3. Results and discussion
3
.
1
. Grape6ine protoplast characteristics The average diameter of leaf protoplasts freshly
isolated from 2-month-old plants was 20 9 10 mm. Viability was estimated to 77 9 6 and protoplast
yield was between 50 and 60 × 10
6
per g of fresh leaves. After 6 days of culture, protoplast viability
was, on average, 65 9 4.5 mean of five different experiments.
Embryogenic cell suspension protoplasts were smaller 5 9 2 mm and they contained lots of
starch grains. Protoplast yield was between 22 and 30 × 10
6
per ml packed cell volume and their viability after isolation was estimated to 84 9 6
mean of four independent replicates.
3
.
2
. Setting optimal permeation conditions Grapevine protoplast electroporation was stud-
ied using a two-step approach. First, we selected appropriate conditions for plasma membrane per-
meabilization by monitoring calcein uptake and protoplast survival in two media differing by their
ionic strength. Indeed, chemical properties of the poration medium are known to affect electrical
membrane breakdown [45]. Second, within these experimental conditions, we selected those yielding
the highest levels of transient gene expression after plasmid uptake.
When considering leaf protoplasts, without elec- troporation, in LS medium Fig. 1A, no fluores-
cence was observed, whereas in HS medium Fig. 1B, 15 of the protoplasts contained calcein, due
to natural passive uptake. When applying an elec- trical field, fluorescence increased with both the
voltage and the capacitance Fig. 1A and B. In LS medium, maximal calcein uptake was observed
at 300 V – 175 mF, 38 of the intact protoplasts were bright fluorescent. Not every protoplast of
the population, however, was transfected under these electrical conditions as protoplasts varied in
size and shape, as well as in the conductive proper- ties of cellular components data not shown. Sur-
vival after treatment at 300 V – 175 mF was about 14 of the non-electroporated control Fig. 1C. In
HS medium, best permeation conditions were 250 or 300 V – 125 mF, with 20 electropermeabilized
protoplasts when taking in account the strong
Fig. 1. Effects of electroporation conditions on grapevine leaf protoplasts. A Calcein uptake in LS medium; B calcein uptake in HS medium; C protoplast survival in LS medium; D protoplast survival in HS medium. Calcein uptake was determined by
counting the number of totally green fluorescent protoplasts out of 100 intact protoplasts per repetition. Survival was estimated immediately after electroporation, as a percent of non-electroporated controls.
Fig. 2. GUS activity in leaf protoplasts electroporated with 20 mg of pVT-GUS plasmid and 30 mg of salmon sperm DNA in LS medium. GUS fluorimetric assay was done 48 h after electroporation, using 5 mg of total proteins. Substrate for glucuronidase
4-MU, 4 methylumbelliferyl b-
D
-glucuronide.
fluorescent background of the non-electroporated controls. Survival was above 65 of the negative
controls Fig. 1D. With embryogenic cell suspension protoplasts,
best electroporation conditions were the same for both media: 200 V – 350 mF, yielding 50 of to-
tally fluorescent protoplasts in LS medium and 37 in HS medium. No passive calcein uptake
was observed.
Because of the less permeation efficiency of HS medium, further study was conducted with LS
medium only. It is characterized by low salt con- tent, and thus higher resistivity. It is less concen-
trated in salts than the pulse medium used by Kovalenko et al. [31] which contained 20 mM KCl
and 6 mM MgCl
2
in 0.6 M sorbitol, pH 6.0. Furthermore, their electroporation apparatus was
different and multiple pulses were applied to the samples, making direct comparisons difficult.
We, then, evaluated transient gene expression in both protoplast populations electroporated with
plasmidic DNA. We selected, from the previous study,
a range
of experimental
conditions combining good levels of protoplast permeation
ca. 30 and moderate mortality ca. 50 sur- vival, both compatible with gene expression: for
leaf protoplasts, one pulse at 150, 174 and 200 V – 150 or 175 mF; for embryogenic protoplasts,
one pulse at 200 V – 100 or 150 mF.
In leaf protoplasts, study of plasmid uptake confirmed that cell viability after electric treatment
under most conditions was sufficient for gene ex- pression. In all experiments done with pVT-GUS
plasmid, GUS activity increased with both the voltage and the capacitance as shown on Fig. 2,
excepted at 200 V – 175 mF. Highest GUS activity was observed at 174 V – 175 mF and 200 V – 150
mF. Using the GFP gene, we showed that 72 h after electroporation at 174 V – 175 mF, 16 9 3.5
of the remaining protoplasts expressed the GFP gene intensively, and 18.5 9 3 at 200 V – 150 mF.
GFP activity was also observed in both electri- cal conditions selected for embryogenic cell sus-
pension protoplasts, with 14 9 2 fluorescent protoplasts. Protoplast viability was estimated to
60 9 8 of the intact protoplasts.
3
.
3
. Inoculation with GFLV particles Analysis of transient protein expression indi-
cated that, for several electrical conditions, mem- brane breakdown was sufficient for plasmid
uptake without irreversibly altering protoplast sur- vival and protein synthesis. Similar study was then
conducted with GFLV particles, in order to evalu- ate optimal conditions of virus inoculation and
multiplication.
Concerning leaf protoplasts, revelation of virus replication assessed through Western blotting is
presented on Fig. 3A. Similar results were ob- tained in three independent experiments: no band
appeared in the non-electroporated protoplast ex- tract, whereas a single band of 38 kDa, corre-
sponding to the P38 protein, was present in the leaf extract of infected Chenopodium. In grapevine
electroporated protoplasts, the same band was
visible in five out of six electrical conditions tested, at about 38 kDa. Because no protein re-
acted in the negative control, the product de- tected after electroporation indicates that virus
penetration and replication had occurred. Proto- plast viability estimated 72 h after GFLV inocu-
lation i.e. 40 9 8 of the remaining protoplasts after electroporation at 174 V – 175mF, was suffi-
cient to allow virus multiplication. For further work, we selected two conditions for which a
significant amount of P38 protein was detected in the protoplast extracts, 274 V – 175 mF and
200 V – 150 mF. This is the first time that a Nepo6irus is successfully inoculated to grapevine
through electroporation. This experiment also in- dicates that, in grapevine protoplasts, conditions
for plasmid and viral particle uptake are almost similar, but not identical, thus justifying the pro-
gressive approach of our study. This shift which remained consistent over the repetitions should
be explained by different physical characteristics size, shape, electrical charges of virus particles
and plasmidic DNA molecules.
Next, we studied virus multiplication over the time, by assaying P38 accumulation in electropo-
rated protoplasts after different periods of cul- ture,
24, 48,
72 h
and 6
days. Virus
multiplication could be occasionally detected as soon as 24 h after electroporation in both se-
lected electrical conditions but, in general, a more intense and consistent reaction of the anti-
body was visible after 48 or 72 h. Therefore, this later period of culture was preferred for further
studies. Six days after electroporation, P38 could not be detected any more, probably because of
the high mortality observed in the protoplast populations several days after virus inoculation.
In Chenopodium protoplasts electroporated with GFLV virus, P38 concentration increased, in a
similar way, from 18 h after electroporation until 96 h [43], and CP detectable after 24 h following
electroporation also accumulated [38]. The effect of ‘protoplast quality’ had a strong effect on
virus multiplication. Plants of different ages 30, 50, 60 and 90 days after the last subculture
were used as protoplast sources that were elec- troporated under normal conditions, 174 V – 175
mF and 200 V – 150 mF. We clearly observed an effect of the age of the mother-plants: in all
cases, virus replication was detected but band intensity decreased with plant ageing, although
viability of freshly isolated protoplasts before electroporation was not significantly affected.
With 90-day-old plants, replication was barely observed. Plants not older than 2 months after
subculture were most amenable for virus intro- duction and multiplication. Virus concentration
has often been reported to be a limiting factor for
virus replication
in plant
protoplasts. Nishiguchi et al. [46] reported a linear relation-
ship between infection rate and virus concentra- tion up to 50 mgml. Okada et al. [23] noticed
that concentrations of TMV particles needed for tobacco protoplast infection were very high 500
mgml, compared to those needed with RNA 10
Fig. 3. Virus inoculation and replication in electroporated grapevine protoplasts. A Leaf protoplasts, effects of electri-
cal parameters 2 mg of virus particles; B cell suspension protoplasts, effects of electrical parameters 2 mg of virus
particles; C ArMV and GFLV – GH inoculation in suspen- sion protoplasts 200 V – 150 mF, 2 and 5 mg of particles.
Western blotting with anti P38 antibody, 72 h after electropo- ration; Ch, GFLV-infected leaves of Chenopodium; NE, non-
electroporated protoplasts incubated in electroporation mix.
Fig. 4. Viral RNA inoculation and replication in electropo- rated grapevine protoplasts, effects of electrical parameters,
A Leaf protoplasts 2 mg of virus particles; B Embryo- genic cell suspension protoplasts 2 mg of virus particles.
Western blotting with anti P38 antibody, 72 h after electropo- ration; Ch, GFLV-infected leaves of Chenopodium; NE, non-
electroporated protoplasts incubated in electroporation mix.
most conditions, but at 200 V – 100 and 150 mF, P38 detection was maximal. This condition was
selected for further work.
3
.
4
. Inoculation with ArMV and GFLV – GH particles
In order to verify that the protocol here devel- oped was not specific of the GFLV-F13 strain, we
electroporated embryogenic cell suspension proto- plasts with 2 and 5 mg of ArMV and of GH strain
of GFLV, at 200 V – 150 mF. Western blot, done 72 h after electroporation with anti-P38 antibody
as described above, is shown in Fig. 3C. Two bands were clearly visible in all four conditions, a
major band at 38 kDa, also found in infected Chenopodium leaves, and corresponding to P38; a
second smaller band probably corresponding to proteolytic processing of P38, as observed in other
work [43]. These results show that the protocol set up in this paper is not specific of a given strain of
GFLV.
3
.
5
. Inoculation with 6iral RNA Electroporation has been widely used to intro-
duce viral
RNA into
protoplasts [47 – 51].
Nishiguchi et al. [46] however, showed that inocu- lation conditions suitable for RNA were different
from those for viral particles. Based on these observations, best conditions for viral RNA up-
take in leaf and cell suspension protoplasts were determined. Results concerning leaf protoplasts
are shown in Fig. 4A. For all six electrical condi- tions examined, one band of 38 kDa was visible as
in infected Chenopodium leaf extract, whereas no product was observed in the negative controls. The
best signals, however, were obtained with 174 V – 150 mF and 150 V – 175 mF, which were then
chosen for further work. These conditions are quite similar to those required for particle inocula-
tion. This is quite different from the results of Nishiguchi et al. [46] which showed that a longer
pulse was necessary at the same voltage to induce TMV particle entry in tobacco protoplasts, com-
pared with RNA uptake.
For inoculation of cell suspension protoplasts with RNA, not all electrical conditions were suit-
able as shown on Fig. 4B. With 10 ng of RNA in the electroporation mix, P38 protein was detected
only at 200 V – 150 mF but never at 200 V – 100 mF. mgml We tested different amounts of GFLV viral
particles: 0.5, 1, 2, and 5 mg. No P38 protein was detected with 0.5 mg of particles. With 1 mg, the
corresponding band was clearly visible. Increasing virus concentration over 2 mg did not increase
band intensity. Viral particles 2 mg were used for further analysis.
The effect of protoplast concentration on repli- cation and detection of P38 was studied, using the
same preparation of protoplasts at different densi- ties, 3.75 × 10
5
, 7.5 × 10
5
and 1.125 × 10
6
proto- plasts per ml, and 2 mg of GFLV particles. Our
results indicated that the lower the concentration of protoplasts, the better the detection of P38 was
on the Western blot. We selected 3.75 × 10
5
proto- plasts per ml for further work.
Concerning cell suspension protoplasts, differ- ent electrical conditions were tested for virus inoc-
ulation, with 2 mg of particles and 7.5 × 10
5
protoplasts per ml. Typical results of Western blotting performed 72 h after electroporation are
shown on Fig. 3B. P38 protein was detected in
4. Discussion
Kategori