Targeted integration by homologous recombination enables in situ tagging and replacement of genes in the marine microeukaryote Diplonema papillatum.

Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well-studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one, Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination in D. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5' and 3' homologous regions longer than 1 kbp were used, achieving N-terminal tagging with mCherry and gene replacement of α- and β-tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein-A tag and used it to generate and express a C-terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging of D. papillatum genes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general.