Amplification of genome-integrated BeYDV replicons by transient expression of Rep in Arabidopsis thaliana

Bean yellow dwarf geminivirus (BeYDV) has been used as a potential vector to improve foreign gene expression, specifically, to achieve higher yields of vaccine proteins in plants. Previously, we have shown that when the BeYDV replication initiator protein Rep was provided in trans, replication and gene expression of GUS were enhanced enormously from a BeYDV expression vector in a transient assay system. In this paper, transgenic lines of Arabidopsis (cv. Columbia) were generated harboring the BeYDV cis-acting elements required for replication. Constructs encoding BeYDV Rep or intronless Rep open reading frames (ORFs) were transiently introduced into transgenic plants via Agrobacterium-mediated infiltration in order to examine the relative levels of replication and expression of the genome-integrated GUS reporter gene. This study shows that expression of Rep protein was regulated in trans from a separate cassette which enabled the rescue, replication and enhancement of the genome-integrated GUS gene in transgenic Arabidopsis. We conclude that Rep expression can be effectively controlled in Arabidopsis plants, and that regulation of Rep expression can result in the amplification of a genome-integrated foreign gene by circumventing the negative effects of gene silencing.

Keywords
Agrobacterium-mediated expression, BeYDV-based vector, gene amplification, GUS expression, Rep expression, transgenic Arabidopsis.