assessment with genetically engineered woody plants expressing virus coat
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).
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.
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
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 DAG
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,
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.
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.
October 1996 - September 1999
Villenave d'Ornon (FR)
Instituto Valenciano de Investigaciones Agrarias
Université de Neuchatel (CH)
Laboratoire de recherche viticole, Moet&Chandon
Comité Interprofessionel du Vin de Champagne
Dipartimento di Protezione delle Piante dalla Malatie
L. da Câmara Machado
Institut für Angewandt Mikrobiologie