the Nepo6irus group. More recently, GFLV inocu- lation of transgenic tobacco expressing the GFLV
CP gene pointed out a delay in tobacco infection [14], suggesting that this strategy could be useful
for the reduction of GFLV spread in grapevine. Thus, this gene has been introduced into several
rootstocks and cultivars [15 – 18]. Tests are under progress in greenhouses and in vineyards, in order
to evaluate the efficiency of the CP-mediated pro- tection strategy for grapevine [19]. Plant screening
however, is slowed down by the fact that GFLV inoculation to grapevine is difficult to master [20],
as sit requires its biological vector, Xiphinema index, for efficient transmission [19].
Several authors have shown that viral resistance of transgenic plants is functional at the single cell
level [21,22]. Electroporation of virus into plant protoplasts constitutes a useful tool to understand
the mechanism of its replication [23,24] and of its inhibition in transgenic plants [25 – 27], as well as
to verify plant tolerance towards virus infection [28 – 30]. In order to evaluate and to compare the
efficiency of the CP transgene and of other antivi- ral strategies in grapevine, we have developed a
technique for direct inoculation of grapevine with GFLV based on protoplast electroporation. So
far, electroporation of grapevine protoplasts has been described by Kolavenko et al. [31] for tran-
sient gene expression only. In a first set of experi- ments, permeation conditions were determined for
the rootstock 41B. We, then, established optimal uptake conditions for GFLV and ArMV, a related
virus also involved in the fanleaf disease, using either particles or viral RNA. This work is the first
case of direct inoculation of grapevine with GFLV and ArMV.
2. Materials and methods
2
.
1
. Virus, RNA and DNA ArMV and GFLV isolates F13 and GH were
routinely propagated on Chenopodium quinoa. Virus and RNA extractions and purifications have
been described previously [32]. Plasmidic DNA was propagated in Escherichia
coli HB 101-p35S, purified by the Qiafilter plasmid maxi kit method Qiagen, Germany, and resus-
pended in TE buffer 10 mM Tris, 1 mM EDTA, pH 8.3 at 1 mgml.
2
.
2
. Plant material Rootstock 41B Vitis 6inifera cv. Chasselas × V.
berlandieri colone no. 233 was used.
2
.
3
. Protoplast isolation Protoplasts were prepared either from in vitro
propagated plants or from embryogenic cell sus- pensions [33].
Well expanded leaves were sliced in 0.5 M man- nitol, 5 mM KCl, 2 mM CaCl
2
· 2H
2
O, 0.4 mM MgCl
2
· 6H
2
O, 0.3 mM 2-[N-morpholino] ethane- sulfonic acid MES, pH 5.7 osmotic pressure,
540 mosmkg and digested in 2 cellulase from Trichoderma 6iride 1 Umg, Fluka and 1 pecti-
nase from Rhizopus sp. 5 Ug, Fluka for 16 h at 23°C in the dark. Mesophyll protoplasts were
isolated and purified according to Chupeau et al. [34], cultured in liquid NN69 medium [35] with 0.6
M glucose and 2.5 mM MES, pH 5.8, at 23°C in the dark.
Embryonic cells 4 – 7 day old were digested in 1 cellulase from Trichoderma 6iride 1 Umg,
Fluka, 1 pectinase from Rhizopus sp. 5 Ug, Fluka, 0.5 Driselase from Basidiomycetes ssp. 1
Umg, Sigma in CPW salts [36] with 0.4 M man- nitol, 5 mM MES for 16 h at 25°C osmotic
pressure, 450 mosmkg. Purified protoplasts see above were cultured in half-strength MS salts
[37], MS vitamins, 0.5 M mannitol, 50 mM su- crose, 50 mg1 casamino acids, 5.4 mM naphtoxy-
acetic acid NOA, 2.5 mM MES, pH 5.8, at 23°C in the dark.
Protoplast viability was estimated by mixing 40 ml of protoplast suspension and 10 ml of Ery-
throsin B 0.4 wv and observation under a light microscope. Results were given as a percentage of
total number protoplasts. In order to evaluate the effects of electroporation conditions on protoplast
survival, viability was first established in both the electroporated samples and the non-electroporated
control as mentioned above, and then, survival rate was calculated as a percent of the non-electro-
porated control. A minimum of two independent repetitions was conducted for viability estimation.
2
.
4
. Protoplast transfection Two electroporation solutions were compared;
‘Low Salt’ medium LS, 0.1 mM CaCl
2
· 2H
2
O,
pH 5.6 [38]; ‘High Salt’ medium HS, 10 mM HEPES, 150 mM NaCl, 5 mM CaCl
2
· 2H
2
O, pH 7.1 [39], both solutions being adjusted to the
proper osmotic pressure with mannitol. Freshly isolated protoplasts were diluted in prechilled
electroporation solution, at 7.5 × 10
5
pml unless otherwise mentioned. Electroporation was per-
formed with the Bio-Rad Gene Pulser II appara- tus set at different voltages and capacitances,
using 400 ml of protoplasts and 400 ml of electro- poration mix per cuvette final protoplast con-
centration for electroporation, 3.75 × 10
5
per ml. All electroporated samples were kept on ice for
a further 30-min incubation and finally diluted in 2.5-ml culture medium. Negative controls con-
sisted of completed samples that were not elec- troporated.
2
.
4
.
1
. Calcein uptake Protoplast suspension 400 ml was mixed to
400 ml of 5 mM calcein in electroporation solu- tion and kept for 5 min on ice. After electropo-
ration and the 30 min incubation, samples were rinsed twice in 14 mM CaCl
2
· 2H
2
O, 4 mM MES, pH 5.7 adjusted with KCl to the proper
osmotic pressure, and centrifuged 5 min at 100 × g, and immediately observed under UV
light. The frequency of permeation was deter- mined by counting the green fluorescent proto-
plasts
out of
a minimum
of 100
intact protoplasts. Only protoplasts exhibiting a bright
intense fluorescence distributed throughout the cell were taken in account. A minimum of two
independent repetitions was done for this study.
2
.
4
.
2
. Plasmid DNA uptake Two different plasmids were used, pVT-GUS
containing the GUS gene under control of the enhanced CaMV ‘70S’ promoter, the non-trans-
lated leader sequence of TMV and the NOS ter- minator
[40]; pCK-GFP-S56C
harboring the
improved GFP S65C coding sequence under con- trol of the enhanced CaMV ‘70S’ promoter, the
non-translated leader sequence of TMV and the CaMV 35S terminator [41].
Plasmid 20 mg, 30 mg of salmon sperm DNA as carrier were successively added to 400 ml of
prechilled electroporation buffer and carefully mixed. Finally, 400 ml of protoplast suspension
were added and a further 5 min incubation was done before electroporation.
Transient gene expression was analysed in a minimum of two independent repetitions. GUS
fluorimetric assay was done 48 h after electropo- ration [42] with total protein extracts 5 mg of
proteins per assay. GFP analysis was performed 72 h after electroporation: samples were ob-
served under blue light 450 – 490 nm. The fre- quency of transient expression was determined
by counting the green fluorescent protoplasts out of a minimum of 100 intact protoplasts, in at
least four repetitions.
2
.
4
.
3
. Virus and RNA inoculation Protoplasts 400 ml at different densities were
carefully mixed with 400 ml of electroporation buffer containing 2 mg of virions, unless other-
wise mentioned, or 10 mg of viral RNA. After electroporation and incubation on ice, proto-
plasts were diluted in their respective culture medium and incubated as described above, for
72 h, unless otherwise specified, before im- munoblotting. Experiments were repeated at least
three times.
2
.
5
. Western blotting Protoplasts were harvested and centrifuged 3
min at 100 × g. The pellet was resuspended in 15 ml of loading buffer 2 SDS, 0.025 Bro-
mophenol Blue, 10 glycerol, 0.06 M Tris – HCl, pH 6.8. Proteins were denatured at 100°C for 5
min. Total proteins from 3 × 10
5
protoplasts were used for Western blotting. Preparation and
characterisation of the rabbit antiserum directed against purified 6HisP38 protein of GFLV iso-
late F13 have been reported previously [43]. P38 protein was chosen because of his stability in
GFLV infected plants Pinck, personal communi- cation. After separation by denaturing polyacry-
lamide
gel electrophoresis
SDS-PAGE, the
proteins to be probed were electro-transferred to Immobilon™ P membrane Millipore 1 h at 8
Vcm. The blot was then probed with the purified antiserum anti-6HisP38 at 130 000 dilu-
tion in PBS buffer, 5 skim milk and 1 Tween 20 for one night at 4°C. The proteins were visu-
alized by an immunoperoxidase method [44]. Positive controls of GFLV revelation consisted
on protein extracts of infected Chenopodium leaves kept frozen at − 20°C, denaturated and
revealed as protoplast extracts.
3. Results and discussion
