Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

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

“…As indicated above, transformation efficiency may depend on the selection marker. We observed in D. papillatum that although puromycin was the most efficient antibiotic (Kaur et al ., 2018), Puro R ‐based selection took longer and the transformation efficiency was somewhat lower compared to Neo R (Faktorová et al ., 2020) or Hyg R (our unpubl. data).…”

“…The V5‐ Neo R fusion flanked by the partial 5′ and 3′ hexokinase UTRs of the trypanosomatid Blastocrithidia sp. p57 (GenBank: MN047315) was inserted into the D. papillatum genome and, despite its random integration, was efficiently expressed (Faktorová et al ., 2020). Importantly, the V5‐ Neo R fusion protein was catalytically active.…”

“…For functional studies of their cellular components, a high‐quality nuclear genome sequence, as well as methods for genetic manipulation, are needed. Although the release of the genome assembly and annotation of D. papillatum is underway (Burger et al ., unpublished), we recently implemented the genetic tools in this species (Kaur et al ., 2018; Faktorová et al ., 2020).…”

“…The development of (high‐throughput) genetic tools in as many planktonic protists as possible will be critical for tackling the functions of at least a small fraction of the over 100 million unique genes from across marine unicellulars (Carradec et al ., 2018). In more than a dozen such lineages across the eukaryotic tree, many impervious to functional studies thus far, the expression of introduced genes was recently demonstrated (Faktorová et al ., 2020). Diplonema papillatum – an easily cultured and comparably fast dividing diplonemid representative – has joined this suite of genetically tractable marine organisms.…”

“…That the gene was expressed a fortuitous consequence of the polycistronic nuclear transcription in diplonemids, a trait that these flagellates share with their sister group kinetoplastids (Clayton, 2016). In sum, until recently, our experiments fell short of targeted integration required for functional studies (Kaur et al ., 2018; Faktorová et al ., 2020).…”