A Simple Protoplast‐Based Method for Screening Potent Artificial miRNA for Maximal Gene Silencing in Arabidopsis

Nannan Zhang1, Dandan Zhang1, Jian‐Feng Li1

1 State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat‐sen University, Guangzhou
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 26.9
DOI:  10.1002/cpmb.28
Online Posting Date:  January, 2017
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The unpredictability of in planta silencing efficiency of artificial micro RNAs (amiRNAs) has greatly limited the application of the amiRNA technology in basic and applied plant research. This unit describes a simple and robust method called the epitope‐tagged protein‐based amiRNA (ETPamir) screen for identifying the most effective amiRNA for silencing a particular Arabidopsis gene. After selecting three to four amiRNA candidates designed by the WMD3 web server for the target gene, the silencing efficiencies of the amiRNA candidates can be compared by co‐expressing individual amiRNAs with the target mRNA encoding an epitope‐tagged target protein in Arabidopsis mesophyll protoplasts. By monitoring target protein levels through immunoblotting with commercial “anti‐tag” antibodies, the most potent amiRNA can be easily identified as the one with minimal target protein accumulation. This empirical method bypasses the complexity of amiRNA silencing mechanisms and the scarcity of plant specific antibodies, and can be applied to a broad range of plant species. © 2017 by John Wiley & Sons, Inc.

Keywords: amiRNA screen; gene silencing; epitope tag; Arabidopsis mesophyll protoplasts

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1:

  • 2 μg/μl DNA purified from:
  • amiRNA‐expressing plasmids
  • Target gene‐expressing plasmid
  • Control gene‐expressing plasmid
  • Biotool Fast Plasmid Maxiprep kit (Biotool, cat. no. B22213)
  • 6× protein loading buffer (Transgene, cat. no. L101‐02)
  • Fetal bovine serum (FBS; Thermo Fisher Scientific, cat. no. 10099‐141)
  • Polyethylene glycol (PEG) solution (see recipe)
  • W5 solution (see recipe)
  • WI solution (see recipe)
  • 2‐ml round‐bottom microcentrifuge tubes (USA Scientific, cat. no. 1620‐2700)
  • 1.5‐ml microcentrifuge tubes (Jet Biofil, cat. no. 160503‐119‐C)
  • 6‐well culture plate (Thermo Fisher Scientific, cat. no. 08‐772‐1)
  • CL2 clinical centrifuge (Thermo Fisher Scientific, cat. no. 004260F)
  • T100 Thermal Cycler (Bio‐Rad, cat. No. 1861096)
  • Vortex dancer II (Leopard Scientific Instruments, cat. no. 202789)
  • Fisher Scientific Isotemp heating block (Thermo Fisher Scientific, cat. no. 11‐715‐305Q)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and immunoblotting (Ni et al., )
NOTE: All steps are conducted at room temperature unless otherwise stated.
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Literature Cited

Literature Cited
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 97:10.2A.1‐10.2A.44. doi: 10.1002/0471142727.mb1002as97.
  Li, J.F. and Zhang, D. 2014. Quantitative analysis of protein‐protein interactions by split firefly luciferase complementation in plant protoplasts. Curr. Protoc. Mol. Biol. 107:20.9.1‐20.9.8. doi: 10.1002/0471142727.mb2009s107.
  Li, J.F., Chung, H.S., Niu, Y., Bush, J., McCormack, M., and Sheen, J. 2013a. Comprehensive protein‐based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25:1507‐1522. doi: 10.1105/tpc.113.112235.
  Li, J.F., Norville, J.E., Aach, J., McCormack, M., Zhang, D., Bush, J., Church, G.M., and Sheen, J. 2013b. Multiplex and homologous recombination‐mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31:688‐691. doi: 10.1038/nbt.2654.
  Li, J.F., Zhang, D., and Sheen, J. 2014. Epitope‐tagged protein‐based artificial miRNA screens for optimized gene silencing in plants. Nat. Protoc. 9:939‐949. doi: 10.1038/nprot.2014.061.
  Ni, D., Xu, P., and Gallagher, S. 2016. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 114:10.8.1‐10.8.37. doi: 10.1002/0471142727.mb1008s114.
  O'Malley, R.C. and Ecker, J.R. 2010. Linking genotype to phenotype using the Arabidopsis unimutant collection. Plant J. 61:928‐940. doi: 10.1111/j.1365‐313X.2010.04119.x.
  Schwab, R., Ossowski, S., Riester, M., Warthmann, N., and Weigel, D. 2006. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121‐1133. doi: 10.1105/tpc.105.039834.
  Yoo, S.D., Cho, Y.H., and Sheen, J. 2007. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2:1565‐1572. doi: 10.1038/nprot.2007.199.
Key References
  Li et al., 2013a. See above.
  Demonstrates the ease and power of the ETPamir screen for pinpointing the most efficient amiRNA for silencing plant gene(s).
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