Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii

A significantly improved viral 2A peptide system for dependable high-level expression of dicistronic genes in Chlamydomonas reinhardtii has been developed. Data are presented demonstrating that use of an especially proficient extended FMDV2A' coding region allows production of two independent protein products from a dicistronic gene with almost complete efficiency. Importantly, results are also presented that demonstrate the utility of this 2A system for efficient high-level expression of foreign genes in C.reinhardtii, which has not previously been reliably achievable in this algal model system. To expand the versatility of the 2A expression system, a number of commonly used selectable marker proteins were assessed for their compatibility with the extended FMDV2A peptide. Additional experiments demonstrate the feasibility and utility of 2A-containing dicistronic systems that rely on a strong conditional promoter for transcriptional control and a low-expression marker gene for selection. This strategy allows easy and efficient delivery of genes of interest whose expression levels require regulation either to mitigate potential toxicity or allow differential expression under controlled experimental conditions. Finally, as an additional practical demonstration of the utility of the extended FMDV2A system, confocal fluorescence microscopy is used to demonstrate that native and foreign proteins of interest bearing post-translational remnants of the extended FMDV2A peptide localize correctly to various cellular compartments, including a striking demonstration of the almost exclusive localization of the Rubisco small subunit protein to the pyrenoid of the C. reinhardtii chloroplast in cells maintained under ambient CO2 concentrations. Significance Statement An improved FMDV 2A system has been developed that allows simultaneous, tightly-coordinated and dependably high-level expression of two different proteins from a single dicistronic gene - with use of a conditional promoter allowing control of gene expression over at least a 100-fold range. Post-translational 2A peptide remnants that remain attached to the N- and C-termini of native and foreign proteins are shown not to affect proper targeting (e.g., targeting of Rubisco to the chloroplast pyrenoid).