Development and evaluation of a Gal4-mediated LUC/GFP/GUS enhancer trap system in Arabidopsis.
BACKGROUND
Gal4 enhancer trap systems driving expression of LacZ and GFP reporters have been characterized and widely used in Drosophila. However, a Gal4 enhancer trap system in Arabidopsis has not been described in the primary literature. In Drosophila, the reporters possess a Gal4 upstream activation sequence (UAS) as five repeats (5XUAS) and lines that express Gal4 from tissue specific enhancers have also been used for the ectopic expression of any transgene (driven by a 5XUAS). While Gal4 transactivation has been demonstrated in Arabidopsis, wide use of a trap has not emerged in part because of the lack of detailed analysis, which is the purpose of the present study.


RESULTS
A key feature of this study is the use of luciferase (LUC) as the primary reporter and rsGFP-GUS as secondary reporters. Reporters driven by a 5XUAS are better suited in Arabidopsis than those containing a 1X or 2X UAS. A 5XUAS-LUC reporter is expressed at high levels in Arabidopsis lines transformed with Gal4 driven by the full, enhanced 35S promoter. In contrast, a minimum 35S (containing the TATA region) upstream of Gal4 acts as an enhancer trap system. Luciferase expression in trap lines of the T1, T2, and T3 generations are generally stable but by the T4 generation approximately 25% of the lines are significantly silenced. This silencing is reversed by growing plants on media containing 5-aza-2'-deoxycytidine. Quantitative multiplex RT-PCR on the Gal4 and LUC mRNA indicate that this silencing can occur at the level of Gal4 or LUC transcription. Production of a 10,000 event library and observations on screening, along with the potential for a Gal4 driver system in other plant species are discussed.


CONCLUSIONS
The Gal4 trap system described here uses the 5XUAS-LUC and 5XUAS rsGFP-GUS as reporters and allows for in planta quantitative screening, including the rapid monitoring for silencing. We conclude that in about 75% of the cases silencing is at the level of transcription of the Gal4 transgene and is at an acceptable frequency to make the Gal4 trap system in Arabidopsis of value. This system will be useful for the isolation and comprehensive characterization of specific reporter and driver lines.
Engineer
Cawas B
CB
Department of Biology, Washington University, Campus Box 1137, 1 Brookings Drive, St. Louis, MO 63130, USA. engineer@biology.wustl.edu
Fitzsimmons
Karen C
KC
Schmuke
Jon J
JJ
Dotson
Stan B
SB
Kranz
Robert G
RG
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D004742:Enhancer Elements, Genetic; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D020868:Gene Silencing; D017930:Genes, Reporter; D023281:Genomics / Q000379:methods; D005966:Glucuronidase / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000378:metabolism*; D019076:Transgenes
D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D008156:Luciferases; D005966:Glucuronidase