Specific gene silencing by artificial MicroRNAs in Physcomitrella patens: an alternative to targeted gene knockouts

Abstract

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.

Figure 1.

Analysis of Physcomitrella lines expressing PpFtsZ2-1-amiRNA and PpGNT1-amiRNA. A, Scheme illustrating the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA overexpression constructs. The modified ath-miRNA319a precursor DNA fragments were cloned into the SmaI and BamHI sites of the pPCV plant expression vector containing a double 35S promoter, nos terminator, and hpt selection marker cassette. Primers that were used for molecular analyses of the transgenic lines are indicated by arrows. B, Secondary structures of foldbacks of the P. patens miR319d precursor (ppt-MIR319d) and Arabidopsis miR319a precursor (ath-MIR319a). The mature miRNA is highlighted in green with uppercase letters. C, PCR screen to identify transgenic lines harboring the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression constructs. WT, Wild type; amiRNA lines, 1, 2, and 3 for PpFtsZ2-1-amiRNA; 1 and 2 for PpGNT1-amiRNA; PpEF1α, control PCRs. D, Expression analysis of PpFtsZ2-1-amiRNA and PpGNT1-amiRNA in Physcomitrella wild type (WT), and lines harboring the PpFtsZ2-1-amiRNA or PpGNT1-amiRNA expression constructs. Fifty micrograms of each RNA was blotted and hybridized with a PpFtsZ2-1-amiRNA and PpGNT1-amiRNA antisense probe, respectively. Hybridization with an antisense probe for U6snRNA served as control. PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression levels were normalized to the U6snRNA control hybridization. Numbers indicate the relative PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression levels. E, Top, 5′ RACE-PCRs for the genes PpFtsZ2-1 and PpFtsZ2-2 from wild type (WT) and line 1 expressing the PpFtsZ2-1-amiRNA; bottom, 5′ RACE-PCR for the gene PpGNT from wild type (WT) and line 1 expressing the PpGNT1-amiRNA. The arrows mark PCR fragments corresponding to the expected size of the cleavage products that were isolated, cloned, and sequenced. The right images show the sequence complementarity of PpFtsZ2-1, PpFtsZ2-2, and PpGNT1 to the amiRNA sequences. The determined cleavage sites within the PpFtsZ2-1 and PpGNT1 mRNAs are marked by vertical arrows and numbers above indicate the number of sequenced products cleaved at this site. [See online article for color version of this figure.]

Figure 2.

Expression analysis of PpFtsZ2-1, PpFtsZ2-2, and PpGNT1, and detection of transitive siRNAs. A, Left, RNA gel blots (20 μg each) from wild type (WT) and PpFtsZ2-1-amiRNA overexpression lines (1–3) hybridized with PpFtsZ2-1 and PpFtsZ2-2 probes; right, RNA gel blots (20 μg each) from wild type (WT) and PpGNT1-amiRNA overexpression lines (1 and 2) hybridized with a PpGNT1 probe. The ethidium bromide-stained gels below indicate equal loading. The hybridization signals were normalized to the rRNA bands, and the PpFtsZ2-1, PpFtsZ2-2, and PpGNT1 expression levels in wild type were set to 1. Numbers indicate the relative PpFtsZ2-1, PpFtsZ2-2, and PpGNT1 mRNA levels. B, Scheme illustrating the generation of transitive siRNAs from amiRNA target cleavage products requiring an RNA-dependent RNA polymerase (RdRP) to generate dsRNA, which is subsequently processed into siRNAs. Black line, mRNA; gray box, amiRNA binding site; curved line, amiRNA. C, Detection of sense and antisense transitive siRNAs produced from PpFtsZ2-1 (left) and PpGNT1 (right) mRNA cleavage products by RNA gel blots hybridized with oligonucleotides derived from regions downstream of the amiRNA binding sites. Hybridization with an antisense probe for U6snRNA served as control.

Figure 3.

Impeded plastid division and formation of macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors. A, Light microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA overexpression line (size bars, 100 μm). B, Confocal laser-scanning microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA overexpression line (size bars, 50 μm). Red, Chlorophyll autofluorescence in plastids. See Supplemental Figure S2 for phenotypes of the other two PpFtsZ2-1-amiRNA lines.