A Region Containing an as-1 Element of Dahlia Mosaic Virus (DaMV) Subgenomic Transcript Promoter Plays a Key Role in Green Tissue- and Root-Specific Expression in Plants

A subgenomic transcript (Sgt) promoter was isolated from the genomic clone of dahlia mosaic virus (DaMV), which is a double-stranded DNA virus of the Caulimoviridae family. The DaMVSgt promoter, which is linked to the heterologous beta-glucuronidase (GUS) reporter gene, was characterized in transient protoplasts and in transgenic tobacco, as well as in Arabidopsis plants. The 5'- and 3'-deletion analysis of a 591-bp DaMVSgt promoter fragment indicated that a 441-bp promoter fragment (-372 to +69 from the transcription start site; TSS) was sufficient for maximal promoter activity. A 141-bp promoter fragment (-72 to +69 from TSS) was the minimal promoter region that also showed relatively strong activity. The three activation sequence-1 (as-1) elements and the border regions were primarily responsible for the promoter activity, as revealed by a finer internal deletion and mutation analysis of the cis-elements and of the immediate border sequence of the activation domain. Electrophoretic mobility shift assay (EMSA), supershift EMSA, DNase I footprinting, Southwestern blotting, and UV cross-linking studies demonstrated the binding of a tobacco transcription factor, TGA1a, that correlated with 2,4-dichlorophenylacetic acid (2,4D)-induced transcriptional activity of the DaMVSgt promoter. Histological GUS staining and the GUS enzymatic assay demonstrated that the 441-bp DaMVSgt4 promoter and 141-bp minimal DaMVSgt4F are 5.5 and 4.6 times, respectively, stronger than the CaMV 35S promoter. The minimal DaMVSgt4F promoter is more active than CaMV 35S in all types of green tissues and roots, without any detectable expression in reproductive tissues and seeds. The DaMVSgt4F promoter may be useful for transgene containment applications.