ABSTRACT

Our proposal addresses environmental safety issues related to transgenic plants expressing virus-derived genes. The main objective of our study is to evaluate under field conditions the incidence of coat protein genes in transgenic grapevines and plums on the dynamics and variability of virus populations, particularly on the development of viable recombinant viruses with potential harmful properties. Experiments will examine whether transgenic plants present additional risks compared to conventional plants. If recombinant viruses are identified, their biological properties will be examined. The potential of heterologous viruses at reverting post-transcriptional gene silencing, and hence at breaking down the engineered protection, will also be determined in transgenic plums, as well as the translocation of transgene transcripts from transgenic rootstocks to nontransgenic scions upon virus infection in grapevines. Identifying potential risks and assessing their impact is the safest approach to protect the environment prior to the release of virus-resistant transgenic crops. Our findings will be disseminated to end-users to increase the perception of new crop protection strategies.

OBJECTIVES

We will assess the potential impact of transgenic plants expressing viral coat protein genes on the diversity of virus populations in the field by:

1. comparing the variability of virus populations in transgenic versus nontransgenic grapevines and plums.
2. analysing whether transgenic plants increase the emergence of viable recombinant viruses beyond that of natural background events.
3. evaluating the biological properties of recombinant viruses.
4. monitoring the stability of post-transcriptional gene silencing, and hence of the engineered protection, upon infection by heterologous viruses.
5. characterising the translocation of transgene transcripts from transgenic rootstocks to nontransgenic scions upon virus infection.

Our proposal addresses environmental safety issues of agricultural biotechnology that will assist EC policy makers in taking scientifically-based decisions for the proper deployment of virus-resistant transgenic crops. It will also contribute to maintaining EC agriculture at a cutting edge of biotechnology and increase the public acceptance of new plant protection strategies.

DESCRIPTION OF THE WORK

Recombination between viral transgene transcripts and challenging RNA viruses has been demonstrated in transgenic plants under greenhouse conditions. This phenomenon has not been demonstrated in the field yet. Our safety assessment studies will determine if viable recombinant viruses can develop in transgenic grapevines and plums that express the coat protein genes of Grapevine fanleaf virus and Plum pox virus (PPV), respectively, and were established in the field in 1996. The coat protein gene of challenging viruses will be analysed by immunocapture-reverse transcription-polymerase chain reaction assays and further characterised, with appropriate controls, by restriction fragment length polymorphism and sequencing. Similar experiments will also be conducted in the greenhouse with transgenic grapevines expressing the coat protein gene of Grapevine virus A or Grapevine virus B. The biological properties and fitness components of recombinant viruses with potential harmful properties will be evaluated in the greenhouse in natural and herbaceous hosts by testing their host range, symptom expression, and vector transmissibility (nematodes, aphids and mealybugs). The occurrence of recombination in nontransgenic plants exposed to single or multiple virus infections, including cross-protected grapevines, will be used as a baseline to determine if recombination occurs in transgenic plants beyond natural background events. We will also evaluate whether reversion of post-transcriptional gene silencing, and hence of resistance to PPV, can be achieved by heterologous viruses in plums. Further, we will examine if virus infection modifies the distribution of transgene transcripts in grapevines. The results of our environmental safety assessment studies will be statistically analysed and evaluated prior to their dissemination to end users (breeders, nurserymen, growers, policy-makers, scientific community and public at large) through web pages, newsletters, recommendations, publications in scientific peer-reviewed and technical journals and a workshop.

DELIVERABLES

Vineyards and orchards with transgenic and nontransgenic plants will be identified and test plants will be selected and propagated. Collections of virus isolates infecting grapevines and plums and viruliferous vectors will be established. Serological and molecular tools will be developed and used in optimised protocols to identify recombinant virus species. Safety assessment data relevant to agricultural practice will be obtained on the impact of transgenic grapevines and plums on the diversity of virus populations and on the effect of heterologous viruses on the stability of resistance to PPV in plums. Data will also be obtained on the translocation of CP transgene products in grapevines. Internal and external webpages will be created for communication.

We will provide training opportunities for junior scientists. Scientific and financial reports will be delivered to assess achievements according to the workplan. Our results will be discussed during a workshop open to the international scientific community and published in newsletters, press releases, and publications.

CONSORTIUM