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[SANET-MG] Transgenic Grapevine Resistant to Fanleaf Virus
5 May 2007
Prof. Joe Cummins
Transgenic Grapevine Resistant to Fanleaf Virus : How safe is it?
Grapevine fanleaf virus (GFLV)is the oldest known virus disease of
grapes. The disease has been reported in the USA and Europe and the
impact varies with different varieties of grapevine, some recording up
to 80% yield losses. The virus is spread from plant to plant by a
species of Dagger nematode. The fanleaf virus is a member of a virus
group called Nepo viruses. The fanleaf virus has two small RNA
chromosomes each of which makes a single messenger RNA molecule and a
single polyprotein which is processed to make a number of proteins
involved with virus replication and the virus capsid (the coat for the
virus). The virus replicates on the inner membrane of the plant cell
cytoplasm (1). The dagger nematode (Xiphinema) attacks the grape root
stocks and carries the virus between the grapevines that it invades,
GFLV can be detected with great sensitivity in the soil nematode (2).
The GFLV chromosome RNA2 bearing the viral coat protein is involved in
transmitting the virus by the dagger nematode Xiphinema
(3).Recombination has been observed between different strains of GFLV
(4). Resistance to GFLV could be achieved either by creating rootstocks
that resisted the nematode or by rootstocks that resisted GFLV.
Presently, the main focus of biotechnology has been top produce GFLV
resistant stocks rather than producing nematode resistant stocks. Dennis
Gonsalves m Marc Fuchs and other researchers patented GFLV resistant
grape vine expressing fanleaf virus coat protein(5) Rootstock
constructs contained at one locus the GFLV coat protein gene driven
by cauliflower mosaic virus promoter and a nopaline synthase terminator
accompanied by a neomycin antibiotic resistance gene with a nopaline
promoter and terminator. At another locus the construct contained a
GUS marker gene allowing rapid identification of transformed grape
stocks accompanied by a GFLV movement protein to deal with the cell to
cell movement of GFLV (6). The GFLV resistant stocks with non-transgenic
scions were tested for recombination under heavy pressure from infected
nematodes but recombinant viruses did not appear over three years of
heavy nematode pressure (7). Fuchs and Gonsalves reviewed the many
transgenic crops made virus resistant using virus coat protein genes.
The way that coat protein genes may interfere with virus replication is
to silence the replication machinery of the virus by inducing production
of a small interfering RNA (RNAi) which suppressed virus replication
(8). Specific genes for GFLV silencing were developed and used to
transform grapevine but fuller tests of the transgenic stocks have not
yet been published (9).
The genes for GFLV resistance are contained within the rootstock
and not present in the scion. There seems little reason to believe that
the major genes could spread to weeds or to other grapevines. There is
some chance that the antibiotic resistance marker genes used to prepare
the transgenic stocks can be transferred to soil microbes. However, the
main concern is the silencing RNAi molecules which are bound to be
transferred throughout the grape vine through the phloem vessels of the
vine in the same way that systemic silencing has been spread across
graft junctions in plants (11,10).
The use of virus coat proteins is now known to activate RNAi
silencing of the viral genes and specific RNAi genes to target
particular viruses are being developed. At this point it is very
important to insure that the novel RNA i genes which are always
producing RNAi in transgenic plants are not harmful to those consuming
the RNAI in food or beverage. RNAi genes are active in both plants and
animals and share homology. It has been observed that oversaturation
of cellular RNAi pathways may lead to fatality in mammals(12). It is
crucial that virus resistance through transgenic food crops be tested
for toxicity to animals. It has been argued that such tests are not
needed because people are always eating some virus infected foods.
However, that argument is not logical because the plants would not be
infected with virus provided that they produced a quantity of RNAi to
silence the virus. In conclusion, the RNAi produced in the rootstock is
bound to be translocated to the foliage and grapes. It is imperative
that the RNAi should be identified and tested for its impact on mammals
and on the environment.
1. Ritzenthaler C, Laporte C, Gaire F, Dunoyer P, Schmitt C, Duval S,
Piequet A, Loudes AM, Rohfritsch O, Stussi-Garaud C and Pfeiffer P.
Grapevine fanleaf virus replication occurs on endoplasmic
reticulum-derived membranes. J Virol. 2002 Sep;76(17):8808-19.
2. Demangeat G, Komar V, Cornuet P, Esmenjaud D and Fuchs M.
Sensitive and reliable detection of grapevine fanleaf virus in a single
Xiphinema index nematode vector. J Virol Methods. 2004 Dec 1;122(1):79-86.
3. Belin C, Schmitt C, Demangeat G, Komar V, Pinck L and Fuchs M.
Involvement of RNA2-encoded proteins in the specific transmission of
Grapevine fanleaf virus by its nematode vector Xiphinema index.
Virology. 2001 Dec 5;291(1):161-71.
4. Vigne E, Demangeat G, Komar V and Fuchs M. Characterization of a
naturally occurring recombinant isolate of Grapevine fanleaf virus.
Arch Virol. 2005 Nov;150(11):2241-55.
5. Gonsalves,D,Xue,B,Krastanova,T,Ling.K and Fuchs,M. Grapevine
fanleaf virus resistance in grapevine expressing grapevine fanleafvirus
coat protein 2003 United States patent 6,667,426
6. Valat, L, Fuchs, M and Burrus, M. Transgenic grapevine rootstock
clones expressing the coat protein or movement protein genes of
Grapevine fanleaf virus: Characterization and reaction to virus
infection upon protoplast electroporation Plant Science [Plant
Sci.].2006 Vol. 170, no. 4, pp. 739-747.
7. Vigne E, Komar V and Fuchs M. Field safety assessment of
recombination in transgenic grapevines expressing the coat protein gene
of Grapevine fanleaf virus. Transgenic Res. 2004 Apr;13(2):165-79.
8. Fuchs M and Gonsalves D. Safety of Virus-Resistant Transgenic
Plants Two Decades After Their Introduction: Lessons fromRealistic Field
Risk Assessment Studies. Annu Rev Phytopathol. 2007 Apr 4; [Epub ahead
Cobanov,P,Wetzel,T,Krczal,G,Jardak-Jamoussi,R and Ghorbel,A. Induction
of silencing in transgenic grapevines ISHS Acta Horticulturae 689: VII
International Symposium on Grapevine Physiology and Biotechnology editor
Williams,L 2005 ISBN 978-90-66057-18-0
10. Tournier B, Tabler M and Kalantidis K. Phloem flow strongly
influences the systemic spread of silencing in GFP Nicotiana benthamiana
plants. Plant J. 2006 Aug;47(3):383-94
11. Ruiz-Medrano R, Xoconostle-Cazares B and Kragler F. The
plasmodesmatal transport pathway for homeotic proteins, silencing
signals and viruses. Curr Opin Plant Biol. 2004 Dec;7(6):641-50.
12. Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR,
Marion P, Salazar F and Kay MA. Fatality in mice due to oversaturation
of cellular microRNA/short hairpin RNA pathways. Nature. 2006 May
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