Spatial control of transgene expression in rice (Oryza sativa L.) using the GAL4 enhancer trapping system

• Published in: The Plant journal : for cell and molecular biology Vol. 41; no. 5; pp. 779 - 789
• Main Authors: Johnson, A.A.T, Hibberd, J.M, Gay, C, Essah, P.A, Haseloff, J, Tester, M, Guiderdoni, E
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-03-2005; Blackwell Science; Blackwell Publishing Ltd; Wiley
• Subjects: Biological and medical sciences; Cells; DNA-Binding Proteins - genetics; ectopic gene expression; enhancer elements; Enhancer Elements, Genetic; Flowers - genetics; Fundamental and applied biological sciences. Psychology; fungal proteins; GAL4‐VP16; Gene expression; gene expression regulation; Gene Expression Regulation, Plant; Genetics; Life Sciences; Molecular and cellular biology; Molecular genetics; molecular sequence data; nucleotide sequences; Oryza - genetics; Oryza sativa; Plant Leaves - genetics; Plant populations; Plant Shoots - genetics; Plants genetics; Plants, Genetically Modified - genetics; Rice; Saccharomyces cerevisiae Proteins - genetics; Seeds - genetics; tissue‐specific promoters; transcription factors; Transcription Factors - genetics; transgenes; tissue-specific promoters; Monocotyledones; Seeds; Genomics; Gene expression; Oryza sativa; Gene; Gramineae; GAL4-VP16; Angiospermae; Green fluorescent protein; Spermatophyta; ectopic gene expression; RIZ; TISSUE-SPECIFIC PROMOTERS; GENOMIQUE; ECTOPIC GENE EXPRESSION
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2005.02339.x

We used enhancer trapping with the GAL4 transcriptional activator from yeast to obtain spatial control of transgene expression in all organs of the model monocotyledonous species rice (Oryza sativa L. cv. Nipponbare). Our T-DNA enhancer trapping cassette consisted of two principle components: (1) the minimal promoter-equipped gal4 gene placed adjacent to the right border, and (2) the green fluorescent protein gene (gfp) fused to the upstream activation sequence element (UAS) to which GAL4 binds and activates expression, so that gfp expression corresponds to gal4 expression. Agrobacterium-mediated integration of the cassette into the rice genome often brings the gal4 gene under transcriptional control of local genomic enhancers and promoters, resulting in gal4/gfp expression patterns ranging in specificity from single-cell types to constitutive expression. We produced more than 13 000 enhancer trap lines with this cassette and screened T0 adult plants (1982 lines), T1 seed (2684 lines) and T1 seedlings (2667 lines) for gfp expression. Approximately 30% of the lines produced GFP, and we identified lines with gfp expression in specific cell types of all major organs of the rice plant. Subsequently, using the GUS reporter gene (uidA), we demonstrated that UAS:geneX constructs can be transactivated in specific cell types where gal4 and gfp are expressed, thus providing an excellent system for the manipulation of gene expression and physiological function in specific cell types of rice.