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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Risk assessment with genetically engineered woody plants expressing virus coat protein gene

Background and objectives (1)

Genetic engineering is an important tool in the development of strategies for the protection of plants against viral infections. Nevertheless, the biosafety risks associated with the introduction of genetically modified organisms (GMOs) into the agricultural industry, need to be considered. One popular approach developed by several scientists working in the area of plant protection, is the use of coat protein (CP) mediated resistance. We focused on the biorisks in plant protection studies using the transgenic woody plant species Vitis and Prunus that express a virus CP gene. Identifying the molecular interactions between a genetically engineered capsid (a viral protecting coat), and the virus challenger genome, is essential for understanding the mechanisms involved in plant protection to virus infection. The potential consequences of transencapsidation (the process of protecting a homologous- or heterologous-viral RNA belonging to the same viral group by a natural or artificial viral CP, forming a homologous or heterologous plant viral particle), and RNA recombination are the main biorisks deriving from the molecular interactions between an engineered CP or a transgene transcript and incoming viruses.

The overall aim was to characterise these phenomena and to assess their biological impacts using natural vectors transmitting transencapsidated particles or recombinant viruses. Natural vectors include aphids for transgenic Prunus expressing plum pox virus CAPSID GENE (PPV CP), nematodes for grapevine harbouring either grapevine fanleaf or arabis mosaic virus CAPSID GENE (GFLV CP, ArMV CP) and mealy bugs for grapevine harbouring grapevine vitivirus A & B CAPSID GENE (GVA CP, GVB CP). These studies are important for transgenic woody perennial crops that remain in fields for over 30 years.

(1) This project was partly a direct follow-on from EC project: EEC8001-CT91-0201 (DGVI).
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  Electron micrographs of VLPs deriving from the expression of PPV CP gene. image Electron micrographs of VLPs deriving from the expression of PPV CP gene.
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Studies of transencapsidation in mixed infection of Vitivirus A and Vitivirus B.

 
Decoration with GVB Pab. image Decoration with GVB Pab.
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Decoration with GVB Pab. image Decoration with GVB Pab.
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Decoration with GVB Pab. image Decoration with GVB Pab.
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Decoration with GVA Mab + Pab. image

Decoration with GVA Mab + Pab.
anti mouse IgG gold
(15 nm).
anti rabbit IgG gold
(10 nm)
.

 
 
 
 
 
 


Approach and methodology

To study these biological risks, two models, Nicotiana & woody plants, were used. Several transgenic woody plants from plums and grapevines expressing CP required for these biological risk studies have been produced (Vitis vinifera; Vitis rupestris, rootstock 3309 Couderc; V. rupestris X V. riparia). Transencapsidation was studied by the comparative use of clones expressing either the native or a modified form of CP. Serological and molecular tools as well as protocols for studying transencapsidation were developed in transgenic woody plants. Recombinant viruses were sought either in transgenic or in non-transformed plants (Nicotiana & woody plants species). These studies were complemented by examination of viable viral RNA derived from RNA recombination species.
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Main findings and outcome

This project has advanced successfully as a result of several factors including the availability of: the transgenic plum and grapevine woody plants (Vitis vinifera, Vitis rupestris, rootstock 3309 Couderc: V. rupestris X V. riparia), which were required for biological risk studies; serological and molecular tools and protocols for studying transencapsidation in transgenic woody plants; PPV deficient for aphid-transmissibility (NAT, i.e. not aphid-transmitted; this is used to design a genomic variant of a naturally aphid transmitted virus) in woody plant species; and cDNA clones for making artificial variants of the virus challenger for recombination studies.

This has enabled the production and characterisation of transgenic grapevines, including those transformed with the native and truncated forms of the GFLV and ArMV CP genes, and GVA and GVB CP genes. In addition, transgenic herbaceous plants, including Nicotiana benthamiana expressing GVA CP, and Nicotiana occidentalis GVB CP, were characterised.

Transencapsidation studies have been carried out with transgenic plums expressing the native form of PPV CP and a modified form PPV imageDAG CP. Risk studies with transgenic plums were carried out under natural conditions in a small scale greenhouse. Phenotypic mixing was detected between ArMV particles and GFLV CP accumulated in transgenic plants (grapevine and Nicotiana benthamiana), and between GVA and GVB particles in doubly-infected herbaceous plants. GVB particles transencapsidated by GVA CP accumulated in transgenic plants were also detected. The biological impacts for these risk studies in confined greenhouse conditions with transgenic plums were also examined.

Recombination studies in herbaceous plant models using artificial variants of the PPV genome have been carried out. Natural recombinants of GFLV-ArMV, as well as potential sites required for the viability of an artificial GFLV-ArMV recombinant were also identified. Infectious full-length clones of GVA & GVB, which are basic tools for making artificial recombinants, were constructed.
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Conclusions

Our transencapsidation studies, carried out in both confined greenhouses and in natural field conditions, show that the use of genetically modified plants needs to be carefully monitored to minimise potential biorisks that these organisms pose in an agricultural context. Studies into recombination have been initiated and are being pursued, and the biological impacts will be evaluated.
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Major publications

Jacquet C., Delecolle B., Raccah B., Lecoq H., Dunez J., Ravelonandro M., “Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants”.
Journal of General Virology, 79, 1998, pp. 1509-1517.

Minafra A., Goelles R., da Câmara Machado A., Saldarelli P., Buzkan N., Savino V., Martelli G.P., Katinger H., Laimer da Câmara Machado M., “Expression of the coat protein gene of grapevine virus A and B in Nicotiana, and evaluation of the resistance conferred to transgenic plants”.
Journal of Plant Pathology, 80, 1998, pp. 197-202.

Olmos A., Cambra M., Esteban O., Gorris M-T., Terrada E., “New device and method for capture, reverse transcription and nested PCR in a single closed-tube”.
Nucleic Acids Research, 27, 1999, pp. 1564-1565.

Belin C., Schmitt C., Gaire F., Walter B., Demangeat G., Pinck L., “The nine C-terminal residues of grapevine fanleaf nepovirus movement protein are critical for systemic virus spread”.
Journal of General Virology, 80, 1999, pp. 1347-1356.

Ravelonandro M., Scorza R., Callahan A., Levy L., Jacquet C., Monsion M., Damsteegt V., “The use of transgenic fruit trees as a resistance strategy for virus epidemics: the plum pox (sharka) model”.
Virus Research, 71, 2000, pp. 63-69.
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imageResearch project
 

Contract number
BIO4-CT96-0773

Period
October 1996 - September 1999

Coordinator
M. Ravelonandro
INRA-Bordeaux
Villenave d'Ornon (FR)

 
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Partners


B. Walter
INRA-Colmar (FR)

G. Llacer
Instituto Valenciano de Investigaciones Agrarias
Moncada (ES)

L. Pinck
CNRS
Strasbourg (FR)

A. Spielmann
Université de Neuchatel (CH)

M-C. Mauro
Laboratoire de recherche viticole, Moet&Chandon
Epernay (FR)

D. Moncomble
Comité Interprofessionel du Vin de Champagne
Epernay (FR)

G. Martelli
Dipartimento di Protezione delle Piante dalla Malatie
Bari (IT)

L. da Câmara Machado
Institut für Angewandt Mikrobiologie
Vienna (AT)

 
 
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