Risk assessment with genetically engineered woody plants expressing virus coat protein gene

• Research unit: EU RFP
• Project number: 96.0303

Key words (English)

Grapevine; nepovirus; coat protein; transformation; transcapsidation; RNA recombination

Alternative project number (English)

EU project number: BIO4CT960773

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Short description (English)

See abstract

Partners and International Organizations (English)

INRA, Bordeaux (F), IVIA, Valence (E), IBMP, Strasbourg (F), LVMH, Epernay (F), CIVC, Epernay (F), DPPM, Ban (I), IAM, Wien (A)

Abstract (English)

The objective of the project is to assess possible risks associated with the use of woody perennial crops (Prunus and Vitis) expressing various viral coat protein transgenes. The main risks to be evaluated are possible transcapsidation and/or recombination between transgene and virus genomic RNA sequences leading to a change in host range of the viruses and/or emergence of novel viruses.
Contribution of the Swiss partner :
1. Transcapsidation of arabis mosaic virus (ArMV) by grapevine fanleaf (GFLV) CP or transcapsidation of GFLV by ArMV-CP in transgenic grapevine plants.
Several IC-RT-PCR experiments were performed on non transgenic grapevine plants doubly infected by the GFLV and the ArMV in order to verify the specificity of the antibodies. Unfortunately, the data obtained demonstrated that the GFLV-CP and the ArMV-CP polyclonal antibodies available were not specific enough for this type of experiments. We therefore did not pursue the transcapsidation experiments.
2. Recombination between GFLV genomic RNA and ArMV-CP mRNA in transgenic grapevine plants
Infection of transgenic plants expressing a virus-derived gene by a related incoming virus corresponds in many ways to a frequently encountered natural situation, i.e. simultaneous infection of non-transgenic plants by two related viruses. Consequently, to evaluate the risks of RNA recombination in virus-infected transgenic plants, one must compare the frequency of its occurrence in transgenic plants to that in doubly infected non-transgenic plants. RT-PCR experiments were performed on doubly infected plants using primers specific to regions upstream and downstream of the CP gene. No recombinant molecules could be detected after a first round of amplification (40 cycles) in 16 independent doubly infected plants. However a band of the expected size was amplified after a second round of PCR reaction in 3 cases. Sequence analysis of the amplified bands confirmed that they were indeed novel chimeric nepovirus CP genes. Preliminary data from a limited number of RT-PCR experiments performed on ArMV-CP transgenic plants infected with GFLV did not lead to detectable amplification of any DNA molecules, but it must be noted that, in this case, a double recombination event within the capsid region would be required for restoring a detectable recombinant molecule.
2. Conclusions/perspectives
Experiments performed on doubly infected non transgenic grapevine plants demonstrated that, under 'natural' conditions, recombination events could occur between two closely related viruses, although at very low frequency. Having in hands three different putative recombinants made of parts of ArMV-CP and GFLV-CP, it would of great interest to check the viability and fitness of these molecules compared to their wild-type counterparts.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 96.0303