INTERACTION BETWEEN THE TOBACCO DNA-BINDING ACTIVITY CBF AND THE CYT-1 PROMOTER ELEMENT OF THE AGROBACTERIUM-TUMEFACIENS T-DNA GENE T-CYT CORRELATES WITH CYT-1 DIRECTED GENE-EXPRESSION IN MULTIPLE TOBACCO TISSUE TYPES

A novel DNA-binding activity, designated CBF, has been identified in n uclear extracts from tobacco leaf, stem and root tissue. CBF interacts specifically with a 30 bp promoter fragment, referred to as cyt-1, of the Agrobacterium tumefaciens T-DNA cytokinin (T-cyt) gene. The T-cyt promoter, although of bacterial origin is active in planta and the 30 bp cyt-1 element is located within a region that is essential for T-c yt promoter activity in leaf, stem and root cells of tobacco plants. G el retardation assays using different synthetic oligonucleotides and m ethylation interference experiments pinpointed the binding site of CBF to a GC-rich sequence ATGCCCCACA within the cyt-1 element. Site-direc ted mutagenesis of the CBF binding site within the T-cyt promoter by u sing PCR resulted in an almost complete loss of T-cyt promoter activit y in transgenic tobacco plants. In a gain-of-function experiment a hex amer of cyt-1 was shown to be able to confer leaf, stem and root expre ssion when fused upstream of a TATA box containing -55 derivative of t he T-cyt promoter. A mutant cyt-1 hexamer, defective in CBF binding, d id not show activity above back-ground levels. These results indicate that binding of CBF to the cyt-1 element is required for cyt-1 directe d gene expression, suggesting that CBF might act as a transcriptional activator. Apart from the ASF-1 binding site of the CaMV 35S promoter, which is also present in the T-DNA nopaline and octopine synthase gen es, the cyt-1 element is the only other identified element reported un til now that in combination with a TATA box is sufficient to drive gen e expression in multiple tobacco tissue types.