Department of Plant Sciences

Rice Enhancer Traps

Alex Johnson

http://129.127.183.5 for the Oryza sativa GAL4/GFP database.

The aim of this BBSRC funded project, a collaborative effort between the laboratories of Dr Mark Tester and Dr Julian Hibberd, is to generate a library of rice (Oryza sativa L. japonica cv. Nipponbare) enhancer trap lines expressing an exogenous transcription factor (gal4) and the green fluorescent protein (gfp) in specific cell-types and tissues.

Working closely with Dr Emmanuel Guiderdoni at the French scientific organization CIRAD (Centre de coopération internationale en recherche agronomique pour le développement, http://www.cirad.fr) in Montpellier, France, we have produced more than 13,000 primary transformants harboring the gal4-gfp construct originally developed by Jim Haseloff (http://www.plantsci.cam.ac.uk/Haseloff/) for enhancer trapping studies in Arabidopsis thaliana. We screened T0 adult plants (1,982 lines), T1 seed (2,684 lines) and T1 seedlings (2,667 lines) to identify lines with gfp expression specific to individual cell-types in roots, leaves and flowers. Expression of gfp in specific tissues (flower, leaf, seed, etc.) averaged 10% while expression frequency at the whole plant level was 29%. The whole plant expression frequency of gfp observed for rice is remarkably similar to the 30% expression frequency reported for Arabidopsis thaliana T1 lines transformed with a similar gal4-gfp construct (http://enhancertraps.bio.upenn.edu) and indicates that the gal4-gfp system provides the same level of enhancer trapping efficiency in both monocots and dicots. Click here for additional details regarding the construction and screening of our gal4-gfp library.

 

We are currently transactivating various genes of interest in specific cell-types of rice by placing genes downstream of the upstream activating sequences element (UAS) of GAL4, and introducing these constructs into selected enhancer trap lines. Binding of GAL4 to the UAS element is what drives expression of GFP in the gal4-gfp enhancer trapping system and, thus, GAL4 should bind to the UAS sequence of these introduced constructs and activate expression of the gene of interest in the same pattern observed for GFP. We recently demonstrated in both calli and plants that GAL4 is capable of activating a UAS:GUS construct in patterns and intensities highly correlated with that of GFP, indicating that targeted expression or manipulation of any gene of interest is possible using this system.

 

Expression patterns of approximately 1500 enhancer trap lines have been documented using stereomicroscopy and, in some cases, confocal microscopy, and the data has been recorded in a FileMaker database which is searchable by expression level, expression pattern, developmental stage screened, as well as other criteria. To access the GAL4/GFP database click http://129.127.183.5. In addition, several searches from the database have been saved as PDF files which can be accessed at the top of this page. Specifically, find the results of three searches for lines with T0 expression patterns (flower, leaf blade and leaf sheath) and four searches for lines with T1 expression patterns (seed, endosperm, seedling root, seedling shoot). The lines are grouped alphabetically, and several lines appear in more than one of the saved searches due to multiple expression patterns within lines. Six lines with root expression patterns that have been analyzed by confocal microscopy can also be viewed in the PDF entitled 'confocal examples'.

The gfp positive enhancer trap lines are currently being multiplied in the greenhouse and T2 seed of roughly 200 lines should be available by June-July 2004, with several hundred more becoming available by December 2004. Requests for seed can be addressed to Alex Johnson at alex.johnson@acpfg.com.au. A database similar to the FileMaker system described here, entitled "Oryza Tag Line" and containing descriptions of these gal4-gfp lines as well as 40,000 other T-DNA insertion rice lines and the genomic regions flanking their integration sites, has been developed at CIRAD and will be publically available starting June 2004 at http://genoplante-info.infobiogen.fr/OryzaTagLine/.