Plant Science 155 2000 203 – 212
GFLV replication in electroporated grapevine protoplasts
Laure Valat
a,b
, Sandrine Toutain
b
, Nadine Courtois
b
, Fabien Gaire
c
, Eric Decout
a
, Lothaire Pinck
c
, Marie-Claude Mauro
b
, Monique Burrus
a,
a
Laboratoire de Biologie et Physiologie Ve´ge´tales URVVC, UPRES EA
2069
, URCA, BP
1039
, F-
51687
Reims, Cedex
2
, France
b
Laboratoire de Viticulture, Moe¨t Chandon,
6
rue Croix de Bussy, F-
51333
Epernay, France
c
Institut de Biologie Mole´culaire des Plantes du C.N.R.S,
12
rue du Ge´ne´ral Zimmer, F-
67084
Strasbourg, Cedex, France Received 9 August 1999; received in revised form 3 December 1999; accepted 8 February 2000
Abstract
Grape6ine fanleaf 6irus GFLV, responsible for the economically important court-noue´ disease, is exclusively transmitted to its natural host in the vineyards through Xiphinema nematodes. We have developed direct inoculation of GFLV into grapevine
through protoplast electroporation. Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions. Permeation conditions were determined by monitoring calcein uptake. Low salt poration medium was selected.
Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation isolate F13. GFLV replication was detected with either virus particles 2 mg or viral RNA 10 ng in both protoplast populations, as shown by
anti-P38 Western blotting. Direct inoculation and replication were also observed with Arabis mosaic 6irus ArMV, a closely related nepovirus, as well as with another GFLV isolate. These results will be valuable in grapevine biotechnology, for GFLV
replication studies, transgenic plant screening for GFLV resistance, and biorisk evaluation. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Grapevine; Electroporation; Grape6ine fanleaf 6irus; Protoplasts; Virus inoculation www.elsevier.comlocateplantsci
1. Introduction
Grape6ine fanleaf 6irus GFLV is a nematode- transmitted virus infecting grapevine and causing
fanleaf degeneration, thus leading to dramatic de- generescence of the whole plant and to yield losses
in grape production. Belonging to the genus Nepo6irus within the family Comoviridae [1],
GFLV is characterized by polyhedral particles and by a genome constituted by two single positive-
sense RNAs. RNA1 of the F13 isolate encodes a 253 kDa polyprotein P1 involved in viral replica-
tion and in polyprotein processing [2]. RNA2 en- codes a 122 kDa polyprotein P2, required for
viral spread in planta, which is cleaved into a 28 kDa N-terminal protein called 2A, a 38 kDa
movement protein referred as 2
BMP
or P38 protein, and a 56 kDa carboxy-terminal coat protein CP
called 2C
CP
[3]. GFLV can only be controlled through plant sanitary selection and soil disinfec-
tion. Most chemicals used to kill the nematodes, however, are noxious to the environment and their
use has been limited in several countries. Different new methods like gene transfer strategy [4] are
currently under investigation in order to control GFLV spread.
Since Powell-Abel et al. [5] demonstrated that tobacco plants expressing the Tobacco mosaic 6irus
CP gene were protected against this virus, incorpo- ration and expression of different CP genes have
provided resistance in several different virus groups [6 – 11]. Bertioli et al. [12] and Brault et al.
[13] reported for the first time such protection in
Corresponding author. Tel.: +
33-326-913318; fax: 33-326- 913427.
0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 2 0 - X
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
