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Biochemical Society Transactions (2007) Volume 35, part 1
Plasmodesmata and intercellular transport
of viral RNA
C. Hofmann, A. Sambade and M. Heinlein1
Institut de Biologie Moléculaire des Plantes, Laboratoire propre du CNRS (Centre National de la Recherche Scientifique) (UPR 2357) conventionné avec
l’Université Louis Pasteur (Strasbourg 1), 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France
Abstract
Cell-to-cell communication in plants involves the symplastic trafficking of informational protein and RNA
macromolecules through cytoplasmic bridges in the plant cell wall known as plasmodesmata. Viruses
exploit this route for the spread of infection and are used as a model to study the mechanisms by which
macromolecules are targeted to the pore. Studies using tobacco mosaic virus have led to the identification
of host components that participate in plasmodesmal targeting of viral RNA and movement protein.
Role and dynamics of plasmodesmata in
intercellular communication
Intercellular communication delivers crucial information
for the position-dependent specification of cell fate and
therefore is an essential biological process during the coordination of development in multicellular organisms. Plants
are characterized by two pathways for intercellular communication: whereas the apoplastic pathway mediates communication via receptor–ligand interactions [1], the symplastic
pathway allows the direct intercellular exchange of macromolecules through cytoplasmic bridges in the cell wall termed
plasmodesmata [2–4]. By being connected to the phloem sieve
elements in the vascular veins and stems, the system of plasmodesmata forms a cell-to-cell and long-distance communication network that enables plants to rapidly disseminate
information and metabolites, thereby co-ordinating cellular
activities at a level above that of the individual cell [4]. The
conductivity of plasmodesmata is under developmental and
physiological control and thus defines cell-to-cell communication within and between ‘supracellular domains’ [4]. In
addition to long-term changes in plasmodesmal conductivity
that have been correlated with conspicuous changes in plasmodesmal structure [5] as well as with the local deposition
and removal of callose [6–8], the plasmodesmata are also
intrinsically dynamic and can be gated by NCAPs (non-cellautonomous proteins) [4]. By causing short-term dynamic
changes in the SEL (size-exclusion limit) of plasmodesmata,
these proteins mediate their own trafficking as well as the
trafficking of a wide range of RNA molecules [3,4,9–15].
Key words: cytoskeleton, macromolecular transport, movement protein, plasmodesmata,
replicase, tobacco mosaic virus.
Abbreviations used: ER, endoplasmic reticulum; MP, movement protein; MPB2C, MP-binding
protein 2C; NCAP, non-cell-autonomous protein; PAPK, plasmodesmal associated protein kinase;
PME, pectin methylesterase; TMV, tobacco mosaic virus; VRC, viral replication complex; vRNA,
viral RNA.
1
To whom correspondence should be addressed (email [email protected]).
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Role of the MP (movement protein) of TMV
(tobacco mosaic virus) in facilitating the
transport of vRNA (viral RNA)
The first NCAP identified was a non-structural protein
encoded by TMV. This protein is known as the MP of the virus
and its early characterization as a vRNA transport protein and
plasmodesma gating factor not only led to the identification
of similar protein functions in other viruses but also provided
the basis for the development of the NCAP concept described
today. Studying the function of this and other viral MPs
as well as of other NCAPs promises to reveal existing mechanisms of macromolecular plasmodesma-mediated
transport that are exploited by viruses for the movement of
their genomes. That the MP of TMV indeed interacts with an
existing mechanism for intercellular transport is indicated by
its rapid intercellular movement upon microinjection into
plant tissues [16]. This protein is particularly suited for
studying the transport of RNA molecules since the CP (coat
protein) of TMV is dispensable for the cell-to-cell movement
of infection, thus indicating that the MP permits the transport of the vRNA in a non-encapsidated form.
Recent and current studies concentrate on addressing the
composition of the vRNA-containing complex that moves
between cells as well as on determining the mechanism by
which this particle is targeted to plasmodesmata. MP probably mediates the transport of vRNA through direct binding,
as is indicated by its ability to bind single-stranded nucleic
acids in vitro, which results in the formation of an unfolded
and elongated RNP (ribonucleoprotein) complex (vRNP)
with apparent dimensions compatible with translocation
through dilated plasmodesmata [17]. In vivo observations
have suggested that vRNA moves between cells in the form
of larger, membrane-associated replication complexes [18].
However, direct in vivo evidence demonstrating that the
movement process involves the formation of an MP–vRNA
complex is still lacking. Recent advances in the development
of quantitative fluorescence microscopy technologies to label
RNA and proteins in vivo and to demonstrate their molecular
Intercellular Signalling in Plants
interactions [19] hold the promise that the in vivo detection
of vRNA and the determination of its interactions with MP
will soon be achieved.
The MP interacts with diverse host factors
Since the cell-to-cell spread of vRNA depends on MP,
information about the movement mechanism can be revealed
through the identification of the binding targets of the
protein. Far-Western and yeast two-hybrid screening led
to the identification of several potential binding partners, a
cell-wall-associated PME (pectin methylesterase), a PAPK
(plasmodesmal associated protein kinase) and calreticulin.
The cell-wall-localized enzyme PME [20,21] was isolated by
a renatured blot overlay assay from cell wall protein fractions
of tobacco. Subsequent yeast two-hybrid analysis confirmed
that the MP binds PME with a domain encompassing
amino acids 130–185. A TMV mutant encoding a MP from
which this region was deleted failed to move cell-to-cell,
suggesting that the interaction of MP with PME is required
for viral cell-to-cell movement [21]. However, although this
interpretation may be correct, one has to be cautious that the
deletion of more than 50 amino acids from the core region of
MP may interfere with viral movement through disruption
of the overall tertiary structure of the protein. Thus, although
several potential roles of PME in the targeting or anchorage
of MP to plasmodesmata have been proposed [21], conclusive
in vivo evidence for such a role remains to be demonstrated.
Importantly, the co-expression of fluorescent-protein-tagged
MP and PME during TMV infection or in transfected cells
did not reveal any significant co-distribution of the two proteins (Figures 1D–1F). MPB2C (MP-binding protein 2C)
has been isolated using a membrane-based yeast screening
system [22] and was shown to have the potential to colocalize with MP on microtubules. Transient expression of
MPB2C causes a decrease in the efficiency by which MP
spreads between cells and, since this effect is correlated with
increased accumulation of MP on microtubules, a role of
MPB2C in controlling virus movement by tethering the MP
to the cytoskeleton has been proposed [22]. PAPK resides at
plasmodesmata and specifically phosphorylates TMV-MP at
its C-terminus in vitro [23]. This finding is consistent with
previous studies showing that the MP is phosphorylated
in vivo and in vitro (reviewed in [24]). However, a TMV
mutant with a large deletion at the C-terminus of MP
can still spread between cells [25] and phosphorylationmimicking mutations in the C-terminus of MP cause a hostspecific reduction in the efficiency of viral spread [26]; this
suggests that such a phosphorylation at the C-terminus
restricts rather than promotes the movement function of
MP [26]. Finally, calreticulin, a calcium sequestering ER
(endoplasmic reticulum)-resident protein, was shown to bind
to MP in vitro and to interfere with TMV movement if
expressed in transgenic plants [27]. As in the case of MPB2C,
overexpression of calreticulin led to enhanced accumulation
of MP on microtubules [27], again revealing a role of the
cytoskeleton during infection.
TMV movement involves interactions of
MP with the cytoskeleton and the ER
Thus what is the role of the cytoskeleton? The cytoskeleton
indeed seems to have an important role, as was shown by
in vivo assays aimed at identifying the targets of the functional protein in cells of spreading infection sites in plants.
Using TMV derivatives expressing functional MP fused to
fluorescent protein, it was demonstrated that the MP accumulates in plasmodesmata and associates with elements of
the ER as well as with microtubules [28,29] (Figures 1A–
1C). MP has also been reported to associate with actin
microfilaments [30], although recent observations seem to
argue against such interactions of MP ([31]; C. Hofmann
and M. Heinlein, unpublished work). Several studies indicate
that the ER provides support for virus replication, whereby
MP contributes to the formation of ER-associated inclusions
that harbour VRCs (viral replication complexes) [29,31–
34]. These ER-associated inclusions contain vRNA, replicase
and MP [29,33], and show actin-dependent mobility in
the cytoplasm [18]. Treatment of plant tissues with actin
antagonist Latrunculin B was reported to reduce TMV
movement and to interfere with the cytoplasmic mobility
of the inclusions, thus suggesting a role of the inclusions
and actin filaments in TMV movement [18]. A role of actin
filaments is further indicated by the observation that
fluorescent-protein-fused TMV 126 kDa replicase protein
associates with actin filaments upon transient expression, a
finding that also suggests that the interaction of VRCs with
actin filaments is replicase-mediated [35].
A role of microtubules in TMV movement has been supported by several in vivo functional studies using TMV
derivatives encoding functional, dysfunctional and temperature-sensitive mutations [25,28,29,37–40]. All temperaturesensitive mutations known to affect the movement function
of TMV are caused by single-amino-acid exchange mutations
in the MP and interfere with the association of MP with
microtubules (but not with its accumulation in plasmodesmata) at non-permissive temperatures ([38]; and V. Boyko
and M. Heinlein, unpublished work). They map to a region
of MP showing structural similarity to the M-loop of
tubulin that is essential for microtubule polymerization and
stability by mediating specific M-to-N-loop interactions
between tubulin molecules in adjacent microtubule protofilaments. The treatment of plant tissues with microtubuledisrupting agents does not strongly interfere with TMV
movement [41,42] most probably because such treatments
may not disrupt all microtubules to an extent required
to interfere with movement of single virus genomes [36].
A role of microtubules is further indicated by the fact
that MP also binds to microtubules in mammalian cells
[31,38], to FtsZ (filamenting temperature-sensitive mutant
Z)-based cytoskeleton in prokaryotes [43], and to preassembled microtubules in vitro [42]. Moreover, vRNA
associates with microtubules in infected protoplasts in a
manner dependent on microtubule-associated MP [33,44].
Nevertheless, although these findings are consistent with
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Biochemical Society Transactions (2007) Volume 35, part 1
Figure 1 Localization patterns of MP–GFP (green fluorescent protein) during infection by TMV-MP–GFP in Nicotiana benthamiana leaf
epidermal tissues and a model for the targeting of MP and vRNA to plasmodesmata
(A–C) Infection site caused by TMV–MP–GFP (A) and localization of MP–GFP to plasmodesmata (B) and microtubules (C). (D–F)
PME and MP are proposed to interact in vivo [21]. However, during infection, there appears to be no obvious co-distribution of
MP–GFP (green) with transiently expressed Nicotiana tabacum PME fused to red fluorescent protein (PME–RFP, red). (G) Model
for the targeting of MP and vRNA to plasmodesmata (PD). The ER–actin network is continuous through plasmodesmata. MP is
targeted to plasmodesmata and increases their size-exclusion limit (gated plasmodesmata). MP can target plasmodesmata
independently of association with microtubules (MTs), whereas the targeting of the MP–vRNA complex requires microtubules
[38]. A putative transmembrane domain [47] anchors MP in the ER membrane which transports the protein to plasmodesmata
with support by actin-driven membrane motility. Upon binding of vRNA and formation of a multimeric MP–vRNA complex,
the microtubule-interacting domain of MP [38], proposed to overlap with the putative transmembrane domain [46,47],
is exposed and mediates microtubule association. Microutubules and associated motor proteins provide support to the
ER-associated transport of the megadalton large MP–vRNA complex to the plasmodesmata channel. Scale bars, 500 µm
(A); 2 µm (B); 5 µm (D–F).
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Intercellular Signalling in Plants
a significant role of microtubules in TMV movement,
the direct observation of RNA-containing particles that
are targeted to plasmodesmata in a microtubule-dependent
manner has not yet been reported. In the context of virus
infection, this important observation is indeed difficult to
obtain, since newly infected cells at the leading front of the
spreading infection site contain only very small amounts
of MP. Although microtubule-aligned and MP-associated
particles have been observed in rare instances [3], better
approaches to observe such particles more consistently are
needed in order to investigate their role in the cell-tocell movement process. Since targeting of MP to plasmodesmata occurs in transgenic plants and therefore does not
require infection or any other viral factor [45], we set
out recently to investigate the localization of MP under
conditions of transient expression. First results indicate that
MP associates with particles within the ER membrane and
that the movements of the particles within the membrane
are both actin- and microtubule-dependent (A. Sambade
and M. Heinlein, unpublished work). Thus, consistent with
previous biochemical evidence [32,42,46,47], the MP may
target plasmodesmata through interactions with both the ER
and the cytoskeleton (Figure 1G).
Recent reports have pointed out that viral replicase is involved in TMV movement [35,48] and silencing suppression
[49,50] and thus illustrated that TMV movement is a complex
phenomenon that depends on more viral factors than just the
MP; it probably also involves viral strategies to successfully
protect the virus against silencing and other defence responses
of the host. Thus, before the mechanism of vRNA movement
can be interpreted correctly and finally be used as a model
for plant endogenous RNA transport, it will be important to
dissect further the cellular processes involved in virus replication and defence.
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