Reactivation of rolB transgene expression in Vitis amurensis Rupr. cells upon retransformation with 2b gene from Cucumovirus isolate NK.

Objective: Studies concerning 2b protein from Cucumovirus showed 2b to effectively repress functioning of the plant silencing complex, current study aimed whether retransformation with 2b gene able to restore silenced transgene expression in plant cells.
Results: A rolB-transgenic cell culture of Vitis amurensis Rupr. that was continuously subcultured during more than 10 years and exhibited decreased transcription of the rolB transgene was retransformed with the 2b gene of Cucumovirus-NK. Three cell lines retransformed with 2b showed a significant up-regulation of rolB expression accompanied with enhancements in their stilbenes content level in more than 2,7-fold compared to parental rolB-transgenic cell line. The mentioned increase in the level of stilbenes content was due to activation of certain stilbene synthase genes expression responsible for stilbenes biosynthesis in V. amurensis cells. Restoration of rolB expression upon 2b-retransformation led to increase in the expression levels of VaSTS2-VaSTS5 and VaSTS7 isoforms.
Conclusions: 2b from CMV-NK can reactivate a silenced transgene expression, even after 10 years of subcultivation, nevertheless, optimization of the methods concerning 2b introduction in plant genomes is necessary to avoid undesirable silencing effects.