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.

Fig. 1. Phylogenetic relationships and transformation status of marine protists.

A schematic view of the eukaryotic tree of life with effigies of main representatives. Color-coordinated species we have attempted to genetically modify are listed below. Current transformability status is schematized in circles indicating: DNA delivered and shown to be expressed (yellow, for details see text and Table 1); DNA delivered, but no expression seen (gray) and no successful transformation achieved despite efforts (blue). The details of transformation of species that belong to ‘DNA delivered’ and ‘Not achieved yet’ categories are described in Supplementary Table 5. mRNA, messenger RNA; FITC–dextran, fluorescein isothiocyanate (FITC)-conjugated dextran.

Fig. 2. Epifluorescence micrographs of transformed marine protists.

Representative images of transformants and wild-type cell lines of ten selected protist species. Colored boxes behind species names refer to phylogenetic supergroup assignments given in Fig. 1. Representative data of at least two independent experiments are shown. The fluorescent images show the expression of individual fluorescent marker genes introduced via transformation for all organisms shown, except in the case of A. amoebiformis. For this, red depicts the natural autofluorescence of photosynthetic pigments in the cell, while the additional green spheres in the transformant fluorescence panel shows introduced GFP fluorescence (see Supplementary Fig. 15c for a trace of these different regions in the cell). Scale bars are as follows: 10 µm for A. amoebiformis, T. pseudonana, A. limacinum, B. saltans, N. gruberi, A. whisleri and S. rosetta; 15 µm for P. marinus; 20 µm for F. cylindrus and 100 µm for P. multiseries.

Fig. 3. Various methods were used to demonstrate successful transformation in different archaeplastid species: luminescence and fluorescence.

a–c, Luminescence (a,b) and fluorescence (by FACS and epifluorescence microscopy) (c) were used to verify expression of introduced constructs in three archaeplastids: O. lucimarinus (a), B. prasinos (b) and M. commoda (c). For the latter, red in the image depicts the natural autofluorescence of photosynthetic pigments in the cell, while green shows introduced eGFP fluorescence and blue shows the DAPI-stained nucleus; the overlay shows colocalization of eGFP and nucleus signals. See Supplementary Fig. 15d for a trace of these different regions in the cell. NS, not significant; trans., transformed. Representative data of at least two independent experiments are shown. For a detailed figure description see Supplementary Notes 2. Source data

Fig. 4. Various methods were used to demonstrate successful transformation in different species: RT–PCR, western blot and sequencing.

a–j, Western blot, RT–PCR or sequencing (in case of Cas9-induced excision by CRISPR) were used to verify expression of introduced constructs in one haptophyte: I. galbana (a), one rhizarian—A. amoebiformis (b), two stramenopiles—F. cylindrus (c) and P. tricornutum (d), three alveolates—K. veneficum (e), P. marinus (f) and A. carterae (g), two discobans—B. saltans (h) and D. papillatum (i) and one opisthokont—A. whisleri (j). Note that nptII/neo is used synonymously with amino 3′-glycosyl phosphotransferase gene (aph(3′)) conferring resistance to kanamycin and neomycin. Representative data of at least two independent experiments are shown. For a detailed figure description see Supplementary Notes 2. Source data

Fig. 5. ‘Transformation roadmap’ for the creation of genetically tractable protists.

a, Vector design and construction for microeukaryotes of interest and a natural community. b, Transformation approaches. Different symbols represent methods (for example chemical, physical or biological) for introducing DNA/RNA/protein into a living cell. c, Protocol. Key methodological steps for successful transformation are listed in an abbreviated form (for particular examples, see Table 1 and text).