Engineering Durable Pathogen Resistance
in Grapevine: GFLV-specific Antibodies
Confer Virus Resistance
create safe, virus-resistant plants by
expressing GFlV-specific recombinant
antibodies. We predicted that highaffinity antibodies specific to the GFlV
coat protein would neutralize the virus
in the early infection stages by interfering with genome encapsidation and
preventing cell-to-cell movement.
Figure 3:
Reduction in the symptoms of systemic GFlV
infection in transgenic T2 N. benthamiana plants
infected with the virus, compared to wild type
(WT)
Background
Grapevine fanleaf virus (GFlV) is one of
the most destructive and widespread
grapevine pathogens, in some cases
causing yield losses of up to 80 %. It is
transmitted by soil nematodes. There
are no natural sources of resistance in
Vitis vinifera, so the control of fanleaf
disease is currently based on sanitary
practices and soil disinfection using
nematicides. Although dissemination
of the virus has been reduced through
these measures, the control of GFlV in
naturally-infected vineyards is still inefficient. The use of nematicides is largely unsuccessful because nematodes can
survive on detached grape roots deep
in the soil, and in many countries the
measure is banned because of environmental toxicity. Therefore, the development of virus-resistant grapevine varieties is likely to be the most effective,
environmentally-friendly and sustainable approach to grapevine fanleaf control.
Aim
Pathogen-specific recombinant antibodies have been used to characterize
pathogen infections and to engineer
resistance. The goal of this study is to
tance was strictly related to scFvGFlVcp-55 accumulation levels (Fig. 4), confirming that the antibody fragment
was functional in planta and responsible for the resistance phenotype. In
addition, transgenic plants conferring
complete protection to GFlV showed
substantially enhanced resistance to
ArMV, making this scFv valuable for
engineering broad resistance to related
nepoviruses.
Approach
The GFlV coat protein was chosen as
the target for GFlV-specific scFv recombinant antibody fragments because it is
the sole determinant of specific transmission by the nematode vector and is
essential for systemic spreading in host
plants. Resistance was evaluated in stably-transformed Nicotiana benthamiana plants accumulating detectable
levels of the GFlV-specific scFv.
Conclusion
Antibody-based pathogen resistance
was used to create nepovirus-resistant
N. benthamiana plants. Therefore, the
scFvGFlVcp-55 antibody is an ideal tool
for the development of virus-resistant
varieties of agriculturally important
crops such as grapevine.
Contact / Ansprechpartner
Results
A single-chain antibody fragment
(scFvGFlVcp-55) was derived from a
monoclonal antibody (Fl3) that binds
specifically to the coat protein of GFlV
(Fig. 1). Specific binding to GFlV isolated from infected N. benthamiana leaves was confirmed by ELISA. The scFv
also recognized the closely related
nepovirus Arabis mosaic virus (ArMV),
indicating the presence of a similar epitope on both viruses. To evaluate the
potential of this scFv to confer antibody-based virus resistance, transgenic
Nicotiana benthamiana plants were
created in which the scFv accumulated
in the cytosol. Recombinant protein
levels of up to 0.1 % total soluble protein (TSP) were achieved. Constitutive
expression of the GFlV-specific scFv
conferred partial or complete protection against GFlV, ranging from
27–100 %, upon challenge with the
viral pathogen (Figs. 2 and 3). Resis-
Dr. Stefan Schillberg
Tel: +49 241 6085–11050
[email protected]
Dr. Greta Nölke
Tel: +49 241 6085–12452
[email protected]
Figure 4:
Relationship between scFvGFlVcp-55 accumulation and reduction in GFlV titer in transgenic T2
N. benthamiana plants infected with the virus
IME Annual Report 2008
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