Journal of General Virology (1993), 74, 24595461.
2459
Printed in Great Britain
Effect of the alfalfa mosaic virus movement protein expressed in
transgenic plants on the permeability of plasmodesmata
A. Poirson, 1. A. P. Turner, z C. Giovane, 1 A. Berna, 1 K. Roberts 2 and T. Godefroy-Colburn ~
11nstitut de Biologie Mol6culaire des Plantes, CNRS, Universit6 Louis Pasteur, 12 rue du G~n6ral Zimmer,
67084 Strasbourg-Cedex, France and 2Department o f Cell Biology, John Innes Institute, Colney Lane,
Norwich NR4 7UH, U.K.
Symplastic transport of different sized fluorescent probes
has been assessed in leaf epidermal cells of transgenic
Nicotiana plants expressing the movement protein (MP)
of alfalfa mosaic virus (AMV). In both N. tabaeum and
N. benthamiana, the size exclusion limit (SEL) of
plasmodesmata increased from Mr 1000, which represents the commonly accepted limit, to over 4.4K.
However, in control plants, movement of a 3K probe
was seen in 11 to 22% of the injections, indicating that
plasmodesmata may on occasion allow the passage of
molecules larger than was previously thought. The
increase of SEL due to the presence of the AMV MP,
although significant, remains insufficient to permit the
passage of viral particles and the possibility of other
mechanisms involved in viral cell-to-cell spread is
discussed.
To invade the inoculated tissue, most plant viruses move
from cell to cell via a symplastic pathway, most likely
through plasmodesmata [for reviews see Hull (1989),
Maule (1991), Deom et al. (1992)]. Plasmodesmata are
dynamic, gatable channels that traverse the cell wall,
providing a means for intercellular communication.
Fluorescent tracers up to only Mr 1000 move symplastically in dye-coupling experiments (Erwee & Goodwin, 1985; Derrick et al., 1990). Wolf et al. (1989)
reported the same result concerning the gating capacity
of tobacco plasmodesmata, suggesting an effective pore
diameter of 0.73 nm. The observed functional limit of
plasmodesmata is therefore well below the size of viral
agents (diameter from 10 to 30 nm for most viruses)
(Hull, 1989). Plasmodesmata must be functionally
altered to facilitate viral cell-to-cell movement. A virusencoded protein called movement protein (MP) has been
implicated in this process. The principal evidence for this
is a reported increase of the size exclusion limit (SEL) of
plasmodesmata, to between 10K and 17K, in transgenic
plants expressing the tobacco mosaic virus (TMV) MP
(Wolf et al., 1989).
In the case of alfalfa mosaic virus (AMV), a 32K
protein, designated P3, appears to be involved in viral
cell-to-cell spread (Dore et al., 1991). In this context, we
have investigated the symplastic transport of fluorescent
dyes to estimate the gating capacity of plasmodesmata
putatively modified in transformed plants expressing P3.
This estimation was made in plant lines obtained from
three hosts of AMV (Nicotiana tabacum cv. Xanthi, cv.
Xanthi NN and N. benthamiana) to assess whether the
effect of P3, if any, was a general phenomenon.
Transgenic plants of N. tabacum cv. Xanthi, genotype
nn or genotype NN, were obtained as previously
described (Erny et al., 1992). In the case of N.
benthamiana, the binary vector pBI 121.1 (Clonetech)
was chosen for plant transformation. The P3 gene was
inserted into the pBI plasmid, replacing the gene coding
for fl-glucuronidase. The procedure leading to plant
transformation and regeneration essentially followed
that given by Jefferson et al. (1987). Plants expressing the
P3 protein are referred to as $3, N3 and B3 for N.
tabacum nn, N. tabacum NN and N. benthamiana,
respectively.
In each series, P3 was assayed by Western blotting as in
Erny et al. (1992). The plant exhibiting the highest production of P3 was selected and its progeny was used for
the microinjection experiments. In each offspring however, 20 to 40 % of the plants did not express the viral
protein at a detectable level and these plants were then
discarded; the level of P3 expression in the remaining
plants was homogeneous and similar to that observed in
regenerated plants by Erny et al. (1992). The amount of
P3 in transgenic plants was at least equivalent to that
found in systemically infected leaves. It was estimated to
be around 100 ng/g of fresh material by quantitative
Western blot assay (Berna et al., 1986), using Escherichia
coli-produced P3 (Schoumacher et al., 1992a) as
standard (data not shown). Controls were represented
by plants transformed with non-recombinant vectors.
0001-1736 © 1993 SGM
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2460
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For dye-coupling experiments, half matured leaves
(the third or the fourth leaf down from the apex) were
excised from 8- to 12-week-old plants. The plant material
was coded to ensure a 'blind' experimental procedure.
Fluorescein isothiocyanate-labelled dextrans
(Fdextrans) with M r values of 3K (Molecular Probes Inc)
and of 4.4K (Sigma) were dissolved in water to a
concentration of 2 to 5 mM and the dye integrity was
checked by thin-layer chromatography. Prior to injection, F-dextran dye was mixed equally with a small
blue-fluorescing probe, cascade blue hydrazide (tripotassium salt, Molecular Probes Inc.). This molecule
(M r 664) was used as an internal control to assess
whether the injection occurred in the vacuolar or in the
cytosolic compartment. Filamented glass microcapillaries (GD-1, 1 mm x 90 mm; Narishige) pulled using a
microelectrode puller (Narishige model PP-83), were
backfilled with the chosen combination of dyes. The
leaves to be injected were fixed, abaxial side up, on a
Petri plate and covered with distilled water. Microinjections were performed using a Narishige micromanipulator (model MO-204). Probes were injected into
leaf epidermal cells under the control of a pressure
injection unit (model PLI-II, Medical Systems Corp.).
Injections of probes were observed using a Nikon
Microphot SA microscope equipped for epifluorescence.
Filter sets used were for fluorescein: excitation 450 to
490 nm, dichroic mirror 510 nm, emission 520 nm; for
cascade blue: excitation 365 nm, dichroic mirror 400 nm,
emission 450 nm.
Positive dye movement was only scored for injections
where fluorescence from both the F-dextran and the
cascade blue probes was seen within 5 min in epidermal
cells surrounding the injected one. As the M r of the
cascade blue allows it to diffuse freely through plasmodesmata (Derrick et al., 1990), no movement of this dye
was recorded after vacuolar injection (about 30 % of the
total number of injections) and the result was then
discounted. At least six to nine cytoplasmic injections
were made for each plant.
Table 1 summarizes the results as the percentage of the
cytoplasmic injections that showed movement of the
F-dextran probes out of the injected cells. In control
plants, no movement of the largest molecule (4.4K) was
observed. Using the 3K tracer, however, the fluorescein
label spread into the neighbouring cells in 11 to 22 % of
injections (depending on the species). Indeed, Wolf et al.
(1989) reported movement of a 3"9K dye in 14% of
injections (one of seven injections) into tobacco mesophyll cells; they nevertheless considered this figure
negligible. Terry & Robards (1987) showed that a 1.1K
F-dextran probe could move symplastically in nectary
trichome cells o f A b u t i l o n striatum, in more than 70 % of
the cases. The conductivity of plasmodesmata was
proposed to be slightly greater than for the other higher
plants. Together with the present results, this implies that
for some plants or some tissues at least, the plasmodesmal SEL might be higher than the widely accepted
figures of 0.8K to 1K (Wolf et al., 1989; and references
therein), most probably lying between these values and
about 3K. Intensive testing with fluorescent probes of M r
values between 1K and 3K would resolve this issue.
In plants expressing the AMV MP ($3, N3 or B3), the
3K F-dextran probe rapidly and freely moved out of the
injected cell (Table 1). With the 4.4K tracer, the
fluorescent label was found to spread into adjacent cells
in 24 % of the cases in $3 plants, whereas dye mobility
was noticed in one of two injections for both N3 and B3
plants.
These results lead us to conclude that P3 modifies the
gating capacity of plasmodesmata in a similar way in all
the plants we studied. The AMV MP alters the
plasmodesmal permeability increasing the SEL to over
4.4K, thus causing a modification functionally equivalent
to that observed after trichome cell infection with
tobacco rattle virus (Derrick et al., 1992). Nevertheless,
this represents a relatively minor functional alteration in
comparison to that induced by the TMV MP, which
enables the passage of 10K probes in non-vascular tissue
(Ding et al., 1992). One has to take into account,
however, that in the latter work, the designation 'nonvascular tissue' did not include the epidermis for which
no specific data were presented. Epidermal cells are
indeed thought to constitute a different symplastic
domain (Erwee & Goodwin, 1985).
Based on a calculation of the Stokes radii of the
F-dextran probes (Peters, 1986), the effective pore
diameter of plasmodesmata would increase to approximately 2-2 nm in the presence of the AMV MP. This value
remains within the limits of the physical width (2.5 nm)
established for the cytoplasmic annulus in natural
tobacco plasmodesmata (Ding et al., 1992). This suggests
that P3 affects the regulation of molecular transport
through plasmodesmata without greatly modifying their
structure, which is consistent with observations made by
electron microscopy on transgenic plans (O. Rohfritsch,
unpublished). The modification of the plasmodesmal
permeability is still insufficient to permit the passage of
viral particles. Other mechanisms must be involved in
this process. It is worth noting that another function for
the viral MP has been proposed. It has been shown that
the TMV MP binds single-stranded nucleic acids in vitro
(Citovsky et al., 1990) and furthermore, electron microscopic studies reveal that these MP-nucleic acid complexes are long, unfolded and very thin (less than 1.5 to
2"0 nm in diameter) (Citovsky et al., 1992). Thus the MP
might interact with both the plasmodesmata and the
viral genome to promote viral cell-to-cell spread. It
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2461
Table 1. Mobility of fluorescent dyes in epidermal cells of transformed
Nicotiana plants*
Percentage of movement of the fluorescent tracer in
N. tabacum cv. Xanthi
Genotype nn
Genotype NN
N. benthamiana
Fluorescent
probe
Control
$3
Control
N3
Control
B3
F-dextran
(3K)
F-dextran
(4.4K)
22
(7 plants)
0
(5 plants)
95
(7 plants)
24
(5 plants)
14
(3 plants)
0
(5 plants)
100
(3 plants)
50
(5 plants)
11
(3 plants)
0
(5 plants)
100
(3 plants)
56
(6 plants)
* Data represent the percentage of cytoplasmic injections (six to nine per plant) that showed
movement of the given probe within 5 man after injection. The number of plants which were tested
is indicated in parentheses.
should be noted that P3 can also bind single-stranded
nucleic acids (Schoumacher et al., 1992a, b). Nevertheless, it has not yet been demonstrated that the
resulting complexes are unfolded and thin enough to be
compatible with the modified effective pore diameter
reported here, and that the nucleic acid-binding ability of
the MP actually reflects an in vivo process.
In this context, it seems important to identify the MP
domain(s) required for plasmodesmal interactions. For
this purpose, contiguous in-frame deletions have been
made along the whole sequence of P3 and the resulting
MP constructs were used for plant transformation.
Further microinjection studies will be done on these
genetically modified plant lines. Nevertheless, the results
presented in this paper already provide further evidence
supporting a role for the viral MP in increasing the SEL
of plasmodesmata.
We thank Dr C. Stussi-Garaud and Dr F. Bernier (University of
Strasbourg, France), Dr R. Overall and T. Holdaway (University of
Sydney, Australia) for stimulating discussion and critical reading of the
manuscript. This work was supported by the Commission of the
European Communities in the context of BRIDGE programme no.
BIOT 900156.
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