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

Plant J. 1993 Sep;4(3):525-34. doi: 10.1046/j.1365-313x.1993.04030525.x.

Abstract

A novel DNA-binding activity, designated CBF, has been identified in nuclear 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-cyt promotor activity in leaf, stem and root cells of tobacco plants. Gel retardation assays using different synthetic oligonucleotides and methylation interference experiments pinpointed the binding site of CBF to a GC-rich sequence ATGCCCCACA within the cyt-1 element. Site-directed mutagenesis of the CBF binding site within the T-cyt promoter by using PCR resulted in an almost complete loss of T-cyt promoter activity in transgenic tobacco plants. In a gain-of-function experiment a hexamer of cyt-1 was shown to be able to confer leaf, stem and root expression when fused upstream of a TATA box containing -55 derivative of the T-cyt promoter. A mutant cyt-1 hexamer, defective in CBF binding, did not show activity above background levels. These results indicate that binding of CBF to the cyt-1 element is required for cyt-1 directed 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 genes, the cyt-1 element is the only other identified element reported until now that in combination with a TATA box is sufficient to drive gene expression in multiple tobacco tissue types.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium tumefaciens / genetics*
  • Base Sequence
  • Binding Sites
  • Core Binding Factors
  • DNA, Bacterial / genetics*
  • DNA-Binding Proteins / metabolism*
  • Gene Expression
  • Genes, Plant
  • Molecular Sequence Data
  • Mutation
  • Neoplasm Proteins*
  • Nuclear Proteins / metabolism
  • Plant Proteins / metabolism
  • Plants, Genetically Modified
  • Plants, Toxic*
  • Promoter Regions, Genetic
  • Tobacco / genetics*
  • Tobacco / metabolism*
  • Tobacco / microbiology
  • Transcription Factors / metabolism*

Substances

  • Core Binding Factors
  • DNA, Bacterial
  • DNA-Binding Proteins
  • Neoplasm Proteins
  • Nuclear Proteins
  • Plant Proteins
  • T-DNA
  • Transcription Factors