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Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants

Nature Protocols volume 9, pages 939–949 (2014)Cite this article

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Abstract

Artificial miRNA (amiRNA) technology offers highly specific gene silencing in diverse plant species. The principal challenge in amiRNA application is to select potent amiRNAs from hundreds of bioinformatically designed candidates to enable maximal target gene silencing at the protein level. To address this issue, we developed the epitope-tagged protein-based amiRNA (ETPamir) screens, in which single or multiple potential target genes encoding epitope-tagged proteins are constitutively or inducibly coexpressed with individual amiRNA candidates in plant protoplasts. Accumulation of tagged proteins, detected by immunoblotting with commercial tag antibodies, inversely and quantitatively reflects amiRNA efficacy in vivo. The core procedure, from protoplast isolation to identification of optimal amiRNA, can be completed in 2–3 d. The ETPamir screens circumvent the limited availability of plant antibodies and the complexity of plant amiRNA silencing at target mRNA and/or protein levels. The method can be extended to verify predicted target genes for endogenous plant miRNAs.

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Figure 1: Flowchart of the ETPamir screens for identifying optimal amiRNAs.
Figure 2: Flowchart of the protein-based validation of predicted target genes for plant natural miRNAs.

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Acknowledgements

We thank the Weigel laboratory for developing the versatile WMD platform and members in the Sheen laboratory for their efforts to test and improve the ETPamir screens. This work has been supported by a Massachusetts General Hospital Executive Committee on Research Postdoctoral Fellowship for Medical Discovery to J.-F.L. and by grants from the US National Science Foundation (grant no. IOS-0843244) and the US National Institutes of Health (grant nos. R01 GM60493 and R01 GM70567) to J.S.

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  1. Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Jian-Feng Li, Dandan Zhang & Jen Sheen

  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Jian-Feng Li, Dandan Zhang & Jen Sheen

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  1. Jian-Feng Li
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J.-F.L. developed the protocol under the guidance of J.S. The article was written by J.-F.L. and J.S. D.Z. prepared Tables 1 and 2 and related references.

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Correspondence to Jian-Feng Li.

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Li, JF., Zhang, D. & Sheen, J. Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants. Nat Protoc 9, 939–949 (2014). https://doi.org/10.1038/nprot.2014.061

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