title: Evaluation and application of a luciferase fusion system for rapid in vivo analysis of RNAi targets and constructs in plants.
abstract: The practical use of RNA-mediated approaches including antisense RNA, ribozymes and siRNAs for specific inhibition of gene expression is limited by lack of simple quantitative methods to rapidly test efficacy in vivo. There have been indications that cotransfer of target::reporter gene fusions with constructs designed against the target sequence, followed by quantification of transient reporter gene activity might be effective. Here, we report detailed testing of the approach in plants, using diverse target::luciferase fusions and antisense or ribozyme constructs. We used quantitative transient luciferase activity (Luc) assays to test antisense constructs against beta-glucuronidase, PR glucanase, vacuolar invertase and cucumber mosaic virus, as well as ribozymes against watermelon mosaic virus and tobacco anionic peroxidase. For constructs previously tested in transgenic plants, the results correspond well with those from the transient expression assay. Target susceptibility was generally not strongly influenced by luciferase fusion, and the assay was not highly dependent on target sequence length. Some sequences reduced Luc activity below the level for reliable quantification, but suitable alternative fusions were readily produced. Transcriptional and translation fusions were effective for 5' target::luc constructs. Translational fusions were more reliable for luc::target 3' constructs. With minimal preliminary work to prepare suitable target::luciferase fusions, the approach appears generally applicable for rapid in vivo validation of effectiveness and specificity of constructs designed for RNA-mediated down-regulation of plant genes.
authors.affiliation: Botany Department/BIOL, University of Queensland, Brisbane, Qld, Australia. r.birch@uq.edu.au
authors.forename: Robert G
authors.initials: RG
authors.lastname: Birch
authors.forename: Bo
authors.initials: B
authors.lastname: Shen
authors.forename: Brett J B
authors.initials: BJ
authors.lastname: Sawyer
authors.forename: Eric
authors.initials: E
authors.lastname: Huttner
authors.forename: William Q J
authors.initials: WQ
authors.lastname: Tucker
authors.forename: Andreas S
authors.initials: AS
authors.lastname: Betzner
mesh_terms: D001681:Biological Assay; D055785:Gene Knockdown Techniques; D005966:Glucuronidase / Q000235:genetics; D008156:Luciferases / Q000378:metabolism*; D010942:Plant Viruses / Q000378:metabolism; D030821:Plants, Genetically Modified; D014176:Protein Biosynthesis; D034622:RNA Interference; D016372:RNA, Antisense / Q000378:metabolism; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D031786:Saccharum / Q000201:enzymology; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D043324:beta-Fructofuranosidase / Q000378:metabolism
chemical_list: D016372:RNA, Antisense; D016337:RNA, Catalytic; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D043324:beta-Fructofuranosidase; D005966:Glucuronidase