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

PROTOCOL

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

Jian-Feng Li1,2, Dandan Zhang1,2 & Jen Sheen1,2

1Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA. 2Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. Correspondence should be addressed to J.-F.L. ([email protected]).

Published online 27 March 2014; http://www.nature.com/doifinder/10.1038/nprot.2014.061

Web End =doi:10.1038/nprot.2014.061

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 23 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.

2014 Nature America, Inc. All rights reserved.

INTRODUCTION

The rapidly expanding genomic information across the plant kingdom stresses an urgent need for reliable and versatile tools to decipher the functions of newly discovered genes and their regulatory networks. Determination of gene functions often requires examination of loss-of-function phenotypes. In the model plant Arabidopsis thaliana, transfer DNA (T-DNA) insertion lines represent the most important resource for loss-of-function mutants. Targeted genome editing tools, including zinc-finger nucleases1, transcription activatorlike effector nucleases2,3 and RNA-guided Cas9 endonucleases46, have recently opened up promising new avenues for generating targeted loss- of-function mutants for Arabidopsis genes lacking T-DNA insertion mutants and for genes in other plant species. However, lethality and complex long-term physiological and developmental consequences associated with stable mutants have imposed limitations in the functional characterization of most genes essential for plant growth and reproduction. It is also more challenging to use T-DNA insertion mutants to study functionally redundant or physically linked genes in plant genomes7. The amiRNAbased method for targeted gene silencing...