Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/18/2005
Publication Date: 7/1/2005
Citation: Cazzonelli, C.I., Burke, J.J., Velten, J.P. 2005. Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco. Plant Molecular Biology. 58(4):465-481. Interpretive Summary: A DNA sequence element from plant geminiviruses (CLE) was isolated and tested for its ability to enhance gene expression in plants. Gene activity in most plant tissues was improved by combining multiple copies of the CLE element but reduced by the introduction of nucleotide base changes within the element's core DNA sequence. A viral protein thought to interact with the CLE DNA element to enhance gene expression was produced in plant cells and found to act in a manner suggesting that it does NOT interact directly with CLE.
Technical Abstract: The conserved late element (CLE) was originally identified as an evolutionarily conserved DNA sequence present in geminiviral intergenic regions. CLE has subsequently been observed in promoter sequences of bacterial (T-DNA) and plant origin, suggesting a role in plant and plant viral gene regulation. Synthetic DNA cassettes harboring direct repeats of the CLE motif were placed upstream from a -46 to +1 minimal CaMV 35S promoter-luciferase reporter gene and reporter activity characterized in Nicotiana species during both transient and stable expression. A single direct repeat cassette of the element (2xCLE) enhances luciferase activity by 2 fold, independent of the element's orientation, while multiple copies of the cassette (4-12xCLE) increases activity up to 10-15 fold in an additive manner. Transgenic tobacco lines containing synthetic CLE promoter constructs enhance luciferase expression in leaf, cotyledon and stem tissues, but to a lesser extent in roots. Single nucleotide substitution at six of eight positions within the CLE consensus (GTGGTCCC) eliminates CLE enhancer-like activity. It has been previously reported that CLE interacts with the AC2 protein (also called AL2 or C2) from Pepper Huasteco Virus a member of the transcriptional activator protein, or TrAP, family. In transient and stable expression systems AC2 expression was found to result in a 2 fold increase in luciferase activity, irrespective of the presence of CLE consensus sequences within the reporter's promoter. These data suggests that the AC2 protein, instead of interacting directly with CLE, functions as either a general transcriptional activator and/or a suppressor of posttranscriptional gene silencing.