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. 2012 Dec 27:3:291.
doi: 10.3389/fpls.2012.00291. eCollection 2012.

The induction of stromule formation by a plant DNA-virus in epidermal leaf tissues suggests a novel intra- and intercellular macromolecular trafficking route

Björn Krenz  1 , Holger Jeske, Tatjana Kleinow
Affiliations

The induction of stromule formation by a plant DNA-virus in epidermal leaf tissues suggests a novel intra- and intercellular macromolecular trafficking route

Björn Krenz et al. Front Plant Sci. .

Abstract

Stromules are dynamic thin protrusions of membrane envelope from plant cell plastids. Despite considerable progress in understanding the importance of certain cytoskeleton elements and motor proteins for stromule maintenance, their function within the cell has yet to be unraveled. Several viruses cause a remodulation of plastid structures and stromule biogenesis within their host plants. For RNA-viruses these interactions were demonstrated to be relevant to the infection process. An involvement of plastids and stromules is assumed in the DNA-virus life cycle as well, but their functional role needs to be determined. Recent findings support a participation of heat shock cognate 70 kDa protein (cpHSC70-1)-containing stromules induced by a DNA-virus infection (Abutilon mosaic virus, AbMV, Geminiviridae) in intra- and intercellular molecule exchange. The chaperone cpHSC70-1 was shown to interact with the AbMV movement protein (MP). Bimolecular fluorescence complementation confirmed the interaction of cpHSC70-1 and MP, and showed a homo-oligomerization of either protein in planta. The complexes were detected at the cellular margin and co-localized with plastids. In healthy plant tissues cpHSC70-1-oligomers occurred in distinct spots at chloroplasts and in small filaments extending from plastids to the cell periphery. AbMV-infection induced a cpHSC70-1-containing stromule network that exhibits elliptical dilations and transverses whole cells. Silencing of the cpHSC70 gene revealed an impact of cpHSC70 on chloroplast stability and restricted AbMV movement, but not viral DNA accumulation. Based on these data, a model is suggested in which these stromules function in molecule exchange between plastids and other organelles and perhaps other cells. AbMV may utilize cpHSC70-1 for trafficking along plastids and stromules into a neighboring cell or from plastids into the nucleus. Experimental approaches to investigate this hypothesis are discussed.

Keywords: chaperone; geminivirus; heat shock protein; movement protein; plastid.

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Figures

FIGURE 1
FIGURE 1
Abutilon mosaic virus-induced cpHSC70-1-containing stromules extending from plastids to the cell periphery. Transient co-expression of test constructs in leaf tissues of locally AbMV-infected N. benthamiana and epi-fluorescence microscopy were carried out according to Krenz et al. (2010, 2011). AbMV infection was established by simultaneous agro-infiltration of infectious DNA A and DNA B clones with the fluorescent protein expression constructs. (A) Merged image of cells expressing NSP:cyan fluorescent protein (CFP), the two split yellow fluorescent protein (YFP)/BiFC constructs of cpHSC70-1 and the plasmodesmata marker PDCB1:mCherry (callose binding protein 1, Simpson et al., 2009) for 4 days post agro-infiltration (dpai). The square in (A) highlights cpHSC70-1-oligomers at chloroplasts and stromules (yellow, arrows) anchoring at the cell periphery (red: PDCB1:mCherry), and is magnified in (B). The separate fluorescence signals superimposed in (A) are shown in (C) YFP, (D) CFP, and (E) mCherry. Note: The plasmodesmata marker PDCB1:mCherry lost its extracellular localization at the neck region of plasmodesmata upon AbMV-infection (compare Figure 3) and is probably distributed throughout the apoplast. NSP:CFP is redirected from the nucleus to the cell periphery, probably the plasma membrane, by presence of MP or AbMV-infection (Zhang et al., 2001; Frischmuth et al., 2007). Bar: 10 μm.
FIGURE 2
FIGURE 2
Abutilon mosaic virus-induced cpHSC70-1-containing stromules grabbing a nucleus. The experimental set-up is the same as stated in Figure 1. (A) Merged image of cells expressing the three test proteins for 4 dpai and (B) magnification of the square in (A) which marks cpHSC70-1-oligomers at chloroplasts and stromules (yellow, arrows) which closely associate to a nucleus (blue: NSP:CFP) near to the cell periphery (blue: NSP:CFP, likely plasma membrane and red: PDCB1:mCherry, apoplast), magnified in (B). The separate emissions merged in (A) are shown in (C) YFP, (D) CFP, and (E) mCherry. N, nucleus; bar: 10 μm.
FIGURE 3
FIGURE 3
Influence of AbMV-infection on localization of the plasmodesmata marker PDCB1. PDCB1:mCherry (Simpson et al., 2009) was expressed in epidermal tissues of N. benthamiana plants either (A) healthy or (B) locally infected with AbMV for 4 days. The experiment was performed as described in Figure 1. (A) Punctate mCherry signals indicate the targeting of PDCB1 to the apoplastic part of the neck region from plasmodesmata according to Simpson et al. (2009). (B) Upon AbMV-infection these plasmodesmata-specific signals disappeared and PDCB1 emerged homogenously in the apoplast. These results showed a virus-induced alteration of the subcellular localization of PDCB1, probably by modifying callose deposition at plasmodesmata. Bar: 10 μm.
FIGURE 4
FIGURE 4
Hypothetical model of AbMV intra- and intercellular trafficking via a plastid network. (A) In healthy plant cells oligomers of the chaperone cpHSC70 locate mainly in small spots at chloroplasts (1), to a lesser amount in small filaments extending from cortical chloroplasts toward the cell periphery (2) and distributed at the cellular margin (3). (B) In AbMV-infected cells homo-oligomers of cpHSC70-1 were found similar to non-infected cells at chloroplasts (1) and to a low extent at the cellular margin (3). However, AbMV-infection establishes the formation of a stromule network interconnecting different chloroplasts (4), but also extending from plastids inward to the nucleus, where they closely attach (5) and outward to the cell periphery/cell wall, assumedly to plasmodesmata (Pd) that transverse the cell wall (6). These stromules exhibit structures where cpHSC70-oligomers appeared mainly in elliptical dilations giving them a “pearls on a string”-appearance. The stromule network might function in intra- and intercellular trafficking of viral nucleoprotein complexes by the interaction of AbMV MP with the chaperone cpHSC70 within stromules (7). The potential underlying transport mechanism is yet unknown, but probably involves membrane fusions or a vesicle formation.
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