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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Assessment of risks induced by virus-derived transgene products in plants, using luteoviruses carrying the green fluorescent protein as a visible reporter

Background and objectives

Potential interactions between viral transgene products and infecting viruses are a serious concern for the environmental safety of transgenic plants expressing plant viral sequences. Interactions that can occur include RNA recombination, heterologous encapsidation (cross-incorporation of viral genetic material into viral particles) and synergism. Luteoviruses are particularly useful in risk assessment studies relating to these interactions. Firstly, they arose by RNA recombination between different ancestor viruses. Secondly, related luteoviruses are known to readily undergo heterologous encapsidation and to also encapsidate unrelated viroid RNA. Finally, synergism is common in mixed infections (with two luteoviruses, or a luteovirus and a potyvirus), leading to increased expression of symptoms and viral titres. Little information is available concerning the probability of the occurrence of undesirable interactions in transgenic plants, making it difficult to draw conclusions about the biorisks of GMO-containing plants. The major bottleneck in obtaining sufficient data on recombination, heterologous encapsidation and synergism is the detection of the RNA interactions described above.

The aims of this project are firstly to assess the probability of these undesirable interactions in transgenic plants using luteoviruses as the model, and secondly, to assess the environmental risks related to the agricultural introduction of such transgenic plants created using viral vectors.

image image GFP expression by the first generation of BWYV-GFP in protoplasts (top right panel and bottom right panel) and in a plant mesophyll cell (left panel).

Approach and methodology

The initial phases of this study are based on the production of transgenic plants expressing poty- and luteoviral sequences as well as full-length infectious clones of Beet western yellow virus (BWYV) and Potato leafroll virus (PLRV), carrying the gene for the jellyfish green fluorescent protein (GFP). Incorporation of a visible reporter gene markedly simplifies and speeds up detection, thus allowing large numbers of virus-infected transgenic plants to be screened. Stable viral chimeras (mutant luteoviruses expressing the GFP gene) can be used to challenge transgenic plants. The fluorescent reporter protein enables rapid screening of a large number of plants by UV light. Thus, events such as heterologous encapsidation, RNA recombination, and synergism are likely to be identified, even if they occur at a low frequency.

Main findings and outcome

A reporter system producing full-length infectious BWYV and PLRV clones and the UV-visible marker (GFP) is being developed. The first generation of BWYV-GFP chimeras has been constructed in which the operon encoding the capsid-associated proteins has been replaced by GFP, or in which part of the minor capsid protein has been replaced by GFP. These constructs are infectious to protoplasts and produced free GFP, which located uniformly throughout the cytoplasm or as fluorescent aggregate bodies. When agro-inoculated into plants, the infections were confined to single cells at the inoculation point. Stability of GFP expression is also being investigated. This is required to rapidly monitor a large number of test plants in a non-invasive manner. This approach permits scientists and breeders to gather sufficient data for statistical analysis, from which the limits of the associated risks can be determined.

This research can be used to examine several important features. Firstly, to assess whether these plants can encapsidate potato spindle tuber viroids. In addition, it should identify RNA recombinations between luteovirus-derived transgenes and infecting BWYV or PLRV, and between Potato virus A (PVA)-derived transgenes and infecting luteoviruses. Secondly, it should identify heterologous encapsidation of BWYV and PLRV in transgenic plants expressing luteoviral capsid-associated proteins. Thirdly, the occurrence of synergism between potyviral transgene products involved in virus movement, and infecting luteoviruses can be identified. Finally, procedures that evaluate the likelihood of these undesirable events can be identified, particularly in relation to naturally occurring mixed infections of the viruses and viroids involved.


These methods should prove to be of enormous value and will be highly applicable to regulatory guidelines in the European Union. In addition, they should encourage European public confidence in the use of transgenic resistance in agriculture. Furthermore, the availability of efficient risk assessment systems will aid breeders in designing and testing second generation transgene constructs in which the risks of recombination and transencapsidation are already minimised.

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Contract number

October 1998 – September 2000

J.F.J.M. van den Heuvel,
F. van der Wilk
Wageningen University and Research Centre (NL)



J.P.T. Valkonen
Swedish University of Agricultural Sciences
Uppsala (SE)

K.E. Richards
Institut de Biologie Moléculaire des Plantes du CNRS
Strasbourg (FR)

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