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

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. This article has 113 total authors; only 32 are listed here

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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

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

Genomic and transcriptomic analysis in the Antarctic ciliate Euplotes focardii: insights regarding the evolution of the molecular basis of cold adaptation

Ciliates provide optimal model systems to study environmental adaptation. Comparative transcriptomic analysis of Euplotes focardii, a strictly psychrophilic ciliate isolated from Antarctic seawater, and the mesophilic congeneric species E. crassus revealed that in E. focardii the majority of the expressed genes code for proteins involved in oxidoreductase activity, as reported for Antarctic fishes and krill. These results confirm that a major problem of Antarctic marine organisms is to cope with increased O2 solubility at low temperatures. They also suggest that an increased defence against oxidative stress likely provides an important evolutionary feature that allowed the adaptation of Antarctic organisms in their oxygen-rich environment. Quantitative PCR showed that expression of Hsp70 genes was induced when E. focardii cells were subjected to oxidative stress, whereas thermal stress did not cause induction. These results argue that E. focardii in its current environment is well protected against reactive oxygen species and are consistent with prior reports of constitutive Hsp70 expression as a defence against cold-induced protein denaturation. E. focardii appears to be poised to cope with the oxidative challenge that is likely to accompany oceanic warming over the next century, but the absence of a temperature-inducible chaperone response may place its proteome at risk. E. focardii genome sequences reported by Lobanov et al. (2017) have been re-assembled by SPAdes with a 25% improvement in the number of contigs, with about 25000 nanochromosomes identified and annotated. Gene families involved in oxidative stress appeared significantly expanded in paralogous genes in E. focardii with respect to E. crassus and E. octocarinatus, thus suggesting gene duplication as mechanism at the basis of molecular cold adaptation. The comparative genome analysis of Euplotes species also revealed a rapid evolution and unusual plasticity of the programmed ribosomal frameshifting, a process that allows the change of the reading frame during translation. This process appears pervasive in Euplotes and it is not conserved in the affected genes of the different species. Attempts were performed to annotate all the genes subjected to frameshifting in E. focardii and unusual sites for +2 frameshifting were found

Trasfection and reverse genetics in marine ciliates with highly amplified nanochromosomes

The study of marine ciliates of the Euplotes genus has provided significant insights into microbial ecology. These organisms have an unusual genetic organization with two genomes, that is, a micronuclear genome representing the germ line, and a macronuclear genome containing single gene nanochromosomes amplified to thousands of copies for their somatic life. Improved understanding of this unique organization has been beneficial in elucidating universal principles in the biology of telomeres. However, the application of transfection and reverse genetics techniques remains a challenge. The development of such techniques would contribute to a better understanding of the complex transposition mechanisms occurring in the new macronucleus after each sexual cycle. In this context, we worked on different methods of transfection and gene silencing by RNAi for Euplotes crassus and E. focardii. The former is mesophilic and has a cosmopolitan distribution, as it has been collected from most of the Earth's oceans, while the latter is a strictly psychrophilic Antarctic species and survives at temperatures from -2 °C to 15 °C. 1. We generated plasmids and artificial nanochromosomes ending with telomeres that contain either a GFP reporter and/or a drug selection marker. 2. We found that E. crassus and E. focardii (which has a slower growth rate) display naturally high resistance to a number of drugs/antibiotics that are typically used for selection in transfection experiments. However, they are much more sensitive to G418, paromomycin and puromycin when grown in a culture medium of 10% artificial seawater and 90% of 0.3 M glucose. 3. We demonstrated the delivery of Cy3-labelled control plasmid to Euplotes by lipofection, electroporation, and microinjection. 4. In order to set up the RNAi technology, we analysed all the steps involved in RNAi by feeding with silencing bacteria. We obtained the silencing of the telomerase gene in E.focardii, which showed a phenotype with visible changes in nuclear structure and with characteristics of senescence. (We would like to acknowledge the Moore Foundation for its financial support.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper