of plant viruses [2]. CP-mediated protection against nepoviruses has been demonstrated in transgenic
Nicotiana benthamiana expressing the ArMV CP gene [3], the Tomato ringspot 6irus CP gene [4], or
the GFLV CP gene [5], and in transgenic N. tabacum expressing the Strawberry latent ringspot 6irus CP
gene [6], or the Grape6ine chrome mosaic 6irus CP gene [7]. The formation of virus-like particles result-
ing from the accumulation of transgenic nepovirus CP in plant tissues was demonstrated for the ArMV
CP in N. benthamiana [8]. Genetic transformation of various grapevine tissues and regeneration of
transgenic plants have been reported for a number of grapevine rootstocks and cultivars [9]. Recents
reports described the regeneration of transgenic grapevine plants expressing a chimeric GFLV CP
gene after transformation of somatic embryos of the rootstock cvs 110 Richter [10,11], Gloire de Mont-
pellier, 3309 Couderc, Millardet de Grasset 101-14 [11], 41B and SO4, and the Vitis 6inifera variety
Chardonnay [12]. In these cases, expression of the GFLV CP transgene leads to the accumulation of
the CP in transgenic tissue detected by ELISA, but the presence of virion-like isometric particles
VLPs was not reported.
In this paper, we describe the expression of a chimeric ArMV CP gene in an annual herbaceous
species, N. benthamiana, and a woody perennial species, V. rupestris. The transgenic N. benthamiana
lines expressed the ArMV CP gene at high levels and accumulation of ArMV CP resulted in the forma-
tion of VLPs in plant tissue. In contrast, none of the transgenic V. rupestris lines accumulated the ArMV
CP at a detectable level. Data from northern blot experiments showed that the apparent lack of
ArMV CP protein accumulation in V. rupestris tissue was correlated to differences in transgene
expression levels andor RNA stability in grapevine tissues compared with N. benthamiana. Little or no
transgene mRNA could be detected in transgenic grapevine tissues. Progeny of ArMV CP-expressing
transgenic N. benthamiana lines showed resistance against ArMV.
2. Materials and methods
2
.
1
. Multiplication and purification of Arabis mosaic 6irus
ArMV isolate Triaca 782 was isolated in 1985 from degenerated grapevine J.-J. Brugger, unpub-
lished results and propagated on Chenopodium quinoa. The virus was purified [13] and viral RNA
extracted as described [14].
2
.
2
. Isolation of the ArMV CP gene The ArMV CP is encoded as part of a polyprotein
by RNA-2 [15]. ArMV RNA-2 cDNA was synthe- sized according to standard procedures Super-
script, Life Technologies and the cDNA was directly used in various PCR reactions. Based on the
published
ArMV-CP sequences
[16 – 18], two
primers were designed to engineer the ArMV CP gene by adding a methionine initiation codon: a
sense upstream primer ARMV-1, homologous to the first 18 nucleotides of the ArMV CP region,
containing in addition an ATG initiation codon and a BclI site at the 5-end TGATCATCCATG-
GGACTTGCTGGTAGAGG, and a reverse dow- stream primer ARMV-2, complementary to the
last 22 nucleotides of the ArMV CP gene, carrying in addition an XbaI site at the 5-end TCTA-
GAAACCTAAACTTTAAAACATGT. The am- plified ArMV CP fragment was then cloned into the
EcoRV site of the pUC-8 vector, creating plasmid pARMV-1 Fig. 1. The ArMV CP gene was
completely sequenced by the PCR cycle sequencing method fmole kit, Promega.
2
.
3
. Construction of a functional chimeric ArMV CP gene
A BclI – XbaI fragment from pARMV-1 contain- ing the complete ArMV CP gene was inserted into
the BamHI and SstI sites of the binary vector pBI121.1, replacing the uidA gene [19]. The
resulting plasmid pCACP-1 contained the ArMV CP gene under the control of the CaMV 35S
promoter and the nopaline synthase terminator Fig. 1.
2
.
4
. Plant transformation The plasmid pCACP-1 was transferred from E.
coli to A. tumefaciens LBA4404 by triparental mating. In vitro-grown leaf explants of N. benthami-
ana were transformed with A. tumefaciens contain- ing
the pCACP-1
plasmid using
standard methods [20]. Putative transformed plants were
selected on media containing kanamycin 100 mgl and cefotaxime 100 mgl. Grapevine transfor-
mation and regeneration were essentially per- formed as described [10].
2
.
5
. Characterization of transgenic plants The ability to form roots on media containing
kanamycin 50 mgl for N. benthamiana and 10 mgl for V. rupestris was used as the first indica-
tor of the transgenic status of the regenerated plantlets, since these amounts of kanamycin com-
pletely inhibited root formation of in vitro-grown wild type N. benthamiana or V. rupestris plants
data no shown. Integration of the transgenes was confirmed by Southern analysis using total DNA
extracted from 0.5 to 1.0 g of fresh leaf tissue of in vitro-grown N. benthamiana or V. rupestris plants
following the procedure described [21]. For South- ern analysis, 3–5 mg of plant DNA was digested
with EcoRI or EcoRV, electrophoresed on a 0.7 agarose gel in 1 × Tris – borate – EDTA buffer, and
blotted onto a charged Nylon membrane Boer- hinger, Mannheim by capillarity transfer under
denaturing conditions 0.4 M NaOH. Transferred DNA were hybridized to
32
P-labelled purified DNA fragment corresponding to the ArMV CP or
the npt II coding region. Presence of the ArMV CP transcript sequences was assayed by northern
blot analysis. Total plant RNA was extracted from in vitro-grown plants of N. benthamiana by
the acid guanidium – thiocyanate – phenol – chloro-
Fig. 1. Construction of the vector pCACP-1. After cDNA synthesis using random hexamers as primers, the coding region of the ArMV CP was specifically amplified using an upstream primer ARMV-1 complementary to the first 18 nucleotides of the CP
coding region according to the published ArMV CP sequence [16 – 18] and carrying in addition a BclI site at its 5-end and a methionine initiation codon, and a dowstream primer ARMV-2 homologous to the last 22 nucleotides of the CP coding region
and carrying an XbaI site at its 5-end. The 1539 bp amplified fragment corresponding to the ArMV CP coding region was cloned into the SmaI site of pUC-8 in order to verify the integrity of the gene by sequencing. After digesting pARMV-1 with BclI and
XbaI, the XbaI ends were filled-in and the fragment containing the ArMV CP gene was gel-purified and ligated to gel-purified plasmid pBI121.1 [19] cut with BamHI and Ecl
136
I. The resulting plasmid pCACP-1 contained the ArMV CP gene under the control of the CaMV 35S promoter p35S and the nopaline synthase terminator tNOS regions, replacing the uidA gene. The
flags stand for the termini sequences of the T-DNA borders. RB, right border; LB, left border; pNOS, nopaline synthase promoter region; nptII, neomycine phosphotransferase II coding region.
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
