title: Functional characterization of the geminiviral conserved late element (CLE) in uninfected tobacco.
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 (2x CLE) enhances luciferase activity by 2-fold, independent of the element's orientation, while multiple copies of the cassette (4-12x CLE) increases activity up to 10- to 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 from Pepper Huasteco Virus (PHV-AC2). PHV-AC2 (also called AL2 or C2) is a member of the transcriptional activator protein, or TrAP, gene family. In transient and stable expression systems PHV-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 PHV-AC2 protein, instead of interacting directly with CLE, functions as either a general transcriptional activator and/or a suppressor of post-transcriptional gene silencing.
authors: USDA-ARS (United States Department of Agriculture-Agricultural Research Services), Lubbock, TX 79415, USA.
authors: Christopher Ian
authors: CI
authors: Cazzonelli
authors: John
authors: J
authors: Burke
authors: Jeff
authors: J
authors: Velten
mesh_terms: D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics; D049454:Firefly Luciferin / Q000032:analysis; D017791:Geminiviridae / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D009154:Mutation; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
chemical_list: C099182:AC2 protein, geminivirus; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049454:Firefly Luciferin; D008156:Luciferases