Abstract
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 (Arabidopsis thaliana) mesophyll protoplasts. We use a luciferase-based reporter of circadian clock activity to optimize and validate this system. Protoplasts transiently cotransfected with promoter-luciferase and gene-specific amiRNA plasmids sustain free-running rhythms of bioluminescence for more than 6 d. Using both amiRNA plasmids available through the Arabidopsis Biological Resource Center, 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 knock down expression of the casein kinase II β-subunit gene family lengthens period, consistent with previous reports of a short period in casein kinase II β-subunit 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.
Highlights
We describe here an in vivo method to rapidly determine gene function using transient expression of artificial microRNAs in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts
Using both artificial microRNAs (amiRNAs) plasmids available through the Arabidopsis Biological Resource Center, 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
To validate the feasibility of this system for chronobiological studies, we monitored clock-driven luminescence rhythms in Arabidopsis mesophyll protoplasts by examining expression of two differently phased clock-controlled reporters after transient transfection
Summary
Protoplasts transiently cotransfected with promoter-luciferase and gene-specific amiRNA plasmids sustain free-running rhythms of bioluminescence for more than 6 d Using both amiRNA plasmids available through the Arabidopsis Biological Resource Center, 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. 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.
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