Over the last decade, plant virus-based vectors have been developed and successfully exploited for high-yield production of heterologous proteins in plants. However, widespread application of recombinant viruses raises concerns about possible risks to the environment. One of the primary safety issues that must be considered is the uncontrolled spread of the genetically engineered virus from experimental plants to susceptible weeds or crops. Using a movement-deficient Potato virus X (PVX)-based

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... ransient gene expression vector which harbors the β-glucuronidase (gus) gene, we established a plant viral expression system that provides containment of the recombinant virus and allows for safe and efficient protein production. By deletion of the viral 25k movement protein gene, systemic spread of the modified virus in non-transgenic Nicotiana benthamiana plants was successfully inhibited. In transgenic N. benthamiana plants expressing the 25K viral movement protein, this deficiency was complemented, thus resulting in systemic infection with the movement-deficient virus. While no differences in virus spread and accumulation were observed compared to infection caused by wild-type PVX in non-transgenic plants, the movement protein transgenic plants exhibited none of the normal symptoms of viral infection. Several biosafety aspects were investigated including the potential for recombination between the defective virus and the movement protein transgene, as well as complementation effects in non-transgenic plants doubly infected with the defective and the wild-type virus. Furthermore, the applicability of the safety system for the production of heterologous proteins was evaluated with gus as a model gene. With respect to the stability of the gus insert and the expression level of the GUS protein, there were no differences between the novel system developed and the conventional PVX-based expression system. Keywords : biosafety / complementation / GUS / movement deficiency / plant virus / Potato virus X / recombinant protein expression / transgenic plants / plant viral expression system * Corresponding author: j.schiemann@bba.de Article published by EDP Sciences and available at http://www.edpsciences.org/ebr or http://dx.doi.org/10.1051/ebr:2005011 U. Manske, J. Schiemann 46 Environ. Biosafety Res. 4, 1 (2005) depending on the host range of the virus. Moreover, the PVX-based expression system with improved biosafety Environ. Biosafety Res. 4, 1 (2005)