Simultaneous analysis of the bidirectional African cassava mosaic virus promoter activity using two different luciferase genes

The expression of geminivirus genes is controlled by bidirectional promoter s which are located in the large intergenic region of the circular DNA geno mes and specifically regulated by virus encoded proteins. In order to study the simultaneous regulation of both orientations of the DNA A and DNA B pr omoters of African cassava mosaic virus (ACMV), they were cloned between tw o different luciferase genes with the firefly luciferase gene in complement ary-sense and the Renilla luciferase gene in virion-sense orientation. The regulation of the ACMV promoters by proteins encoded by the complete DNA A, as well as by the individually expressed transactivator (TrAP) or replicat ion-associated (Rep) proteins was assessed in tobacco and cassava protoplas ts using dual luciferase assays. In addition, the regulation of the DNA A p romoter integrated into tobacco genome was also assessed. The results show that TrAP activates virion-sense expression strongly both in cassava and to bacco protoplasts, but not in transgenic tobacco plants. In contrast to thi s, DNA A encoded proteins activate virion-sense expression both in protopla sts and in transgenic plants. At the same time they reduce the expression o f the complementary-sense Rep gene on DNA A but activate the expression of the complementary-sense movement protein (MPB) gene on DNA B. The degree of MBP activation is higher in cassava than in tobacco protoplasts, indicatin g that the plant host also influences the promoter strength. Transient tran sformation experiments using linearized DNA indicate that the different reg ulation of the ACMV DNA A promoter in protoplasts and transgenic plants cou ld be due to different DNA curvature in free plasmids and in genes integrat ed in plant genomic DNA.