainly tonoplast labeling. (B) Confocal image of pUBQ3:GFP-MtSYP22–expressing 14 DAI nodule, showing tonoplast labeling. No signal is observed on the symbiosomes (arrow). (C) pLB:GFP-MtSYP22 expression in the infected cells of the fixation zone of 14 DAI nodules. GFP-MtSYP22 appears on the tonoplast and dot-like structures, but not on the symbiosomes. (D) In 5-week-old pLB:GFP-MtSYP22–expressing nodules, GFP-MtSYP22 can be observed over the senescent symbiosome membrane (arrow) in the basal part of the nodule. (E) Confocal image of pUBQ3:At-γ-TIP-GFP–expressing 14-d-old nodule, showing labeling of the tonoplast, but not of the young differentiating symbiosomes. (F) pUBQ3:At-δ-TIP-GFP–expressing 14-d-old nodule, showing also tonoplast but not symbiosome labeling. (G) Confocal image of pLB:GFP-MtVTI11 expression in the infected cells of the fixation zone of 14 DAI nodules. In contrast with tonoplast labeling, symbiosomes are not labeled. (H) Immunolocalization of MtVTI11 on 8-week-old wild-type nodule tissue showing the labeling of symbiosome membranes in senescent nodules. Rhizobia in (B) to (F) are expressing mRFP (red). It, infection thread. Bars = 500 nm in (A) and 200 nm in (B) to (D).

Figure 8.

Symbiosomes Retain Plasma Membrane Identity throughout Their Development. (A) Confocal image of pUBQ:GFP-SYP132–expressing root. GFP-SYP132 marks the plasma membrane and occasionally accumulates in spot in the plasma membrane (arrow). (B) and (C) pE12:GFP-SYP132–expressing nodules show GFP-SYP132 on the plasma membrane and infection thread (it) membrane as well as just formed symbiosomes ([B]; arrow) and early stage symbiosomes ([C]; arrow). (D) pLB:GFP-SYP132–expressing nodule showing GFP-SYP132 marking the symbiosome membrane (arrow) in fully infected cells of the fixation zone. Rhizobia in (B) to (D) are expressing mRFP (red). Bars = 20 μm (A) and 10 μm in (B) to (D).