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BREAKTHROUGH TECHNOLOGIES

Specific Gene Silencing by Artificial MicroRNAs in Physcomitrella patens: An Alternative to Targeted Gene Knockouts^{1,[C],[W],[OA]}

Plant Biotechnology, Faculty of Biology (B.K., R.R., W.F.), Freiburg Initiative for Systems Biology (R.R., W.F.), and Centre for Biological Signaling Studies (R.R.), University of Freiburg, 79104 Freiburg, Germany; and Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen, Germany (S.O., D.W.)

MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNAs processed from nuclear-encoded transcripts, which include a characteristic hairpin-like structure. MiRNAs control the expression of target transcripts by binding to reverse complementary sequences directing cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism, which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome necessitates the development of alternative techniques to speed up reverse genetics analyses and to allow for more flexible inactivation of genes. To prove the adaptability of amiRNA expression in Physcomitrella, we designed two amiRNAs, targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the gene PpGNT1 encoding an N-acetylglucosaminyltransferase. Both amiRNAs were expressed from the Arabidopsis (Arabidopsis thaliana) miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the amiRNAs and an efficient knock down of the cognate target mRNAs. Furthermore, chloroplast division was impeded in PpFtsZ2-1-amiRNA lines that phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the initial amiRNA signal by secondary transitive small interfering RNAs, although these small interfering RNAs do not seem to have a major effect on sequence-related mRNAs, confirming specificity of the amiRNA approach.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wolfgang Frank (wolfgang.frank{at}biologie.uni-freiburg.de).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.108.128025

^* Corresponding author; e-mail wolfgang.frank{at}biologie.uni-freiburg.de.

Received August 13, 2008; accepted August 22, 2008; published August 27, 2008.

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				C. Addo-Quaye, J. A. Snyder, Y. B. Park, Y.-F. Li, R. Sunkar, and M. J. Axtell Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome RNA, December 1, 2009; 15(12): 2112 - 2121. [Abstract] [Full Text] [PDF]