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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.
References
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 
of print]doi:10.1146/annurev.phyto.45.062806.094434    
9.   
Reustle,G,Ebel,R,Winterhagen,P,Manthey,T,Dubois,C,Bassler,A,Sinn,M,  
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 
25;441(7092):537-41
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