Gene silencing by artificial microRNAs in Chlamydomonas

Abstract

SummaryChlamydomonas reinhardtii is a unicellular green alga. It is a model system for studying functions of the chloroplast, basal body and flagella. The completion of the Chlamydomonas genome sequence makes it possible to use reverse genetic approaches in this organism. Chlamydomonas contains a set of endogenous microRNAs (miRNAs) that down-regulate their target gene expression through mRNA cleavage. Here we developed an artificial miRNA-based strategy to knock down gene expression in Chlamydomonas. Using an endogenous Chlamydomonas miRNA precursor as the backbone, we constructed two artificial miRNAs (amiRNAs) targeting the MAA7 and RBCS1/2 genes, respectively. When overexpressed, these two amiRNAs could cleave their respective targets precisely at the predicted sites, resulting in greatly decreased accumulation of MAA7 and RBCS1/2 transcripts and expected mutant phenotypes. We further showed that the two amiRNAs could be produced simultaneously from a dimeric amiRNA precursor. We anticipate that the amiRNA technology developed in this study will be very useful in assessing the functions of individual genes and in genome-wide approaches.

Non-Thermal Radio Frequency Stimulation Inhibits the Tryptophan Synthase Beta Subunit in the Algae <i>Chlamydomonas reinhardtii</i>

“…Zhao et al have shown that the siRNA machinery functioning in Chlamydomonas, siRNA against MAA7 reduces the mRNA levels of MAA7 [4]. This leads to a reduction of the conversion of prodrug 5-FI to the highly toxic 5-FTryptophan.…”

“…To optimize the Nativis technology we targeted a well characterized enzyme in Chlamydomonas, the beta subunit of tryptophan synthase (MAA7) [4]. Zhao et al have shown that the siRNA machinery functioning in Chlamydomonas, siRNA against MAA7 reduces the mRNA levels of MAA7 [4].…”

“…We inhibited the synthesis of the enzyme tryptophan synthase beta subunit (MAA7) by applying the signal derived from a published siRNA (Zhao et al 2009). With lower levels of MAA7, Chlamydomonas reinhardtii can grow in the presence of the prodrug 5-Fluoroindole (5-FI), because less 5-Fluoroindole can be converted to the toxic 5-Fluoro-L-tryptophan (5-FT).…”

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AbstractTo demonstrate the ability of the Nativis signal transduction technology (Butters et al 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas reinhardtii.We inhibited the synthesis of the enzyme tryptophan synthase beta subunit (MAA7) by applying the signal derived from a published siRNA (Zhao et al 2009). With lower levels of MAA7, Chlamydomonas reinhardtii can grow in the presence of the prodrug 5-Fluoroindole (5-FI), because less 5-Fluoroindole can be converted to the toxic 5-Fluoro-L-tryptophan (5-FT).

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Isolation and characterization of pigment deficient insertional mutants in the chlorophyte Chlamydomonas reinhardtii

“…RNA silencing technique has been successfully used to suppress endogenous transcript levels in Chlamydomonas [11][12][13] but it is difficult to obtain a complete loss-of-function RNA-silenced strain and there may be off-target effects induced by the introduced construct [6,10].…”