Viral infection enables phloem loading of GFP andlong-distance trafficking of the protein
Gadi Peleg, Dikla Malter and Shmuel Wolf*
Institute of Plant Sciences and Genetics in Agriculture and Otto Warburg Minerva Center for Agricultural Biotechnology, The
Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, PO Box 12, Rehovot 76100,
Israel
Received 8 February 2007; revised 7 March 2007; accepted 12 March 2007*For correspondence (fax +972 8 9462385; e-mail [email protected]).
Summary
It is generally accepted that viral systemic infection follows the source-to-sink symplastic pathway of sugar
translocation. In plants that are classified as apoplastic loaders, the boundary between the companion cell–
sieve element (CC–SE) complex and neighboring cells is symplastically restricted, and the potential passage of
macromolecules between the two domains has yet to be explored. Transgenic tobacco plants expressing
green fluorescence protein (GFP) and cucumber mosaic virus (CMV)-encoded proteins fused to GFP under the
control of the fructose-1,6-bisphosphatase (FBPase) promoter were produced in order to localize the encoded
proteins in mesophyll and bundle sheath cells and to explore the influence of viral infection on the functioning
of plasmodesmata interconnecting the two domains. GFP produced outside the vascular tissue could
overcome the symplastic barrier between the CC–SE complex and the surrounding cells to enter the
vasculature in CMV-infected plants. Grafting of control (non-transgenic) tobacco scions to CMV-infected
FBPase-GFP-expressing root stocks confirmed that GFP could move long distances in the phloem. No
movement of the gfp mRNA was noticeable in this set of experiments. The ability of GFP to enter the
vasculature and move long distances was also evident upon infection of the grafting plants with other viruses.
These results provide experimental evidence for alteration of the functioning of plasmodesmata intercon-
necting the CC–SE complex and neighboring cells by viral infection to enable non-selective trafficking of
macromolecules from the mesophyll into the sieve tube.
Keywords: coat protein, cucumber mosaic virus, movement protein, plasmodesmata, tobacco.
Introduction
Following viral replication in infected plant cells, systemic
infection requires the short-distance cell-to-cell movement
of viral genomes and their long-distance transport through
the vascular system. This systemic transport is governed by
a series of mechanisms involving virus and plant factors
(Lazarowitz and Beachy, 1999; Lucas and Gilbertson, 1994;
Scholthof, 2005). From its initial entry into plant cells, the
virus’s infectious material is transported to neighboring cells
via plasmodesmata. The plasmodesmata, together with
phloem conductivity, create a symplastic continuity that
enables the traffic of macromolecules and viral genomes
between cells and organs. However, in order to move long
distances, viruses have to overcome symplastic restric-
tion(s) imposed by plasmodesma-mediated selective traf-
ficking that forms symplastic domains. Accumulation of
cowpea chlorotic mottle virus in virus-resistant soybean
plants (Goodrick et al., 1991) or tomato aspermy virus in
tomato plants (Thompson and Garcıa-Arenal, 1998) is evi-
dent in bundle sheath cells but not in phloem parenchyma
cells, indicating the presence of a symplastic barrier to viral
movement at this boundary. A more distinct domain is the
sieve element–companion cell (SE–CC) complex in the
phloem tissue. The extremely low abundance, and sugges-
ted altered function, of plasmodesmata interconnecting the
SE–CC complex and neighboring cells (Ding et al., 1996,
1998) raise questions regarding the mechanism by which the
viral cargo overcomes this barrier and enters into the sieve
tube system.
Cucumber mosaic virus (CMV) is a single-stranded
positive-sense RNA virus divided into three segments
ª 2007 The Authors 165Journal compilation ª 2007 Blackwell Publishing Ltd
The Plant Journal (2007) 51, 165–172 doi: 10.1111/j.1365-313X.2007.03128.x
(Palukaitis et al., 1992). RNA-1 and 2 encode the 1A and 2A
proteins involved in RNA replication. RNA-2b encodes a
suppressor of post-transcriptional gene silencing, and RNA-
3 encodes the 3a gene as well as the coat protein (CP) gene,
which is also translated from sub-genomic RNA. Each of the
five encoded proteins participates in viral movement, but
both 3a protein and CP have been shown to be indispens-
able for cell-to-cell and long-distance movement of CMV
(Canto et al., 1997). The CMV 3a protein has the capacity to
bind single-stranded RNA (Li and Palukaitis, 1996), can
interact with plasmodesmata to alter their size exclusion
limits, and facilitates the traffic of viral RNA from cell to cell
(Canto et al., 1997; Ding et al., 1995; Kaplan et al., 1995). It
was therefore defined as a movement protein (MP). Immu-
nolocalization studies of the CMV MP in infected Nicotiana
clevelandii plants revealed that, even in minor veins, the
protein localizes mainly to plasmodesmata interconnecting
the CCs and SEs (Blackman et al., 1998). Expression of CMV
MP fused to green fluorescent protein (GFP) under a CC-
specific promoter (from commelina yellow mottle virus)
established that the fused protein moves through the
plasmodesmata interconnecting the CCs and SEs (Itaya
et al., 2002). Interestingly, the ability of the fused protein to
move outside the vascular tissue was organ- or develop-
mental stage-specific, indicating that CMV MP can mediate
its cell-to-cell movement by interaction with plasmodesmal
factors in a specific manner.
Earlier studies indicated that functional CP is also essen-
tial for cell-to-cell (Kaplan et al., 1998; Nagano et al., 2001)
and long-distance (Taliansky and Garcia-Arenal, 1995)
movement, but its mode of operation has yet to be explored.
The requisite co-existence of CP and MP for the short- and
long-distance movement of CMV led to speculation that
their interaction is required for viral infection (Blackman
et al., 1998; Canto et al., 1997). As CMV virions were not
detected in plasmodesmata interconnecting CCs and SEs of
infected plants, it was suggested that the virus moves into
the SE as a ribonucleoprotein complex that contains the viral
RNA, CP and MP (Blackman et al., 1998).
Despite ample information on the influence of viral
movement on plasmodesmata interconnecting mesophyll
cells, the mode by which viral components interact with
plasmodesmata interconnecting the SE–CC complex with
neighboring cells is still unknown. One might expect that
loading of the virus into the phloem would be associated
with alteration of the functioning of these plasmodesmata.
However, it is still not clear which viral protein(s) are involved
in the modification of these specific plasmodesmata, and
whether such a modification influences the traffic of endog-
enous macromolecules between the two distinct domains.
In this study, we produced transgenic plants expressing
GFP and CMV-encoded proteins fused to GFP under the
control of the fructose-1,6-bisphosphatase (FBPase) promo-
ter, which is specific to mesophyll cells (Lloyd et al., 1991).
We found that, upon viral infection, GFP that is produced
outside the vascular tissue can overcome the symplastic
barrier between the SE–CC complex and the surrounding
cells to enter the vasculature and move long distances in the
phloem. Our findings suggest that viral infection disrupts the
selectivity of macromolecular trafficking into the phloem.
Results
Tissue-specific expression of free GFP, CMV-MP:GFP, and
CMV-CP:GFP in transgenic tobacco plants
The FBPase promoter was employed to express free GFP,
CMV-MP:GFP or CMV-CP:GFP in transgenic tobacco plants
outside the boundary of the plant vasculature (Figure 1a).
These transgenic plants provide an experimental system to
study the role of the viral proteins in cell-to-cell and long-
distance transport of macromolecules. Detailed PCR analy-
sis (representative analyses are presented in Figure 1b–d)
indicated that 6, 12 and 11 independent tobacco lines
contained the genes coding for free GFP, CMV-CP:GFP and
CMV-MP:GFP, respectively.
Tissue and cellular localization of the fused proteins
CMV-MP:GFP and CMV-CP:GFP and free GFP in transgenic
tobacco plants
Fluorescence microscopy indicated that, when expressed
under the control of the FBPase promoter, free GFP and both
CMV-MP:GFP and CMV-CP:GFP were localized predomin-
antly to mesophyll cells with no expression in the epidermis
(Figure 2). Free GFP was localized to the cytoplasm and
nuclei (Figure 2a,b), CMV-MP:GFP was specifically localized
to the cell periphery, with bright fluorescent dots indicating
targeting to the plasmodesmata (Figure 2c,d), and CMV-
CP:GFP was localized predominantly to the nuclei. Bright
circles within the nuclei indicated specific localization of
CMV-CP:GFP to the nucleolus (Figure 2e,f).
Interestingly, when expressed under the control of the
FBPase promoter, CMV-MP:GFP was also detected between
vascular parenchyma cells or between vascular parenchyma
and bundle sheath cells (Figure 3a), while no fluorescent
signals from CMV-CP:GFP were detected in any of the
phloem cells (Figure 3b).
Phloem loading of GFP in virally infected tobacco plants
Transgenic plants expressing free GFP, CMV-MP:GFP and
CMV-CP:GFP under the control of the FBPase promoter
were infected with CMV to determine whether systemic
viral movement would enable traffic of the various
proteins into the vascular system. CMV infection caused a
significant reduction (and often elimination) of GFP fluor-
escence in plants containing CMV-MP:GFP or CMV-CP:GFP
166 Gadi Peleg et al.
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 165–172
(Figure S1c). Bright fluorescence was observed in plants
expressing free GFP (Figure 4b). Perhaps the most signifi-
cant outcome, in this set of experiments, were the bright
fluorescent signals within the vascular system of plants
expressing free GFP under the control of the FBPase pro-
moter as a result of CMV infection (Figure 4c,d). These
results indicate that the viral infection significantly alters
the function of plasmodesmata interconnecting the SE–CC
complex with the neighboring cells, thereby enabling the
traffic of GFP from the mesophyll into the vascular system.
In this respect, it is interesting to note that GFP fluorescence
was also detected in the epidermis of FBPase-GFP-expres-
sing CMV-infected plants.
Grafting experiments were employed to verify that the
GFP present in the vasculature of CMV-infected plants can
move long distances within the sieve tubes. As indicated in
Figure 4(e,g) GFP fluorescence was detected in control
FBPase promoter
FBPase promoter
FBPase promoter
GFPCMV-MP
EcoRI/BamHI XbaI XbaIBamHI
GFPCMV-CP
EcoRI/BamHI SmaI SalIBamHI
EcoRI/BamHI XbaI
GFP
SalI
(a)
(b)
(c)
(d)
Figure 1. Tobacco transformation.
(a) Structure of the chimeric binary plant expression cassettes used for plant
transformation.
(b)–(d) PCR analyses of leaves from various transgenic N. tabacum lines. (b)
Transgenic plants containing gfp (lanes 1–5) and non-transgenic control line
(lane 6).
(c) Transgenic plants containing cmv-cp:gfp (lanes 1–3 and 5), a transgenic
line that does not contain the gene (lane 4) and non-transgenic control line
(lane 6).
(d) Transgenic plants containing cmv-mp:gfp (lanes 1–5) and non-transgenic
control line (lane 6). M, molecular marker.
(a) (b)
(c) (d)
(e) (f)
(g) (h)
Figure 2. Cellular localization of free GFP (a, b), CMV-MP:GFP (c, d), CMV-
CP:GFP (e, f), in transgenic plants expressing the various gene constructs
under the control of the FBPase promoter, compared with control non-
transgenic plants (g, h).
GFP expression is demonstrated in spongy mesophyll (a, c, e, g) and palisade
(b, d, f, h) cells of young mature leaves. Fluorescence was detected and
imaged by confocal laser scanning microscopy. Bar = 10 lm.
Viral infection and phloem loading of proteins 167
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 165–172
tobacco scions, which did not contain gfp, when grafted
onto CMV-infected transgenic plants expressing GFP under
the control of the FBPase promoter. No fluorescence signal
was detected in a control tobacco plant grafted onto FBPase-
GFP non-infected transgenic rootstock (Figure 4f,h).
To further explore the general influence of viral infection
on phloem loading and long-distance transport of GFP,
similar grafting plants were inoculated with either tobacco
mosaic virus (TMV) or potato virus Y (PVY). Western blot
analyses confirmed the presence of GFP in the scion (sink
leaves) of control non-transgenic plants grafted onto
FBPase-GFP rootstocks infected with TMV, whereas GFP
was absent from control scions grafted onto healthy FBPase-
GFP-expressing rootstocks (Figure 5). GFP was also detec-
ted in control (non-transgenic) scions following infection of
FBPase-GFP stocks with PVY (data not shown).
The presence of GFP in the scion of control non-transgenic
tobacco plants may result from movement of the protein
translated in the rootstock expressing the gene, or from
traffic of the gfp mRNA. The shoot apex, upper stem
segment and young sink leaf of the control scions were
subjected to RT-PCR analyses to explore whether gfp
transcripts might be moving long distances. As indicated
in Figure 6, gfp transcripts were not detected in any tissue of
the control scions, indicating that the gfp mRNA cannot
move long distances from the transgenic rootstock.
Discussion
To explore the influence of viral proteins on phloem loading
of macromolecules, expression of the target molecule out-
side the vascular tissue must be assured. A tissue-specific
promoter (FBPase) was employed to express GFP and CMV-
encoded MP and CP fused to GFP as markers for phloem
loading and long-distance trafficking of macromolecules.
When expressed in transgenic tobacco plants under the
control of the FBPase promoter, all three proteins were
detected mainly in the spongy mesophyll and palisade cells
of source leaves. This tissue-specific expression is in agree-
ment with an earlier study in which the GUS reporter gene
was expressed under the control of the same promoter in
tobacco plants (Lloyd et al., 1991). In that study, GUS
expression was evident in photosynthetically active and
meristematic cells. As expected, fluorescence decoration of
the cell wall was observed in plants expressing CMV-MP:GFP
(Figure 2c,d), suggesting that the fused protein was targeted
to plasmodesmata. The specific localization (fluorescence) of
the fused protein on cell walls interconnecting neighboring
cells and not those facing air spaces (Figure 2c) provided
further support for interaction of the fused protein with
plasmodesmata. In contrast, CMV CP was localized exclu-
sively to the nuclei. Bright fluorescence signals in the center
of the nuclei suggested that the CP is localized specifically to
the nucleolus (Figure 2e). Targeting of virus-encoded CPs to
the nucleolus has been observed in tobacco plant cells
infected with tobacco etch virus (Langenberg and Zhang,
1997), in N. benthamiana epidermal cells infected with a
TMV derivative expressing the GFP-tagged ORF3 protein
(CP-like protein) of groundnut rosette virus (Kim et al., 2004),
and in cells infected with CMV (Lin et al., 1996). Mutational
analysis identified a particular sequence within the potato
leafroll virus CP that is involved in nucleolar targeting (Haupt
et al., 2005), while micro-injection and transient expression
experiments have demonstrated localization of the tomato
yellow leaf curl virus CP fused to GFP to nucleoli of
N. benthamiana mesophyll cells (Rojas et al., 2001). It has
been proposed that CP is involved in the shuttling of viral
nucleic acids between the nucleoplasm and the cytoplasm;
however, the biological significance of its nucleolar local-
ization is still unknown. Functional analyses of mutants in
the nucleolus localization domain of the groundnut rosette
virus ORF3 protein have indicated a correlation between its
nucleolar localization and its ability to form cytoplasmic
ribonucleoprotein particles and transport viral RNA long
distances via the phloem (Kim et al., 2004). These latter
results suggest involvement of the nucleolus in the control of
long-distance transport of RNA and possibly proteins.
Movement of free GFP between epidermal cells and from
the epidermis to mesophyll cells has been observed follow-
ing biolistic bombardment of plasmid encoding the gene
(a) (b) Figure 3. Cross-sections of third-order veins of
mature transgenic tobacco leaves expressing
CMV-MP:GFP (a) or CMV-CP:GFP (b) under the
control of the FBPase promoter.
In CMV-MP:GFP-expressing plants (a), GFP fluor-
escence is seen in the vascular parenchyma cells
or between phloem parenchyma and bundle
sheath cells (see arrows), compared with ab-
sence of the CMV-CP:GFP in the vascular area (b).
Typical autofluorescence is detected in xylem
elements. Fluorescence was detected and im-
aged by confocal laser scanning microscopy.
Bar = 10 lm. BS, bundle sheath; C, companion
cell; P, phloem parenchyma; S, sieve element; X,
xylem.
168 Gadi Peleg et al.
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 165–172
onto sink leaves (Itaya et al., 1998; Oparka et al., 1999).
During the sink-to-source transition, the capacity of the GFP
to move from cell to cell decreased substantially, and this was
associated with the developmental shift from simple to
branched forms of plasmodesmata (Itaya et al., 1998; Oparka
et al., 1999). However, when fused to either CMV-MP (Itaya
et al., 1998) or TMV-MP (Crawford and Zambryski, 2001), cell-
to-cell traffic of the 57 kDa fused protein was found in source
leaves as well. In the current study, localization of free GFP
and CMV-CP:GFP was restricted to mesophyll and bundle
sheath cells (Figures 2 and 3), but CMV-MP:GFP also decor-
ated the walls of the vascular parenchyma cells (Figure 3).
The presence of CMV-MP:GFP in vascular parenchyma cells
suggests that the MP can also interact with and move
through plasmodesmata interconnecting the bundle sheath
(a) (b)
(c) (d)
(e) (f)
(g) (h)
Figure 4. Influence of CMV infection on the transport of GFP into the vascular
tissue in transgenic tobacco plants expressing GFP under the control of the
FBPase promoter.
(a) Infected (left) and healthy (right) young source leaves.
(b) GFP fluorescence in spongy mesophyll cells of infected plants.
(c, d) Cross- and longitudinal sections, respectively, of a third-order vein of a
young mature leaf infected with CMV. Arrows indicate GFP fluorescence in
companion cells.
(e) GFP fluorescence in companion cells (see arrow) of a young leaf of control
non-transgenic tobacco scion grafted onto an infected FBPase:GFP-expres-
sing rootstock.
(f) No fluorescence signal, except for the typical autofluorescence of xylem
cells, was detected in a control non-transgenic tobacco scion grafted onto a
non-infected FBPase-GFP transgenic rootstock.
(g) GFP fluorescence in bundle sheath (see arrow) of a young leaf of a control
non-transgenic tobacco scion grafted onto an infected FBPase:GFP-expres-
sing rootstock.
(h) No fluorescence signal was detected in a control non-transgenic tobacco
scion grafted onto a non-infected FBPase-GFP transgenic rootstock.
For (e)–(h), longitudinal sections were performed on the third-order vein of a
young leaf (number 2 from the shoot apex). Typical autofluorescence is
detected in xylem elements in (c), (d) and (f). Fluorescence was detected and
imaged by confocal laser scanning microscopy. C, companion cell; X, xylem.
Figure 5. Presence of GFP as indicated by immunoblots.
GFP is present in the sink leaf of a non-transgenic control scion (1) and the
source leaf of FBPase-GFP transgenic rootstocks (2) infected with TMV, and is
present in the source leaf of non-infected FBPase-GFP-transgenic rootstock (3)
but absent from the sink leaf of non-transgenic control non-infected scion (4).
Figure 6. Presence of the gfp transcript as indicated by RT-PCR analyses.
The gfp transcript is present in healthy (lane 1) and CMV-infected (lane 2)
source leaves of FBPase-GFP rootstock, but absent from the sink leaf, stem
segment and apex of control non-transgenic scions in non-infected plants
(lanes 3, 5 and 7, respectively) and CMV-infected plants (lanes 4, 6 and 8,
respectively). Lane 9 is a positive control comprising plasmid containing the
gfp gene. Lanes 10 and 11 are negative controls comprising reaction mixture
with no cDNA template or in which the reverse transcriptase was absent,
respectively. The presence of the actin gene was seen in all plant tissues
(lower panel). M, molecular marker.
Viral infection and phloem loading of proteins 169
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 165–172
and vascular parenchyma cells of source leaves. In this
respect, it is interesting to note that this boundary provides a
symplastic barrier for the trafficking of some viruses (Good-
rick et al., 1991; Thompson and Garcıa-Arenal, 1998).
CMV infection was induced to explore the influence of all
viral components on phloem loading of macromolecules.
GFP expression was evident in CCs of CMV-infected FBPase-
GFP-expressing plants (Figure 4c,d). The presence of GFP in
the phloem of plants expressing this gene under the control
of the FBPase promoter indicates that CMV infection disen-
gages the symplastic barrier between the SE–CC complex
and neighboring cells. Moreover, grafting experiments
established that, upon CMV infection, GFP could enter the
sieve tube and move long distances to a non-transgenic
control tobacco scion in which gfp was not present (Fig-
ure 4e,g). Phloem loading and long-distance movement of
GFP upon infection with viruses from distant families (TMV
and PVY) suggest that the ability to interact with and alter the
functioning of plasmodesmata interconnecting the SE–CC
complex and neighboring cells is a general characteristic of
plant viruses.
Interestingly, no GFP fluorescence was observed in CMV-
infected plants expressing either CMV-MP:GFP or CMV-
CP:GFP. It is logical to assume that elimination of GFP
fluorescence in these plants was due to silencing of the
virally encoded mRNA via a post-transcriptional mechanism
(Baulcombe, 1999). Inoculation of these plants with either
TMV or PVY did not cause a significant reduction in GFP
fluorescence (Figure S1). These results support the assump-
tion that the reduction in expression of the CMV-encoded
proteins fused to GFP is only due to CMV infection. Never-
theless, plants infected with CMV exhibited typical symp-
toms, indicating that the viral genes were not silenced
effectively. It may well be possible that partial silencing was
sufficient to prevent detection and monitoring of GFP
fluorescence, but was not sufficient to prevent rapid replica-
tion of the viral genome. On the other hand, we cannot rule
out the possibility that the disappearance of GFP fluores-
cence was due to another, as yet unknown, mechanism.
No movement of CMV-CP:GFP or CMV-MP:GFP outside
the domain in which they were expressed was observed in
PVY- or TMV-infected plants. One possible explanation for
the non-movement of the virally encoded proteins fused to
GFP may relate to their cellular localization. Targeting of
CMV-encoded proteins to the nucleolus or plasmodesmata
(in the case of CP or MP, respectively) may result in
anchoring of those proteins to the cellular organelles,
thereby preventing (or inhibiting) their cell-to-cell and
long-distance movement. In this case, cell-to-cell and long-
distance trafficking of non-specific proteins occurred for
cytosolic proteins only. Alternatively, we cannot rule out the
possibility that the size exclusion limit of plasmodesmata
interconnecting the phloem with neighboring cells is not
sufficient to allow phloem loading of the fused proteins.
Tobacco plants are classified as apoplastic loaders, which
are characterized by a small number of plasmodesmata
interconnecting the bundle sheath/phloem parenchyma
cells and the SE–CC complexes. Intercellular and inter-organ
communication are required for the plant to function as a
whole organism, and it is therefore logical to assume that the
role of these plasmodesmata is to enable the long-distance
exchange of information molecules (Oparka and Turgeon,
1999; Wolf and Lucas, 1997). On the other hand, plasmodes-
mata interconnecting these two domains must be extremely
selective with regard to the passage of macromolecules in
order to enable distinct cellular processes in specialized
tissues. Unique protein profiles in the phloem sap of
cucurbits (Walz et al., 2004) and Brassica napus (Giavalisco
et al., 2006) reflect the selectivity of protein transport into the
sieve tube. Our data indicate that this restricted communi-
cation pathway is exploited by plant viruses to establish
systemic infection, altering the functioning of plasmodes-
mata interconnecting the two boundaries and allowing non-
selective trafficking of endogenous macromolecules.
Using a 2D gel electrophoresis method, we recently
compared the protein profiles in the phloem sap of healthy
and CMV-infected melon plants (Malter and Wolf, unpub-
lished data). We were able to identify 10–20 additional
proteins in the phloem sap of CMV-infected plants, exhib-
iting an increase of 5–10% in their phloem sap protein
content compared with healthy plants. These results further
support the hypothesis that CMV infection releases the
plasmodesma-imposed restriction of macromolecule traf-
ficking. Nevertheless, one should remember that the altered
protein profile in CMV-infected melon plants may be due to
traffic of endogenous proteins from the CCs and not all the
way from the mesophyll into the sieve tube. The fact that
only a small number of additional proteins were found in the
phloem sap of infected melon plants suggests that the plant
maintains some degree of selectivity, even during viral
infection. A current study is aimed at characterizing the
nature of those proteins that are present in the sap of virally
infected versus healthy plants.
Experimental procedures
Reagents and enzymes
DNA restriction enzymes and all nucleic acid modification enzymeswere obtained from Boehringer Mannheim (http://www.roche.com/home.html), unless otherwise specified. Cefotaxime (claforan) waspurchased from Laboratoires Roussel (http://www.sanofi-aventis.com). All other reagents were obtained from Sigma Chemical Co.(http://www.sigmaaldrich.com/) and Bio-Rad (http://www.bio-rad.com/).
Plasmids and tobacco transformation
The gfp (Chiu et al., 1996) was constructed by PCR using primers5¢-GGATCCGGACCTCCTCCTGGAATGGTGAGCAAGGGCGAG-3¢ and
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5¢-GAGCTCTTACTTGTACAGCTCGTCC-3¢, thus adding a spacer(Gly-Pro-Pro-Pro-Gly) to the gfp gene, and BamHI and SacI sites atthe 5¢ and 3¢ ends of the gene, respectively. The cmv-mp and cmv-cpgenes were fused to the gfp gene using PCR as follows. The geneswere obtained by PCR from the RNA-3 clone of CMV (Fny strain).cmv-mp was excised using primers 5¢-CCATGGCTTTCCAAGGTAC-3¢ and 5¢-GGATCCAAGACCGTTAACCACC-3¢, thus creating NcoI andBamHI sites at the 5¢ and 3¢ ends of the gene, respectively. cmv-cpwas excised using primers 5¢-AAAACCATGGACAAATCTGAATCA-3¢and 5¢-AAAAGGATCCGACTGGGAGCACTCCA-3¢, also creating NcoIand BamHI sites at the 5¢ and 3¢ ends of the gene, respectively.
The three constructs were first fused into the bacterial expres-sion vector pET29a (Novagen, http://www.clinafa.com/html/NVG/home.html) at the NcoI, BamHI and SacI sites, and expressed inEscherichia coli BL21 as CMV-MP:GFP or CMV-CP:GFP fusionproteins. The fusion proteins were examined by SDS–PAGE andfluorescence microscopy (data not shown).
The amplified gfp was digested with BamHI and HindIII at the 5¢and 3¢ ends, respectively, and was ligated into pBluescript (pBS) atthe same restriction sites. The amplified cmv-mp:gfp gene wasdigested with XbaI and SalI at the 5¢ and 3¢ ends, respectively, andligated into pBS at the same restriction sites. The amplified cmv-cp:gfp gene was digested with SmaI and XbaI at the 5¢ and 3¢ ends,respectively, and then ligated into pBS at the same restriction sites.The three genes were inserted into the binary vector pBinPlus (pBP;van Engelen et al., 1995) containing the FBPase promoter (Lloydet al., 1991), kindly provided by Professor Sonnewald (Lehrstuhl furBiochemie, Friedrich-Alexander Universitat Erlangen-Nurnberg,91058, Erlangen, Germany). The gfp gene was inserted as an XbaI/SalI fragment, cmv-mp:gfp was inserted as an XbaI/XbaI fragment,and cmv-cp:gfp was inserted as a SmaI/SalI fragment.
The three binary vectors containing the genes coding for GFP,CMV-MP:GFP and CMV-CP:GFP were transformed into Agrobacte-rium strain LBA4404. Transgenic plants were produced by conven-tional methods (Horsch et al., 1985). Briefly, leaf discs of Nicotianatabacum (Xanthi nc) were inoculated with the recombinant Agro-bacterium cells, using kanamycin (300 mg l)1) and claforan(500 mg l)1) as selective agents. GFP-expressing plants were selec-ted using a fluorescent microscope (model BH-2; Olympus, http://www.olympus-global.com/). Rooted transformants were grownunder sterile conditions at 25�C, and then removed to soil mixturein a greenhouse with natural sunlight.
Grafting experiments and virus inoculation
Non-transgenic tobacco plants were grafted onto rootstocks (with5–7 source leaves) of FBPase:GFP transgenic tobacco plants (Fig-ure S2). Seven to 10 days after grafting, the rootstock’s youngestsource leaf was inoculated with CMV (strain Fny) using carborun-dum as an abrasive and 500 ll inoculum containing 40 lg virus.Control grafts, including non-transgenic rootstock, were subjectedto a similar inoculation procedure with H2O. Additional virusinfections included TMV and PVY using similar procedures.
Confocal laser scanning microscopy
For cross-sections, cut leaves were embedded in 7% low-meltingagarose (Conda, http://www.condalab.com), after which they weresectioned (60–80 lm width) using a vibratom (VT1000S, Leica,http://www.leica.com). GFP expression in phloem cells was visu-alized in cross-sections of third-order veins (the first-order vein isthe midrib which branches at frequent intervals to form second-order veins, and these are branched to form the third-order veins).
The pattern of GFP expression was detected by confocal laserscanning microscope (CLSM model LSM510; Zeiss, http://www.zeiss.com/) using blue laser (25 mW argon) excitation light(488 nm).
GFP detection gel blots
An alternative method for the identification of GFP in grafting plantswas based on Western blot analyses. Tissue samples were takenfrom young, healthy and virally infected sink leaves of control (non-transgenic) scions and from source leaves of FBPase:GFP trans-genic tobacco rootstocks.
Plant tissue (1:4 w/w) was homogenized in extraction buffer (9 M
urea, 75 mM Tris buffer, pH 6.8, 4.5% v/v SDS and 7.5% v/v b-mercaptoethanol). After grinding, samples were left at roomtemperature for 30 min, boiled for 5 min, and then centrifuged at10 000 g for 10 min. The supernatant was brought to a finalconcentration of 10% v/v glycerin. Equal amounts of protein wereseparated on 12% SDS–polyacrylamide gels containing 0.3% bis-acrylamide at 200 V and 18 mA for 2 h. Proteins were transferred toa nitrocellulose membrane and GFP was detected using polyclonalanti-GFP antiserum (1:100; Clontech, http://www.clontech.com/).
RNA isolation and RT-PCR analyses
Total RNA was extracted from 100 mg of the shoot apex, stemsegments and a young sink leaf of CMV-infected and healthy control(non-transgenic) tobacco scions of the same age, using Tri-reagent(Sigma) according to the manufacturer’s recommendations. TotalRNA extracted from source leaves of FBPase:GFP transgenic to-bacco rootstocks served as a positive control. RNA was quantifiedusing an RNA/DNA analyzer (GeneQuant II, Pharmacia Biotech,http://www.apbiotech.com). From 2 lg total RNA per sample,cDNA was prepared using a 22-mer-encoded oligo(dT) primerand Superscript II reverse transcriptase (Lifetechnologies, http://www.gentaur.com).
Two specific primers within the conserved region of the gfp, 5¢-AAAATGGTGAGCAAGGGCGAG-3¢ and 5¢-AAAGTACAGCTCGTCCATGCC-3¢, forward and reverse, respectively, were used for theidentification of gfp mRNA in the various plant tissues. For the sameset of cDNA samples, another PCR analysis was performed for thehousekeeping gene actin, serving as an internal probe for the mRNAlevel. Parallel RT-PCR analyses were performed, including controlsin which the reverse transcriptase was omitted to verify thatgenomic template was absent from the reaction mixture.
Acknowledgements
This paper is a contribution from the Uri Kinamon Laboratory.G.P. and D.M. were supported by a scholarship from theKinamon Foundation. Support of this project by the GermanFederal Ministry of Education and Research (BMBF) within theframework of German–Israeli Project Cooperation (DIP) is gratefullyacknowledged.
Supplementary Material
The following supplementary material is available for this articleonline:Figure S1. Cellular localization of CMV-CP:GFP in transgenictobacco plants expressing the gene construct under the control ofthe FBPase promoter following viral infection.
Viral infection and phloem loading of proteins 171
ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 51, 165–172
Figure S2. Graft union of a control Xanthi tobacco plant ontransgenic rootstock expressing GFP under the control of theFBPase promoter.This material is available as part of the online article from http://www.blackwell-synergy.com.
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