Beyond CAT, BSD, and URA: SUL, A novel selectable marker for Cyanidioschyzon merolae

The unicellular red alga Cyanidioschyzon merolae is a valuable model organism for studying pre-mRNA splicing, stress adaptation, and biotechnological applications. However, the limited availability of selectable markers has constrained its potential in genetic engineering. In this study, we evaluated the sul1 gene, which encodes a sulfadiazine-resistant variant of dihydropteroate synthase, as a new selectable marker (SUL) for C. merolae transformation. SUL has previously been used for this purpose in plants and green algae. We analyzed the sensitivity of C. merolae to sulfadiazine and determined the concentration that effectively inhibited cell growth. To test the effectiveness of SUL as a selectable marker , we designed a transformation construct containing SUL directed to the algal mitochondria through a native targeting peptide, along with mVenus to visualize transformation. We integrated the construct into a neutral genomic locus via homologous recombination. Fluorescence microscopy confirmed stable mVenus expression, and sulfadiazine selection successfully enriched transformed cells. As a demonstration of the utility of this marker, we rescued the large-cell phenotype of a cell division cycle-like kinase 2 (CmClk2) mutant by replacing the CAT-marked kinase domain deletion with the SUL-marked native kinase domain, thereby restoring CmClk2 function and recycling the CAT marker. The deletion phenotype provides evidence for a conserved cell-cycle regulatory role for CmClk2 in C. merolae . Beyond establishing SUL as an effective selectable marker, this highlights how SUL facilitates functional genetic studies of essential cellular regulators. • DHPS mutant sul1 is a novel selectable marker for genetic transformation in Cyanidioschyzon merolae . • Growth of Cyanidioschyzon merolae shows dose-dependent sensitivity to sulfadiazine. • Sulfadiazine selection enables targeted gene insertion and fluorescent protein tagging. • CLK2 kinase domain deletion results in large cells, demonstrating that CmClk2 regulates cell size before cell division.