The role of P25 interacting transcriptional regulator VIP1 in activation of transcription.

Abstract: Rhizomania, caused by beet necrotic yellow vein virus (BNYVV), has been considered as an economically important disease around the world because of the extreme reduction in sugar beet yield and sugar content in affected plants. The spread of rhizomania all over the world, including the emergence of resistance- breaking virus isolates, have been become a major concern for the plant pathologists and plant breeders aiming at improving sugar beet resistance to   BNYVV as well as better understanding sugar beet-virus interactions during disease development. The main focus of this project is to elucidate the role of P25-interacting partner, the VIP1 transcription factor, in activation of transcription.  The isolation of the gene-of-interest (VIP1) was performed by RT-PCR on total RNA preparations extracted from root tissue of sugar beet (Beta vulgaris ssp. vulgaris).  The isolated gene of interest was cloned using gateway system into a binary expression vector and the obtained construct was then transformed into Agrobacterium tumefaciens for analysing transient expression in the experimental host (Nicotinana benthamiana).  Dual-luciferase promoter activity assay was performed on isolated leaf discs co-expressing P25 and VIP1 and compared to appropriate controls.  Six promoter constructs were tested. However, we observed an increase in luciferase activity (1.8-4.2-fold) upon co-expression of P25 and VIP1 only for two constructs tested, although the increase was not supported by Student’s t-test at 0.05 significance level. Nevertheless, the luciferase activity assay data for these two constructs were consistent with RNA-seq and RT-qPCR data obtained previously showing upregulation of the expression of these two specific sugar beet genes during BNYVV infection in sugar beets.     Thus, the results support our hypothesis that the interaction of the virus virulence factor P25 with VIP1 transcription factor is needed to activate transcription of certain genes in the nucleus for the virus benefit.