Introduction of a leaky stop codon as molecular tool in Chlamydomonas reinhardtii

Abstract

Expression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct.

Transit Peptides Often Require Downstream Unstructured Sequence for Efficient Chloroplast Import in Chlamydomonas reinhardtii

“…This study was seeded by the observation that the full-length RCA1-cTP fails to generate reliable chloroplast localization of a Venus fluorescent reporter, expressed from a bicistronic expression vector ( Onishi and Pringle, 2016 ; Caspari, 2020 ), in Chlamydomonas ( Figure 1A ): fluorescence signal in the Venus channel was visible from across the cell, rather than being restricted to the chloroplast, the compartment labeled by chlorophyll autofluorescence. Given that successful targeting using the RBCS2-cTP had previously been reported when extended by 23 residues downstream of the cleavage site in Chlamydomonas ( Razzak et al, 2017 ) and by 24 in vascular land plants ( Comai et al, 1988 ), and extending the RCA1-cTP by 23 residues had equally restored targeting ( Garrido et al, 2020 ), a series of successive extensions was prepared.…”

Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

“…Native cTPs were tested by inserting the respective genomic sequences upstream of a Venus fluorescent reporter in a bicistronic expression vector (Onishi and Pringle, 2016; Caspari, 2020). Neither RBCA nor RBCS cTPs enabled import into the chloroplast (Fig.…”

Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts

“…S2, Supplementary text). Localization was obtained using stable Chlamydomonas expression lines, generated by introducing DNA sequences encoding peptides upstream of the Venus coding sequence in a bicistronic expression vector (Caspari, 2020) using Gibson assembly, with transformation cassettes integrated into the Chlamydomonas nuclear genome at random sites via electroporation. Micrographs for three biological replicates (i.e., independent insertion lines) per construct are displayed in Fig.…”

“…K is for killing, R is for targeting. (A) Indicated peptides were inserted upstream of a Venus fluorescent protein reporter in a bicistronic expression system for Chlamydomonas (Caspari, 2020). AR P : hybrid HSP70a-RBCS2 promoter, i1: RBCS2 established by an NMR-study 1104 (Krimm et al, 1999), since no 1105 helix could be predicted by our 1106 approach.…”

“…Transformants (≥24 per construct) selected for Paromomycin resistance were grown in 200µl TAP in 96-well plates under 50µmol photons m -2 s -2 for 3-5 days and then screened for Venus expression in a fluorescence plate reader (CLARIOstar, BMG Labtech) as described previously (Caspari, 2020).…”