form method [22] following the recommendations of the supplier reagent commercialized by Life
Technologies as Trizol. For grapevine, total nucleic acids were isolated as described [21] and
total RNA were subsequently purified by the acid guanidium – thiocyanate – phenol – chloroform
method procedure. About 10 mg of RNA were separated by electrophoresis on a denaturing
formaldehyde – agarose gel, transferred to a nylon membrane, and probed with a digoxigenin-labeled
purified DNA fragment corresponding to the ArMV CP region. ArMV CP mRNAs were visual-
ized by chemiluminescent detection Boehringer Mannheim. The R1 progeny of transgenic N.
benthamiana plants were screened for kanamycin resistance kan
R
phenotype by germinating seeds at least 100 seeds for each individual lines on
agar plates containing MS medium supplemented with
400 mgl
kanamycin sulfate.
Seeds were surface-sterilized in 10 commercial bleach
for 20 min and rinsed twice in sterile distilled water. Lines producing 100 kan
R
seeds were used for further analysis. For grapevine, primary
transformants were
propagated in
vitro from nodal cuttings according to standard proce-
dures.
2
.
6
. Expression of the ArMV CP gene in transgenic plants
Expression of the ArMV CP gene in transgenic plants was first determined by enzyme linked im-
munosorbent assay ELISA, using a rabbit anti- serum to ArMV virion essentially as described
[13]. Leaf material 0.3 – 0.5 g from in-vitro grown N. bentamiana or V. rupestris were ground in 3 ml
of extraction buffer 0.5 M Tris – HCl, 2 PVP, 1 PEG 6000, 0.14 M NaCl, 0.05 Tween, 0.02
NaN
3
, pH 8.2. Optical densities were measured at 405 nm 2 h after addition of the substrate p-nitro-
phenylphosphate at 1 mgml.
2
.
7
. Electron microscopy Crude leaf sap from N. benthamiana plants
grown under greenhouse conditions 4 – 6-week- old or from 2-week-old seedlings were negatively
stained with phosphotungstic acid and viewed on carbon-coated,
Formvar-filmed grids
using a
Philips 300 electron microscope according to [23,24].
2
.
8
. Protection experiments R2 seedling progeny from self-fertilized R1
transgenic N. benthamiana producing 100 kan
R
seeds were used to evaluate resistance to ArMV infection. Similar numbers at least 15 plants of
transgenic and control non-transformed plants consisting
of 6-week-old
greenhouse grown
seedlings were dusted with Carborundum and were mechanically inoculated with a 1:50 diluted
crude sap from systemically ArMV-infected C. quinoa leaves. The inoculum was prepared in 0.01
M phosphate buffer pH 7.0 containing 0.01 M sodium diethylthiocarbamate. Because ArMV CP
expressing transgenic N. benthamiana lines con- tained serologically detectable amount of ArMV
CP, the extent of infection was checked 2, 4 and 8 weeks
post-inoculation by
back transmission
of a 1:100 dilution of inoculated N. benthamiana upper leaf homogenate to healthy C. quinoa.
ArMV infection in C. quinoa was monitored by ELISA 9, 11 and 14 days after back-transmission.
The percentage of infected N. benthamiana plants was calculated as the percentage of infected C.
quinoa measured 14 days post back inocula- tion.
3. Results
3
.
1
. Construction of the pCACP-
1
transformation 6
ector The structure of the pCACP-1 transformation
vector is illustrated in Fig. 1. The integrity of the ArMV CP and the correct addition of the ATG
initiation were verified by sequencing. Comparison of the CP deduced amino acid sequence of
isolate
Triaca 782
with that
of published
ArMV sequences of [8,16] reveals a very high degree of homology 93 and 97 identity, respec-
tively.
3
.
2
. Characterization of regenerated plants Seventeen putative transgenic N. benthamiana
lines transformed with the pCACP-1 plasmid were regenerated. Genomic DNA isolated from a subset
of seven kanamycin resistant N. benthamiana plants was digested with the restriction enzymes
Fig. 2. Molecular characterization of seven transgenic N. benthamiana plants transformed with the ArMV CP gene. A Southern blot analysis of DNA isolated from N. benthamiana plants. Total DNA 3 – 5 mg prepared from an untransformed control plant
wt, or transgenic lines 65-1, 65-2, 65-3, 65-5, 65-6, 65-8 and 65-10 was digested with EcoRI or EcoRV, electrophoresed, and blotted onto Nylon membrane. The probe was a 1.5-kb fragment corresponding to the entire ArMV CP region solid bar in B.
32
P-labelled lambda DNA was also included in the hybridization mixture to reveal the position of the molecular weight marker fragments. M, lambda DNA cut with HindIII used as molecular weight marker; C, 40 pg of plasmid pCACP-1 cut with EcoRV.
In the EcoRV digest, the expected 1.1 kb ArMV-CP internal fragment is indicated by an arrow. B The top line shows a schematic representation of the T-DNA from the plasmid pCACP-1 integrated in the plant genome. The symbols used are the
same as in Fig. 1. The size of the expected internal EcoRV fragment and the minimum expected sizes for the EcoRI or EcoRV border fragments are shown below.
EcoRI or EcoRV, and characterized by Southern analysis using a 1.5 kb fragment corresponding to
the entire ArMV CP as probe Fig. 2B. In the EcoRI digest, one to three fragments hybridized to
the probe in each plant Fig. 2A. The bands represent right border fragments Fig. 2, allowing
the determination of T-DNA insertion loci and showing that, with the exception of plants 65-5
and 65-6, all the plants gave a specific hybridiza- tion pattern, demonstrating that they arose from
independent transformation events. The use of EcoRV allowed the detection of both an internal
T-DNA fragment of 1.1 kb and border fragments corresponding to the junction of the T-DNA with
the plant genomic DNA Fig. 2A. Results from both EcoRI and EcoRV digests showed that three
lines contained at least one apparently intact single T-DNA copy lines 65-1, 65-5 = 65-6, and 65-10,
one line contained at least two T-DNA copies line 65-3, one line contained at least three T-DNA
copies line 65-2 and one line contained a single incomplete or rearranged copy of the ArMV CP
gene line 65-8, in which the expected 1.1 kb internal fragment was missing. Inheritance of the
kanamycin resistant trait by plating seeds on selec- tive medium showed that the ratio of resistant to
sensitive R2 seedlings was 3:1 from a self for all the lines analyzed, except line 65-3 ratio 15:1,
demonstrating that T-DNA insertion at a single locus occured in most of the lines, except line 65-3
2 loci.
Only seven transgenic V. rupestris lines trans- formed with pCACP-1 could be regenerated. Hy-
bridization results of five kanamycin resistant V. rupestris plants are shown in Fig. 3, in which a
1.5-kb fragment corresponding to the entire ArMV CP was used as probe. Data for EcoRV
digests Fig. 3 showed that, with the exception of lines 030502 and 030505, which appeared to be
identical based on their hybridization patterns, all transgenic lines arose from independent transfor-
mation events. In addition, all have at least one intact copy of the ArMV CP gene. Line 030502
= 030505 contained a very high number of T-
Fig. 4. ELISA detection of the ArMV CP in the R2 progeny of six transgenic N. benthamiana lines and in four transgenic
V. rupestris lines. The OD450 nm was measured 2 h after addition of the substrate p-nitrophenylphosphate. Ext. buf,
extraction buffer; N.b. wt, untransformed N. benthamiana plant; 03wt, healthy V. rupestris plant; 03ArMV, ArMV-in-
fected V. rupestris plant.
Fig. 3. Molecular characterization of five transgenic V. rupes- tris plants transformed with the ArMV CP gene. Southern
blot analysis of DNA isolated from V. rupestris plants. Total DNA 3 – 5 mg prepared from an untransformed control plant
wt, or transformants 030501, 030502, 030503, 030505 and 030506 digested with EcoRV, electrophoresed and blotted
onto Nylon membrane. Hybridization conditions were as described in Fig. 2B. M, lambda DNA cut with HindIII used
as molecular weight marker. For a schematic representation of the T-DNA from the plasmid pCACP-1 integrated in the
plant genome, see Fig. 2B. The expected 1.1 kb ArMV CP internal fragment is indicated by an arrow.
DNA inserts seven to ten copies, probably as- sembled as complex tandem andor inverted repeat
units, whereas lines 030501 and 030506 contained at least one insert and line 030503 probably two
inserts. Results from Southern blots using other probeenzyme combinations data not shown
confirmed these findings and revealed that out of the seven regenerated transgenic V. rupestris lines,
only four arose from independent transformation events lines 030501, 030502, 030503 and 030506.
3
.
3
. Expression of the ArMV CP gene Expression of the ArMV CP transgene in trans-
genic N. benthamina and V. rupestris was first tested by ELISA using a rabbit antiserum specific
to ArMV virions Fig. 4. Six of the seven trans- genic N. benthamiana lines analyzed accumulated
the ArMV CP at various levels, whereas no ex- pression was detected in untransformed N. ben-
thamiana plants. As expected, line 65-8 did not accumulate the ArMV CP at detectable levels,
since molecular analysis showed that this line con- tained an incomplete ArMV CP gene. In contrast,
no detectable amount of ArMV CP could be detected by ELISA in four V. rupestris lines con-
taining the same ArMV CP gene Fig. 4. An electron microscopy study was undertaken to test
for the presence of VLPs in transgenic plants, as reported previously [10]. Virion-like structures
consisting of empty shells could be detected in the three N. benthamiana lines analyzed 65-2, 65-3,
65-5 Fig. 5a. Confirmation of the identity of the VLPs was performed by immunodetection using
a rabbit polyclonal antiserum specific to ArMV virions Fig. 5b.
In contrast, VLPs were not detected in V. rupes- tris despite several attempts of enrichment by su-
crose gradient. To determine whether the lack of ArMV CP
accumulation in transgenic V. rupestris was due to transgene expression level, we compared the
ArMV CP gene expression by northern blot in transgenic V. rupestris and N. benthamiana plants
using approximately the same amount of total RNA 10 mg Fig. 6. The expected mRNA tran-
script was detected in most of the transgenic N. benthamiana lines analyzed, with the exception of
line 65-8 whereas none was detected in a control plant wt. A band of the expected size was also
visible in the four transgenic V. rupestris lines 030501, 030502, 030503, 030506, but the hy-
bridization
signal was
weaker than
that found in transgenic N. benthamiana plants, partic-
ularly in line 030506. No band corresponding to the ArMV CP mRNA was detected in an
untransformed grapevine
plant wt,
and in
ArMV-infected V. rupestris lane wt + ArMV. In the latter case, the expected 3760 nt band corre-
sponding to the genomic ArMV RNA-2 was clearly visible.
3
.
4
. Protection experiments Homozygous N. benthamiana R2 progeny were
used for protection studies. Preliminary experi- ments using an inoculum consisting of a 1:10, 1:50
and 1:100 diluted crude leaf sap of ArMV-infected Chenopodium quinoa demonstrated that the 1:50
concentration was sufficient to obtain more than 90 infection of control plants. This dilution was
therefore used in further studies. Two weeks post- inoculation, 69 of the non-transformed plants
were infected, while the percentage of infection of the six independent transgenic lines was signifi-
cantly lower from 11 for lines 65-1 and 65-3 up to 55 for lines 65-5 and 65-8 Fig. 7. Four
weeks
post inoculation,
almost 85
of the non-transformed plants were infected, whereas
this percentage
remained significantly
lower for most of the transgenic lines. Two lines 65-1
and 65-2
showed a
significant resistance
phenotype, with only 13 65-1 or 28 65-2 of infection 4 weeks post inoculation. Three lines
65-3, 65-8 and 65-10 had a low level of re- sistance 55 to 60. One line 65-5 showed a
high percentage of infection not significantly dif- ferent from the non-transformed plants. After 8
weeks, all the transgenic lines were infected at the same level as the control plants, demonstrating
that the protection phenotype was overcome over time.
Fig. 5. Electron microscopy of negatively stained VLPs in crude leaf sap of transgenic N. benthamiana: a without prior
incubation with an ArMV specific antiserum; b with prior incubation with a rabbit antiserum specific to ArMV virions.
Bar = 100 nm.
Fig. 6. Northern blot analysis. Total plant RNA were ex- tracted from in 6itro grown plants of N. benthamiana or V.
rupestris. The probe was a 1.5 kb DNA fragment correspond- ing to the entire ArMV CP region labelled by PCR according
to the recommendations of the supplier of the labeling kit Boerhinger Mannheim. Two run-off transcripts correspond-
ing to the first half Tr1 = 806 nt and the entire ArMV CP Tr2 = 1537 nt were synthesized in vitro and 10 pg of each
transcript were loaded as size markers. The position of the expected 1596 nt ArMV CP and the ArMV RNA-2 mRNA
are shown with an arrow. Wt, untransformed plant; 65-1, 65-2, 65-5, 65-8, 65-10, independent transgenic N. benthami-
ana lines; 030501, 030502, 030503, 030506, independent trans- genic
V. rupestris
lines; wtArMV,
ArMV-infected untransformed V. rupestris plant.
Fig. 7. Protection experiments in N. benthamiana plants ho- mozygous for the ArMV CP gene. Each curve represents at
least 15 plants. Six-week-old greenhouse grown seedlings were dusted with Carborundum and were mechanically inoculated
with 1:50 diluted crude sap from systemically ArMV-infected Chenopodium quinoa leaves. Inoculated plants were observed
daily for the development of systemic symptoms. Because ArMV CP expressing transgenic N. benthamiana lines con-
tained serologically detectable amount of ArMV CP, the extent of infection was tested 2, 4 and 8 weeks post-inocula-
tion by back transmission to C. quinoa. The percentage of infected N. benthamiana plants was deduced from the percent-
age of infected C. quinoa measured 14 days post back inocula- tion by ELISA.
with the same chimeric gene did not accumulate the ArMV CP product at detectable levels and no
VLPs were produced in grapevine cells. The lack of ArMV CP accumulation in transgenic V. rupes-
tris may have several reasons. Data from southern blotting experiments showed that all transgenic V.
rupestris lines had at least one apparently intact copy of the transgene.
However, transgene expression studies by north- ern blot analysis demonstrated that the ArMV CP
transcript accumulated at lower level in V. rupes- tris compared to N. benthamiana. Several hypothe-
sis could account for the apparent reduced level of ArMV CP in transgenic grapevine tissues. First,
the rate of transgenes transcription driven by the CaMV 35S promoter might be significantly lower
in grapevine compared to other dicot plants, but this can be ruled out because several reports have
shown good expression of various transgenes in grapevine using this promoter [9,10]. Second, fail-
ure to extract RNA of good quality from grapevine may lead to partial or complete degra-
dation of the transgene RNA. However, recent northern blot data obtained after successive hy-
bridizations of the same blot to a control house- keeper gene N. benthamiana actin gene, then to a
specific ArMV CP sequence showed only a slight degradation of the grapevine RNA samples which
could not account for the differences observed. Third, transgene RNA turn-over may be higher in
grapevine cells, resulting in lower steady-state level and thus reduced translation rate. Fourth, the
genetic state of the transgenes present in the grapevine or the N. benthamiana genome may
have a significant influence on gene expression. Indeed, all the expression experiments were per-
formed on R2 progeny of N. benthamiana which were homozygous for the transgenes. In contrast,
the V. rupestris lines were vegetatively propagated and were therefore hemizygous for the transgenes.
This particular state might influence gene expres- sion level. Finally, the involvement of gene silenc-
ing at the transcriptional or post-transcriptional level cannot be ruled out.
Transgenic expression of nepovirus CP genes has been reported in herbaceous species for five
nepoviruses ArMV [8], TRSV [4], GFLV [5], GCMV [7] and SLRV [6]. In these studies, the
transgenic CP could readily be detected by ELISA in crude plant extracts of most of the transgenic
lines analyzed. Interestingly, VLPs formation was
4. Discussion
