Journal of General Virology (2003), 84, 985–994
DOI 10.1099/vir.0.18885-0
Immunodetection and fluorescent microscopy of
transgenically expressed hordeivirus TGBp3
movement protein reveals its association with
endoplasmic reticulum elements in close proximity
to plasmodesmata
E. N. Gorshkova,1 T. N. Erokhina,2 T. A. Stroganova,3 N. E. Yelina,1
A. A. Zamyatnin Jr,1 N. O. Kalinina,1 J. Schiemann,4 A. G. Solovyev1 and
S. Yu. Morozov1
Correspondence
Sergey Morozov
[email protected]
1
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow
State University, Moscow 119899, Russia
2
M. M. Shemyakin & Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
Sciences, 16/10 Miklukho-Maklaya Str., Moscow 117997, Russia
3
Institute of Microbiology, Russian Academy of Sciences, 7 Prospect 60 Let Oktyabrya,
Moscow 117811, Russia
4
Institute of Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for
Agriculture and Forestry, Messeweg 11/12, D-38104 Braunschweig, Germany
Received 8 October 2002
Accepted 5 December 2002
The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV,
genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green
fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies
(mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs
efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic
Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that
PSLV TGBp3 and GFP–TGBp3 had a tendency to form large protein complexes of an unknown
nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and
probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal
cells in transgenic plants demonstrated that GFP–TGBp3 localized to cell wall-associated
punctate bodies, which often formed pairs of opposing discrete structures that co-localized with
callose, indicating their association with the plasmodesmata-enriched cell wall fields. After
mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared
within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static,
most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm.
INTRODUCTION
The positive-stranded RNA genome of Poa semilatent virus
(PSLV, genus Hordeivirus) consists of three RNAs, termed a,
b and c. RNAs a and c of hordeiviruses are essential for virus
genome replication and encode two viral replicase components and a regulatory protein, whereas RNA b encodes the
coat protein and three proteins involved in virus movement
(Lawrence & Jackson, 1999; Lawrence et al., 2000; Savenkov
et al., 1998; Solovyev et al., 1996). Genes of the hordeivirus
movement proteins (MPs) are organized in the evolutionarily conserved gene module called the ‘triple gene block’
(TGB), which is also found in some other plant viruses of
the proposed families Tubiviridae and Potexviridae (Herzog
0001-8885 G 2003 SGM
et al., 1998; Koenig et al., 1998; Lawrence & Jackson, 2001a;
Lough et al., 1998; Morozov & Solovyev, 1999; Morozov
et al., 1989). All three TGB-encoded proteins, referred to as
TGBp1, TGBp2 and TGBp3 according to their position, are
necessary for cell-to-cell movement of virus (Beck et al.,
1991; Gilmer et al., 1992; Herzog et al., 1998; Krishnamurthy
et al., 2002; Petty & Jackson, 1990).
Cell-to-cell movement of plant RNA viruses is generally
thought to involve specific ribonucleoproteins (RNPs) composed of virus RNA and proteins (reviewed in Carrington
et al., 1996; Citovsky & Zambryski, 1991; Haywood et al.,
2002). In hordeiviruses, and presumably in other TGBcontaining viruses of the proposed family Tubiviridae
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985
E. N. Gorshkova and others
(genera Hordeivirus, Pecluvirus, Pomovirus and Benyvirus),
the movement-related RNPs are formed by the TGBp1
protein, which has RNA-binding properties and an RNA
helicase activity in vitro (Bleykasten et al., 1996; Brakke et al.,
1988; Cowan et al., 2002; Donald et al., 1997; Kalinina et al.,
2001; 2002). The two other TGB products of hordei- and
potexviruses, TGBp2 and TGBp3, are highly hydrophobic
and represent integral membrane proteins (Morozov et al.,
1989, 1990, 1991; Solovyev et al., 1996, 2000).
In plant cells transiently expressing TGBp2 protein, it is
associated mostly with the endomembrane system, particularly the tubular network of cortical endoplasmic reticulum
(ER) and Golgi-like structures (Cowan et al., 2002; Solovyev
et al., 2000). In a transient expression assay, TGBp2 of Potato
virus X (PVX) was able to modify the size-exclusion limit of
plasmodesmata (Tamai & Meshi, 2001). Moreover, TGBp2
and TGBp3 of PVX may even traffic from cell to cell in some
host plants (Krishnamurthy et al., 2002). TGBp3 was
localized to cell wall-associated membrane bodies (Cowan
et al., 2002; Solovyev et al., 2000; Zamyatnin et al., 2002).
Importantly, TGBp3 was able to change the localization of
TGBp2, targeting this protein to TGBp3-containing peripheral bodies. The TGBp3-directed subcellular targeting
seems not to involve sequence-specific interaction of the
TGBp2 and TGBp3 molecules and can also occur in mammalian cells, suggesting involvement of an universal mechanism for membrane protein transport (Solovyev et al., 2000;
Zamyatnin et al., 2002).
In hordeiviruses and other TGB-containing viruses of the
family Tubiviridae, TGBp2 and TGBp3 specifically target
TGBp1, and presumably TGBp1-formed movementcompetent RNPs, to plasmodesmata-associated sites (Erhardt
et al., 1999; 2000; Lawrence & Jackson, 2001b). This intracellular transport of TGBp1 is believed to be driven by the
TGBp3 subcellular targeting signal (Solovyev et al., 2000).
However, the mode of TGBp3 functioning is still obscure
and questions remain concerning the molecular mechanisms of protein subcellular sorting to peripheral cell compartments directed by TGBp3 and possible additional
movement-related TGBp3 functions.
Attempts at TGBp3 immunodetection in plants infected with
the type hordeivirus, Barley stripe mosaic virus (BSMV), have
failed (Donald et al., 1993). The lack of an immunodetection
system has impeded characterization of TGBp3 synthesized
during infection and complicated studies of its possible
interactions with host protein and lipid components and its
compartmentalization. In this paper, we have used subcellular fractionation, immunodetection and confocal microscopy to localize the PSLV TGBp3 protein in transgenic
Nicotiana benthamiana expressing the wild-type and GFPfused protein.
METHODS
The two hydrophilic regions of PSLV TGBp3 were expressed in E. coli as
fusions with mouse dihydrofolate reductase (DHFR). The 59-terminal
region of the PSLV TGBp3 gene encoding a hydrophilic segment located
at aa 1–61 and the central region encoding aa 82–139 were amplified by
PCR using pQE-GFP-18K as the template. Both PCR products were
digested with BamHI and XbaI and cloned into similarly digested
pQE40 (Qiagen). The expression vectors carrying the sequences of the
N-terminal and central hydrophilic regions of TGBp3 were designated
pQE-DHFR-111 and pQE-DHFR-35, respectively (Fig. 1).
BSMV mutant P684 containing the PSLV TGB and PVX.GFP, the PVX
derivative expressing GFP, have been described previously (Fedorkin
et al., 2001; Solovyev et al., 1999).
Expression of cloned genes in E. coli and protein purification. Recombinant pQE-based expression vectors were transformed
into E. coli strain M15[pREP4] (Qiagen). Induction of protein expression in the bacteria carrying vectors for GFP- and DHFR-fused
polypeptides was carried out essentially as described previously
(Solovyev et al., 1999). Purification of the proteins on Ni–NTA resin
(Qiagen) was according to manufacturer’s instructions. Before
immunization of mice, the protein was dialysed against 0?5 % SDS.
Monoclonal antibodies and PEPSCAN analysis. Four 8-week
old BALB/c mice were each immunized intraperitoneally with protein
suspension in water containing 0?5 % SDS (50–60 mg PSLV GFP–
18K) and boosted twice with the same dose at 2-week intervals. A
final boost was given by the intraperitoneal route with 50 mg of the
antigen without Freund’s adjuvant. Three days later, the splenocytes
were harvested and fused with the mouse plasmacytoma cell line
Sp2/0 using 45 % PEG 1500 (Erokhina et al., 2000). Hybridomas
secreting the specific monoclonal antibodies (mAbs) were identified
by indirect ELISA. These hybridomas were cloned twice under conditions of limiting dilution.
Overlapping sets of synthetic octapeptides, progressively moving along
by two residues and covering the 20 C-terminal residues of GFP plus aa
1–58 (N-terminal hydrophylic segment) and the aa 81–143 (central
hydrophylic segment) of PSLV TGBp3, were prepared on derivatized
cellulose sheets (SPOTs kit; Genosys) using activated F-moc amino
acid residues, as described (Erokhina et al., 2001).
Production of transgenic plants expressing PSLV TGBp3 and
its GFP fusion protein. Transgenic plants expressing the 18K pro-
tein or its translational fusion with the GFP gene (GFP–18K) were
generated with the binary vectors pBin-18K and pBin-GFP-18K,
which carried 35S promoter expression cassettes from the plasmids
pRT-18K and pRT-GFP-18K, respectively (Solovyev et al., 2000).
N. benthamiana leaf discs were transformed with Agrobacterium
tumefaciens AGL carrying vectors pBin-18K and pBin-GFP-18K and
transformants were regenerated on a kanamicin-containing selective
medium. R0 plants were self-pollinated and R1 progeny were analysed
by Northern blotting for the presence of 18K-specific mRNAs
(Fig. 2).
Subcellular fractionation and Western blot analysis. Leaf
Construction of recombinant clones. For expression of GFP, the
18 kDa PSLV TGBp3 protein (18K) or its translational fusion with
986
GFP gene (GFP–18K) in E. coli we used vector pQE30N, which is a
derivative of pQE30 (Qiagen) with an NcoI site introduced into the
plasmid polylinker (Solovyev et al., 1999). The 18K gene was excised
from plasmid pRT-GFP-18K (Solovyev et al., 2000) with NcoI and
XbaI and GFP–18K was excised from pRT-GFP-18K by partial digestion with NcoI and complete digestion with XbaI. The genes were
then cloned into similarly digested pQE30N. The GFP gene was isolated as an NcoI–XbaI fragment from pRT-GFP-S65T (Solovyev et al.,
2000) and ligated into NcoI/XbaI-digested pQE30N (Fig. 1).
tissue (100 mg) was ground into a fine powder with liquid nitrogen
in 2 ml buffer A (400 mM sucrose, 100 mM Tris/HCl, 10 mM KCl,
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Journal of General Virology 84
Detection of hordeivirus TGBp3
Fig. 1. Maps of pQE-based expression cassettes for the GFP fusion with the PSLV 18K TGBp3 protein (GFP–18K) and DHFR
fused with the N-terminal and central hydrophilic regions of 18K. Boxes with diagonal hatching show the hydrophilic regions of the
PSLV 18K protein. Boxes with vertical hatching represent the two hydrophobic transmembrane segments of the protein. T5, phage
T5 promoter; RBS, ribosome binding site; 6 His, a six-histidine tag fused to the N terminus of the expressed proteins. Restriction
sites used for cloning are indicated.
5 mM MgCl2, 10 mM b-mercaptoethanol, 1 mM PMSF, pH 7?5).
The slurry was filtered through one layer of Miracloth and the filtrate
was either centrifuged at 30 000 g for 30 min to yield a pellet of combined P1 plus P30 fraction (MEM fraction) enriched in organelles
and miscellaneous membranes and S30 fraction (soluble protein fraction), or centrifuged for 10 min at 1000 g to yield a pellet fraction
(P1) enriched in nuclei. In the latter case, the supernatant was centrifuged at 3000 g for 10 min to obtain pellet fraction P3 composed of
chloroplast fragments and mitochondria. The resulting supernatant
(S3) was centrifuged at 30 000 g for 30 min to give a P30 fraction
enriched in cell membranes. The Miracloth residue was washed in
buffer A containing 2 % Triton X-100 followed by centrifugation
(10 000 g) and resuspension in buffer A and this cycle was repeated
until the pellet became white to yield the cell wall (CW) fraction. All
fractions was resuspensed in 1?56 Laemmli sample buffer containing
either 9 M urea or as described in the figure legends.
For further subcellular fractionation on sucrose gradients, the S3
fraction from transgenic plants (~600 ml) was loaded on a 12 ml linear
25–55 % (w/w) sucrose gradient made in the extraction buffer, which
contained either 5 mM or 0?1 mM magnesium chloride. Immunoblot
analysis was performed using mAbs against the PSLV GFP–18K fusion
protein or polyclonal antiserum raised against the intrinsic ER protein
AtSEC12 (Bar-Peled & Raikhel, 1997) from Arabidopsis thaliana
(RoseBiotech).
Unfractionated leaf extracts (TE preparation) were prepared by adding
200 ml 1?56 Laemmli sample buffer, containing either 9 M urea or as
described in the figure legends, to 100 mg of plant tissue ground to a
fine powder with liquid nitrogen.
Fractionated proteins were transferred from 8–12 % gradient SDSpolyacrylamide gels to nitrocellulose membrane (Immobilon-P).
Reactions were revealed with the enhanced chemiluminescence
immunoblotting system (Amersham).
Fluorescent microscopy. Fluorescence was detected using a Zeiss
Fig. 2. Northern blot analysis of transgenic plants expressing the
18K protein or the GFP–18K fusion protein with an 18K-specific
probe. Lanes 1 and 2, 18K-expressing transgenic plants of lines
R3 and R15, respectively. Lanes 3 and 4, GFP–18K-expressing
transgenic plants of lines R1 and R5, respectively. C, control nontransformed plants. Approximate sizes of the detected mRNAs
are indicated.
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Axioscope 20 fluorescence microscope, as previously described
(Zamyatnin et al., 2002). Callose was visualized in leaf cells with
0?1 % (w/v) sirofluor-containing aniline blue (Merck) in 1 M glycine,
pH 9?5, as previously described (Smith & McCully, 1978; Stone et al.,
1984). To induce plasmolysis, leaf pieces were incubated in 0?5 M
mannitol solution for 5 min. Particle bombardment of N. benthamiana leaves was performed using the flying disc method with a
high-pressure helium-based PDS-1000 system (Bio-Rad), as described
by Morozov et al. (1997).
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E. N. Gorshkova and others
RESULTS
Monoclonal antibodies and epitope mapping
Using the expression vector pQE-GFP-18K (Fig. 1), the
6-His-tagged GFP-fused TGBp3 of PSLV was expressed in
E. coli and affinity-purified. Immunization of mice with the
6-His-GFP–18K fusion resulted, after cloning, in 26 mAbproducing hybrid clones, which were tested by indirect
ELISA and immunoblots with purified GFP and two recombinant proteins, DHFR-111 and DHFR-35, representing
DHFR fusions of the N-terminal and central hydrophilic
regions of the PSLV TGBp3, respectively (Fig. 1). Four mAbs
(3B4, 3B6, 3B9 and 3B12) gave a strong positive reaction with
GFP but not with DHFR-111 or DHFR-35 and three clones
(mAbs 2D3, 2D4 and 2D5) gave a strong reaction with GFP–
18K and weaker signals with both GFP and DHFR-111,
whereas mAb 1D4 bound only to DHFR-35 (data not shown).
Two mAbs, 1D4 and 2D5, showing different specificity to
GFP, DHFR-111 and DHFR-35, were selected for peptide
scanning analysis. Two overlapping sets of synthetic peptides
were used. To locate the epitope of mAb 2D5, a peptide set
was taken spanning the GFP–18K fusion protein region,
which included the 20 C-terminal residues of GFP and the 58
N-terminal residues of PSLV TGBp3. This mAb strongly
reacted with the peptide sequence ELYKGSMA located at the
junction of GFP (the six C-terminal GFP residues are shown
in bold) and the N-terminal hydrophilic segment of the
PSLV 18K protein. The C-terminal half of this octapeptide
was identical to the DHFR-111 sequence at the junction of
DHFR and PSLV TGBp3. This could explain the reaction of
mAb 2D5 with DHFR-111 in addition to GFP and GFP–18K
(Figs 1 and 3, and data not shown). To locate the epitope of
mAb 1D4, a peptide set was used that spanned aa 81–143 of
PSLV TGBp3. The mAb 1D4 strongly reacted with the
peptide sequence YVKGGY positioned in the central
hydrophilic region (aa 87–92) just downstream of the first
hydrophobic stretch of PSLV TGBp3 (data not shown).
Transgenic plants expressing the PSLV TGBp3
and its GFP fusion protein
In both the pBin-18K and pBin-GFP-18K transformants,
single bands of the expected sizes, approximately 0?5 kb for
the 18K-transgenic plants and 1?3 kb for the GFP–18Ktransgenic plants, were detected by Northern blot in several
plant lines (Fig. 2). Analysis of these lines by Western blotting with monoclonal antibodies to PSLV 18K demonstrated
expression of proteins of the expected sizes, either an 18 kDa
protein in 18K-transgenic lines, or a 45 kDa protein in the
GFP–18K-transgenic lines (Fig. 3A; Fig. 4). Based on these
analyses, four transgenic lines were selected for further work.
Lines R3 and R15 were used for the 18K transgene and lines
R1 and R5 for GFP–18K transgene (Fig. 2). For each transgene, similar results were obtained for the two lines. Therefore, only data for lines R1 and R15 are presented below.
Immunodetection of PSLV TGBp3 in transgenic
and infected plants
Initially, we analysed N. benthamiana plants infected with the
chimeric BSMV/PSLV virus P684, which carries the PSLV
Fig. 3. (A) Immunodetection of the PSLV 18K and GFP–18K proteins with mAbs 1D4 and 2D5 in non-transformed
N. benthamiana infected with the recombinant hordeivirus P684 and GFP–18K-transgenic N. benthamiana. MEM, S30 and
CW fractions were analysed. PVX.GFP, total protein extracts of N. benthamiana infected with a PVX-based vector expressing
GFP. The position of protein molecular size markers is shown on the right. (B) Stability of high molecular mass complexes
formed by GFP–18K following boiling and treatments with b-mercaptoethanol (b-ME) and different concentrations of urea. The
position of GFP–18K in the gel is indicated.
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Detection of hordeivirus TGBp3
agreement with the prediction that hordeivirus TGBp3 has
the properties of an integral membrane protein (Morozov
et al., 1989; Solovyev et al., 1996). Interestingly, treatment
with 2 % Triton X-100, a non-ionic detergent, which releases
membrane-bound MPs (Huang & Zhang, 1999; Reichel &
Beachy, 1998), was not sufficient to extract PSLV TGBp3
from the P1 fraction (Fig. 4).
Fig. 4. Extraction of the 18K protein from the P30 pellet with
0?1 M Na2CO3, 1 M NaCl or 2 % Triton X-100. The position of
the protein in the gel is indicated.
TGB region in place of that of BSMV (Solovyev et al., 1999).
For sample preparation, inoculated leaves were first taken at
5 days post-inoculation (p.i.) when the chlorotic symptoms
appeared. Subsequently, inoculated and systemically infected
leaves were harvested at later steps of infection. Samples were
fractionated to yield the MEM (P1 plus P30), S30 and CW
fractions. Neither mAb 2D5 nor 1D4 was able to detect the
PSLV TGBp3 protein in any subcellular fraction at any
infection stage tested, even when a sensitive enhanced
chemiluminescent detection system was used (Fig. 3A).
Previously, we have demonstrated that PSLV TGBp3
protein-induced peripheral membrane bodies contained a
resident ER fluorescent protein, suggesting that these bodies
could originate from an ER compartment (Zamyatnin et al.,
2002). To verify the origin of the peripheral bodies biochemically, the P3 fraction of the GFP–18K transgenic plants
was fractionated on a 20–55 % sucrose gradient (Fig. 5). As a
molecular marker, we used antibodies to the ER-specific
membrane protein AtSEC12 (Bar-Peled & Raikhel, 1997)
from A. thaliana. If subcellular fractionation was performed
in the presence of 0?1 mM Mg2+, the GFP–18K protein and
its high molecular mass aggregates were found in a number
of fractions forming a distinct peak (Fig. 5, lower panel,
fractions 11–14), which largely coincided with the AtSEC12containing peak (Fig. 5, lower panel, fractions 10–13). On
the other hand, the plasma membrane-specific aquaporin
PIP1b was found mostly in other gradient fractions (data not
shown). These observations showed that GFP–18K is mainly
To test whether mAbs 2D5 and 1D4 were able to recognize
GFP–18K expressed in plants, the leaves of transgenic
N. benthamiana plants were analysed by Western blotting. As
a control, PVX.GFP-infected N. benthamiana was used. It
was found that both mAbs 2D5 and 1D4 readily detected
GFP–18K in the MEM and CW fractions (Fig. 3A and 4).
However, the protein did not appeared in the soluble protein
(S30) fraction (Fig. 3A). In the control, as expected, GFP was
readily detected in extracts from PVX.GFP-infected plants
probed with mAb 2D5 but not with mAb 1D4 (Fig. 3A).
Importantly, substantial amounts of GFP–18K (Fig. 3) and
18K (Fig. 4) were found in the form of high molecular mass
bands with molecular masses greater than 175 kDa. These
aggregates were stable following b-mercaptoethanol treatment and boiling but dissociated in 9 M urea (Fig. 3B).
PSLV TGBp3 is an integral membrane protein
associated with the ER
To determine whether the PSLV TGBp3 and its complexes
were peripherally associated with or embedded into membranes, the P30 fraction was subjected to different biochemical treatments. The results showed that TGBp3 could
not be extracted from the P30 pellet by treatment with
1?0 M NaCl or 0?1 M Na2CO3 (Fig. 4). Similar results
were obtained for GFP–18K (data not shown). This is in
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Fig. 5. Western blot analysis of fractions from a sucrose
density gradient. Membrane preparations with 5 mM MgCl2
(+Mg2+) or 0?1 mM MgCl2 (2Mg2+) from transgenic plants
expressing GFP–18K were separated on a 25–55 % sucrose
gradient. Fractions were analysed with antibodies to 18K (mAb
1D4) and to the ER-specific protein AtSEC12, as indicated on
the left. Fraction numbers are indicated above each lane.
Sedimentation was from left to right.
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E. N. Gorshkova and others
associated with the ER membranes. If subcellular fractionation was performed in the presence of 5 mM Mg2+, there
was a slight shift of the peak compared with 0?1 mM Mg2+
and the appearance of a minor peak for both GFP–18K and
AtSEC12 (Fig. 5, upper panel, fractions 17–18). It is known
that low Mg2+ concentration releases ribosomes attached to
the ER, and a change of membrane mobility has also been
observed in other studies using sucrose-gradient fractionation of the plant ER in the presence or absence of EDTA (for
example, see Pedrazzini et al., 1997). Moreover, membrane
ER proteins used as markers for the sucrose-gradient fractionation of plant and yeast endomembranes often show
minor peaks of ER-containing fractions (for example, see
Ballensiefen et al., 1998; Sterling et al., 2001).
Non-fused TGBp3 from transgenic plants behaved similarly
to GFP–18K in sucrose gradients (data not shown). These
data suggested that transgenically expressed PSLV TGBp3 is
bound to membranes originating from the ER.
Fluorescent microscopy of GFP–18K expressed
in transgenic plants
The GFP–18K-expressing plants were examined using confocal laser scanning microscopy. In single optical sections
traversing the middle of the epidermal cell layer, the
GFP fluorescence was found in cell wall-associated bodies
(Fig. 6B), similar to that observed previously in cells expressing the GFP–18K protein (Solovyev et al., 2000; Zamyatnin
et al., 2002). In projections of a series of optical sections,
small GFP–18K-containing bodies were also visible on the
upper and lower cell surfaces (Fig. 6A), also consistent
with the transient expression data (Solovyev et al., 2000).
Therefore, the distribution of GFP–18K in transgenic
Fig. 6. Fluorescent microscopy of TGBp3 fusions and callose. (A–C) Fluorescent microscopy of epidermal cells of transgenic
plants expressing GFP–18K showing protein localization to cell-wall-associated bodies (A, B), which appear at higher
magnification as twin structures located at the opposite sides of the cell wall (C). (D) Staining of plasmodesmata-associated callose
depositions with sirofluor. (E) Localization of GFP–18K-containing bodies in the cytoplasm after plasmolysis of an epidermal cell of
a GFP–18K-expressing transgenic plant. Superposition of a bright field image and an image of GFP fluorescence is presented.
Arrows indicate the position of the plasma membrane. (F–H) Co-localization of DsRed–18K with callose depositions. (F) DsRed
fluorescence. (G) Callose staining. (H) Superposition of images in (F) and (G). Scale bars: (A) 10 mm; (C) and (D) 2 mm; (E) 7 mm;
(F) 4 mm.
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Detection of hordeivirus TGBp3
N. benthamiana plants was similar to that found in particle bombardment experiments. However, large cell-wallassociated bodies typically found in cells with high levels of
transiently expressed GFP–18K were not found in the GFP–
18K-transgenic plants. This observation is in agreement with
data showing that the size of the peripheral bodies correlates
with TGBp3 expression levels (Zamyatnin et al., 2002).
Examination of GFP–18K-expressing epidermal cells of the
transgenic plants under higher magnification revealed that
the peripheral fluorescent bodies located at the cell wall
represented twin structures consisting of pairs of disconnected bodies located on opposite sides of the cell wall
(Fig. 6C). Therefore, when the protein is expressed in the
transgenic plant cells, the peripheral bodies formed in two
neighbouring cells localized just opposite one another.
Similar twin bodies were found in plant tissues infected with
a tobacco mosaic virus vector expressing a GFP-fused TGBp3
protein of Potato mop-top virus (PMTV) (Cowan et al.,
2002). The structural basis for formation of the twin bodies
could be provided by plasmodesmata, assuming association
of TGBp3-containing bodies with opposite neck regions of
plasmodesmata in the same pit field (Blackman & Overall,
2001; Cowan et al., 2002; Erhardt et al., 2000; Lawrence &
Jackson, 2001b).
TGBp3 is localized to cytoplasmic structures
close to plasmodesmata
To verify the hypothesis that the GFP–18K-containing
peripheral bodies were associated with plasmodesmata, we
attempted to visualize plasmodesmata-associated callose
with sirofluor, a specific fluorescent dye (Smith & McCully,
1978; Stone et al., 1984). Fluorescent microscopy of nontransgenic control plants treated with sirofluor-containing
aniline blue demonstrated staining of discrete structures at
cell walls. At higher magnifications, these structures sometimes appeared as twin bodies located at opposite sides of the
cell wall (Fig. 6D), which was similar to previously described
callose staining (Oparka et al., 1997). Phenotypically, the
plasmodesmata-associated callose depositions visualized by
the sirofluor staining were strikingly similar to the GFP–
18K-containing peripheral bodies found in transgenic
plants (Fig. 6C).
As sirofluor has an emission maximum similar to that of
GFP, their fluorescence cannot be easily distinguished.
Therefore, in experiments with sirofluor staining we used
DsRed–18K – the 18K protein tagged with DsRed, a red
fluorescent protein isolated from Discosoma sp. As described
previously, localization of DsRed–18K is similar to that of
GFP–18K (Zamyatnin et al., 2002). To compare the subcellular localization of DsRed–18K and callose, the leaves of
N. benthamiana were bombarded with pRT-DsRed-18K and
then treated with sirofluor-containing aniline blue solution.
Using epifluorescence microscopy, DsRed–18K-containing
peripheral bodies were located in the vicinity of the
plasmodesmata-related sirofluor-stained callose depositions
(Fig. 6F–H). Interestingly, not all the plasmodesmata-related
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structures visualized by this treatment were associated with
DsRed–18K-containing peripheral bodies. Moreover, different sirofluor-stained structures were similar in size to each
other, whereas the size of the 18K-containing peripheral
bodies varied considerably (Fig. 6F–H and data not shown).
To distinguish between binding of the TGBp3-containing
peripheral bodies to the cell wall, plasma membrane or
cortical cytoplasm elements, the leaf portions of transgenic
N. benthamiana plants expressing GFP–18K were subjected
to plasmolysis by mannitol treatment and epidermal cells
were observed under the microscope. After the treatment,
gaps between cell walls and plasma membranes were visible in the bright field (Fig. 6E). Fluorescent microscopy
revealed that, after plasmolysis, GFP–18K-containing bodies
appeared within the cytoplasm but not cell walls (Fig. 6E,
arrows). Importantly, the GFP–18K-containing bodies,
which are normally static in the cells of transgenic plants,
became motile after plasmolysis and displayed random
motion in the cytoplasm (data not shown).
DISCUSSION
Earlier microscopic studies of fluorescent protein fusions
with TGBp3 transiently expressed in bombarded epidermal
leaf cells provided evidence for TGBp3 association with
specific membrane bodies near the cell wall (Cowan et al.,
2002; Solovyev et al., 2000; Zamyatnin et al., 2002). In this
paper, to characterize further the subcellular association of
TGBp3, we have conducted biochemical analysis of subcellular fractions and fluorescent microscopy of transgenic
N. benthamiana plants expressing the PSLV TGBp3 or its
fusion with GFP.
Although TGBp3 of PVX was detected by immunological
methods in infected plants as a component of the CW
fraction (Hefferon et al., 1997), immunodetection of TGBp3
in hordei- and benyvirus infections failed (Donald et al.,
1993; Niesbach-Klösgen et al., 1990). In this paper, we were
also unable to detect the presence of PSLV TGBp3 in virusinfected plants (from 5 to 18 days p.i.) with sensitive mAbs
combined with an enhanced chemiluminescence immunoblotting system (Fig. 3). Most probably, TGBp3 is expressed
in amounts too low to be detected in infected leaf tissues.
Indeed, it was found that, in BSMV-infected protoplasts,
TGBp3 was expressed at a tenfold lower level compared with
TGBp2 (Zhou & Jackson, 1996).
Immunodetection of the PMTV GFP–TGBp3 expressed
from a tobacco mosaic virus-based vector has demonstrated
its association with the CW and membrane fractions (P1 and
P30) (Cowan et al., 2002). In agreement with these data, it
was found that PSLV GFP–18K (Fig. 3A) and 18K (Fig. 4
and data not shown) in transgenic plants were exclusively
associated with the MEM (P1 and P30) and CW fractions.
Complementation studies using these transgenic plants
argue in favour of functional equivalence of transgenically
expressed GFP–18K and 18K. In both types of transgenic
plants, cell-to-cell and systemic movement of BSMV mutant
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E. N. Gorshkova and others
P684 containing the PSLV TGB was inhibited (data not
shown) in accordance with findings that independent
expression of TGBp3 results in blockage of TGB-mediated
virus cell-to-cell movement (Bleykasten-Grosshans et al.,
1997). Moreover, despite the lack of complementation of
P684 with disabled TGBp3 gene, both types of transgenic
plants efficiently complemented cell-to-cell and longdistance movement of PVX with disabled TGBp3 gene
(data not shown).
An unusual property of 18K and GFP–18K is the ability to
form high molecular mass aggregates in SDS-PAGE with
4–6 M urea, dissociating in 9–12 M urea (Figs 3 and 4).
These protein bands may correspond to natural TGBp3
forms and represent homomultimeric protein complexes
or aggregates with host components. Indeed, TGBp3 was
recently found to self-interact and thus, has the potential
to form homomultimers (Cowan et al., 2002). However,
integral membrane proteins may form high molecular
mass aggregates that are artefacts of the protein boiling
procedure in a standard Laemmli buffer. The SDS-resistant
thermic aggregation is not observed when SDS/HEPES
buffer and dithiothreitol are used instead of Tris and bmercaptoethanol in the gel loading buffer (Sagne et al.,
1996). However, such replacement had no obvious effect
on the TGBp3 aggregates (data not shown). Additionally,
SDS-resistant thermic aggregation results in the almost complete disappearance of a monomeric protein band with a
concomitant accumulation of aggregates (Sagne et al., 1996),
which is not the case with TGBp3 (Figs 3 and 4). Therefore,
it is likely that PSLV TGBp3 expression in transgenic
plants resulted in the formation of specific homomultimeric protein complexes. Recently, the high molecular
mass aggregates were also detected in plants expressing
PMTV GFP–TGBp3 fusion protein (Cowan et al., 2002).
When an isolated P30 fraction from PSLV TGBp3-expressing
transgenic plants was treated with agents that are known to
solubilize peripheral membrane proteins, TGBp3 remained
associated with the pellet (Fig. 3B). Moreover, the strong
anionic detergent SDS and the non-ionic detergent Triton
X-100, known to efficiently solubilize integral membrane
MPs (Huang & Zhang, 1999; Reichel & Beachy, 1998), were
not sufficient for extraction of the bulk of the PSLV TGBp3
from the P30 fraction (Fig. 3B and data not shown),
suggesting that it is not only hydrophobic interactions with
cell membranes that govern TGBp3 binding with the rough
membrane fraction.
To characterize the potential association of the TGBp3induced bodies with cell membranes, sucrose-gradient fractionation of the cell membranes from transgenic plants was
performed (Fig. 5). Immunodetection of the membraneembedded resident ER protein AtSEC12 (Bar-Peled &
Raikhel, 1997) showed that, as expected from microscopic
data (Zamyatnin et al., 2002), PSLV TGBp3 accumulated in
the ER-derived membrane fraction (Fig. 5).
The regular distribution of fluorescent opposing bodies in
992
transgenic plants expressing GFP–18K (Fig. 6C) suggested
that TGBp3 might be incorporated into membrane compartments near the plasmodesmata orifice (plasmodesmata
neck region). Our data on the co-localization of callose
deposits and TGBp3-induced bodies strongly support this
hypothesis (Fig. 6F–H) (see Botha & Cross, 2000; Blackman
& Overall, 2001; Cowan et al., 2002). Fluorescent microscopy
of N. benthamiana expressing GFP–18K revealed that the
peripheral bodies are pulled away from the plant cell wall
together with the plasma membrane during mannitolinduced plasmolysis (Fig. 6E). Concomitantly, the TGBp3containing fluorescent bodies in plasmolized cells became
motile, displaying stochastic motions in the cytoplasm (data
not shown). This is in contrast to the static behaviour of the
peripheral bodies in normal epidermal cells of bombarded
and transgenic plants that express GFP–TGBp3 (data not
shown and Solovyev et al., 2000). It was found that a membrane sucrose synthase that is bound to the plasma membrane and cell wall remains wall-associated after plasmolysis
(Amor et al., 1995). In contrast, our finding that the GFP–
18K-containing bodies became motile suggested that they
may be physically associated with the desmotubule, which
can be damaged or even torn after plasmolysis (Tilney et al.,
1991), rather than with the cell wall or plasma membrane.
Taking into account the localization of TGBp3 to plasmodesmata and its possible association with the desmotubule, a
mechanism for TGBp3-induced intracellular protein transport in virus infection can be hypothesized. At the sites of
their synthesis in the ER membrane, the TGBp2 and TGBp3
proteins might form specific compartments, which are then
transported using the targeting signal of TGBp3 to a specific
plasmodesmal receptor by diffusion along the ER network
(without exiting the ER), similar to lipids found to be able to
transverse plasmodesmata (Cantrill et al., 1999; Grabski et al.,
1993; Zamyatnin et al., 2002). Alternatively, TGBp3 may
form specific ER-derived membrane containers (vesicles or
tubules) that incorporate TGBp2 and are delivered to the
neck region of plasmodesmata and fused there to cortical ER
(see Brandizzi et al., 2002; Cowan et al., 2002; Jiang et al.,
2002; Solovyev et al., 2000; Stephens & Pepperkok, 2001;
Vitale & Denecke, 1999). Therefore, TGBp3 may function as
a part of a membrane transport pathway for translocation of
virus RNA, TGBp1 and TGBp2 to the orifices of plasmodesmata microchannels (see also Haywood et al., 2002;
Lough et al., 1998; Lucas, 1999; Tamai & Meshi, 2001).
ACKNOWLEDGEMENTS
This work was supported by Volkswagen-Stiftung, Federal Republic of
Germany, by the Russian Foundation for Basic Research (RFBR), by the
Russian Governmental programme ‘Integration’ and by the German–
Russian Inter-governmental programme for cooperation in agricultural
sciences.
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