Robust Transgene Expression from Bicistronic mRNA in the Green AlgaChlamydomonas reinhardtii

Abstract

The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

Evidence supporting an antimicrobial origin of targeting peptides to endosymbiotic organelles

“…In this bicistronic system, the STOP-codon of the fluorescent reporter and the initiation codon of the selectable marker are separated by only six nucleotides (TAGCAT), which is sufficient to ensure robust expression of both genes in Chlamydomonas reinhardtii. Compared to classical expression systems where the selectable marker is driven by a separate promoter, bicistronic expression results in a much higher fraction of recovered transformants showing expression of the gene of interest (Onishi and Pringle 2016).…”

“…To assess TP-activity of AMPs we used a bicistronic expression system based on ribosome re-initiation as described by Onishi and Pringle (2016) with coding sequences for candidate peptides inserted upstream of a Venus fluorescent reporter (Figure 7 and Figure 9). In this bicistronic system, the STOP-codon of the fluorescent reporter and the initiation codon of the selectable marker are separated by only six nucleotides (TAGCAT), which is sufficient to ensure robust expression of both genes in Chlamydomonas reinhardtii.…”

“…Chlamydomonas reinhardtii cells derived from wild-type strain T222+ were grown in mixotrophic conditions in Tris-acetate-phosphate (TAP) medium (Harris 1989) under ~30 μmol photons m-2 s-1 at 25°C, either in 200 μL in 96-well plates for 3-4 days or in agitated flasks of 200 mL. Constructs were transformed into strain T222+ by electroporation and transformants, selected for paromomycin resistance, were screened for high Venus expression in a fluorescence plate reader (CLARIOstar, BMG labtech) as described in Onishi and Pringle (2016).…”

“…Constructs were made by inserting sequences coding for candidate peptides directly upstream of the Venus start codon in plasmid pMO611, kindly provided by the Pringle lab. pMO611 is a derivative of the published bicistronic expression plasmid pMO449 (Onishi and Pringle 2016) in which translation of the eight first RBCS codons ahead of the Venus coding sequence was prevented by mutating the start codon to CTG. To increase expression of the AMP constructs, we modified plasmid pMO611 further by inserting RBCS2 intron 2 within Venus, 73 bases downstream of the initiation codon.…”

Cleavage-furrow formation without F-actin inChlamydomonas

“…Previous descriptions of cytokinesis in Chlamydomonas have been based on light and electron micrographs of fixed cells (30, 53, 54). To observe the process in living cells, we expressed the plasma-membrane ATPase PMH1 (57) tagged with mNeonGreen (mNG) and observed cells by time-lapse microscopy. As expected, cleavage furrows ingressed primarily from one pole of each imaged cell (Fig.…”