Jeongsik Kima and David E. Somersa,b*
aDepartment of Plant Cellular and Molecular Biology/Plant Biotechnology Center Ohio State University
1060 Carmack Road Columbus, OH 43210, USA (J.K., D.E.S.)
bDivision of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, 790-784, South Korea (D.E.S.)
1 This research was supported by NSF grant IOS-0748749 to D.E.S and by the World Class University Project (Project No. R31-2008-000-10105-0) of Ministry of Education, Science and Technology (Korea).
Rapid assessment of the effect of reduced levels of gene products is often a bottleneck in determining how to proceed with an interesting gene candidate. Additionally, gene families with closely related members can confound determination of the role of even a single one of the group. We describe here an in vivo method to rapidly determine gene function using transient expression of artificial microRNAs (amiRNAs) in Arabidopsis thaliana mesophyll protoplasts. We use a luciferase-based reporter of circadian clock activity to optimize and validate this system. Protoplasts transiently co-transfected with promoter-luciferase and gene-specific amiRNA plasmids sustain free-running rhythms of bioluminescence for more than six days. Using both amiRNA plasmids available through the ABRC, as well as custom-design of constructs using the Weigel amiRNA design algorithm, we show that transient knockdown of known clock genes recapitulates the same circadian phenotypes reported in the literature for loss-of-function mutant plants. We additionally show that amiRNA designed to knockdown expression of the casein kinase 2 beta subunit (CKB) gene family lengthens period, consistent with previous reports of a short period in CKB overexpressors. Our results demonstrate that this system can facilitate a much more rapid analysis of gene function by obviating the need to initially establish stably transformed transgenics to assess the phenotype of gene knockdowns. This approach will be useful in a wide range of plant disciplines when an endogenous cell-based phenotype is observable or can be devised, as done here using a luciferase reporter.