Journal of General Virology(1991), 72, 209-211. Printedin Great Britain
209
The tobacco mosaic virus 30K movement protein in transgenic tobacco
plants is localized to plasmodesmata
D. Atkins, 2 R. Htlll, 2 B. Wells, 1 K. Roberts, 2. P. Moore 3 and R. N. Beachy 3
~Department of Virus Research and ZDepartrnent of Cell Biology, John Innes Institute, Norwich NR4 7UH, U.K. and
3Department of Biology, Washington University, St Louis, Missouri 63130, U.S.A.
Transgenic tobacco plants expressing a gene encoding
the tobacco mosaic virus (TMV) movement protein
(30K) were studied using immunocytochemical techniques. The movement protein was shown to be localized
within or on most of the plasmodesmata observed in
the transformed plant. These results are consistent
with the idea that the movement protein interacts with
the plasmodesmata to facilitate the cell-to-cell spread
of TMV.
The passage of a plant virus from cell to cell is assumed to
be via the intercellular connections known as plasmodesmata. The structure of these junctions (for review see
Robards & Lucas, 1990) in the 'normal state' provides a
barrier to the intercellular passage of the virus owing to
the size limitations of the channel neck constriction
(Gibbs, 1976). The channels must therefore be modified
to enable virus particles or nucleoproteins to pass.
One of the most comprehensively studied plant viruses
is tobacco mosaic virus (TMV) and there is strong
evidence for the involvement of a viral gene product, the
movement protein (MP), in cell-to-cell spread of the
virus (for a review see Hull, 1989). It is thought that the
MP may facilitate the spread of the virus from cell to cell
by modifying plasmodesmatal permeability. Analysis of
potential MP/plasmodesmatal interactions has included
the localization of the MP in TMV-infected plants. This
study has shown that the protein is transiently associated
with the plasmodesmata at the early stages of infection
(Tomenius et al., 1987). In addition, the plasmodesmata
of transgenic plants expressing the MP were shown to
have an increased permeability limit (Wolf et al., 1989;
Deom et al., 1990).
It was of interest to determine the localization of the
MP in transgenic plants which constitutively express the
MP gene at a high level and that have plasmodesmata of
modified permeability. Furthermore, analysis of transgenic plants was expected to identify the localization of
the MP within the plant cell independently of other viral
products normally present during infection.
Transgenic tobacco plants and antiserum to an
oligopeptide corresponding to a region near the carboxy
terminus of the MP were as described in Deom et al.
(1987). Two plant lines were examined, the MP gene-
expressing line 277 and the control transgenic plant line
306 which contains the transformation vector without
the MP gene. Proteins were extracted and fractionated
from the older leaves, which had previously been shown
to contain the greatest amounts of MP (Deom et al.,
1990), using a method described by Godefroy-Colburn et
al. (1986). The extracted proteins were analysed by SDSPAGE and transferred to nitrocellulose filters by semidry electroblotting before being probed with the MP
antiserum (Deom et al., 1987) which was detected using
goat anti-rabbit IgG-alkaline phosphatase conjugate
and the chromogenic substrate nitroblue tetrazolium.
The Mr of proteins analysed by SDS-PAGE and
immunoprobing were determined by comparison with
0000-9660 © 1991 SGM
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83K
49K
34K
19K
Fig. 1. CellwallfractionproteinsfromMP gene-expressingtransgenic
line 277 and control transgenic line 306 plants separated by SDS-PAGE and analysedby probing with the anti-MP serum.
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210
Short communication
Fig. 2. Representative micrographs of plasmodesmata in the cell walls of leaf cells from transgenic plants (277) that express the MP of
TMV. The material has not been osmicated in order to preserve antigenicity and so the contrast, particularly in membranes, is low.
Immunogold labelling of the MP shows that gold grains are localized over plasmodesmata. The 'signal to noise' ratio is high, with
negligible gold found over the cell wall, vacuole and other cell organelles. V, vacuole; C, cytoplasm; W, wall. Bar marker represents
200 nm.
samples of pre-stained protein markers (Bio-Rad) electrophoresed in parallel.
Probing of the separated plant protein extracts with
anti-MP antiserum revealed significant labelling of MP
in the cell wall fraction from line 277 plants (Fig. 1). The
detection of this band was blocked completely by
preabsorption of the antiserum with the antigenic
oligopeptide thus confirming the antigenic identity of the
protein detected. As no signal was detected with the
preabsorbed antiserum even after prolonged incubation,
it was considered unnecessary to probe sections with this
antibody preparation. These results confirmed that the
line 277 plants were expressing the protein and were
therefore suitable for localization studies.
Leaves of plant lines 277 and 306 were prepared for
sectioning by low temperature embedding of nonosmicated material in London resin and analysed by
immunogold cytochemistry as described by Linstead et
al. (1988). Ultrathin sections were cut, blocked, washed
and incubated with a 1:100 dilution of the anti-MP
serum for 1 h at room temperature. Gold labelling was at
similar temperatures using a 1 : 30 dilution of goat antirabbit I g G antiserum conjugated to 15 nm gold particles
(Janssen Auroprobe). Immunostained sections were
examined in a J E O L J E M 1200EX electron microscope.
Immunogold analysis of sections of spongy mesophyll
and epidermal cells from plant line 277 revealed a
significant amount of gold labelling within or on the
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Short communication
(a)
:
~
: ~ i: ¸~
211
(b)
i¸
Fig. 3. Micrographs of plasmodesmata in both longitudinal (a) and transverse (b) section from the control transgenic plant 306. The
diameter of the channel is smaller than those shown in the transgenic line 277 plants; plasmodesmata did not label with anti-MP serum.
Bar marker represents 200 nm.
plasmodesmatal structures observed (Fig. 2). Of the
more than 100 plasmodesmata examined, over 90 ~ were
labelled. When a similar number of plasmodesmata were
examined in sections from the control plants, line 306,
none were labelled which suggests that the effect was not
caused by the transformation vector itself. The percentage of labelled plasmodesmata in the MP-expressing
transgenic plants is comparable to the 83 ~ labelling seen
at 24 h after TMV infection of normal plants (Tomenius
et al., 1987). Examination of the cell wall regions of a
typical section showed that less than 5 ~ of the area was
occupied by plasmodesmata whereas 9 0 ~ (10/11) of the
observed gold particles were localized over these
structures. Analysis of other parts of the cell very
occasionally identified randomly distributed gold particles and no localized concentrations were detected. In
addition, the central cavities of the plasmodesmata
observed in plant line 277 were larger in diameter and
differed in morphology to those of the control plant line
(Fig. 3). These ultrastructural modifications are currently under investigation.
These observations demonstrate for the first time that
the MP that accumulates in the transgenic plants is
localized within or on the plasmodesmata. The MP
localizes to the plasmodesmata in the absence of other
TMV proteins and this fact suggests that the MP alone
may function to open the plasmodesmata and permit the
cell-to-cell spread of TMV in a normal infection. In
addition, the localisation is consistent with the proposed
MP-mediated modification of the plasmodesmatal permeability of these transgenic plants (Deom et al., 1990).
Our results argue against models of plasmodesmatal
modification that involve viral components other than
the MP, and also strongly suggest that the MP alone
interacts specifically and directly with a plasmodesmatal
receptor that is responsible in turn for channel gating.
The observations, however, raise the question of how
these plants maintain a metabolic balance with 'open
gated' plasmodesmata, and additional experiments will
be necessary to address this question.
D.A. was in receipt of a grant from the Agricultural and Food
Research Council of the U.K. Other support was provided by a grant
from the National Science Foundation (DMB8717012) to R.N.B.
P.M. was supported by a Fellowship from the American Cancer
Society.
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(Received 8 May 1990; Accepted 4 October 1990)
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