Authors
Gabriela Dujovny, Adrián Valli, María Calvo and Juan Antonio García^1
1Centro Nacional de Biotecnología-CSIC, Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain

Abstract
The control of replication can facilitate a viral amplicon to reach high expression levels by enabling the virus to escape host defence mechanisms and reducing the deleterious effects of viral infection. We have developed a novel system to regulate amplicon expression by controlling the temperature of plant growth. Nicotiana benthamiana plants were transformed at two different temperatures with a cDNA copy of the Plum pox potyvirus genome harbouring the open reading frame 2 of Porcine circovirus 2 between the nuclear inclusion protein b and coat protein coding sequences. Although transformation at 27 ° C mainly yielded nonexpressing amplicons, lines with a tight control of amplicon expression were obtained. Viral replication was not detected in these plants when germinated at 28 ° C, but was observed when the plants were shifted to 20 ° C. In lines transformed at 24 ° C, although the amplicon was expressed at 28 ° C, viral accumulation was low and caused only minor growing defects. Viral replication was enhanced in these plants by shifting the temperature to 20 ° C; under such conditions, the amplicon reached higher and more persistent expression levels than in plants transformed at 27 ° C. These results demonstrate the utility of temperature regulation to control viral amplicon expression.

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