Targeted integration by homologous recombination enables in situ tagging and replacement of genes in the marine microeukaryote Diplonema papillatum
Language English Country England, Great Britain Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
Grant support
16-18699S
Czech Grant Agency - International
LL1601
ERC CZ - International
GBMF4983.01
Gordon and Betty Moore Foundation - International
ERD Funds OPVVV16_019/ 0000759
the Czech Ministry of Education - International
NSERC; RGPIN-2019-04024
the Natural Sciences and Engineering Research Council of Canada - International
CZ.02.1.01/0.0/0.0/16_019/0000759
project Centre for research of pathogenicity and virulence of parasites r.n. - International
- MeSH
- Euglenozoa genetics MeSH
- Homologous Recombination MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
Czech Academy of Sciences Institute of Parasitology Biology Centre Czech Republic
Faculty of Sciences University of South Bohemia Cˇeské Budějovice Czech Republic
See more in PubMed
Adl, S.M., Bass, D., Lane, C.E., Lukeš, J., Schoch, C.L., Smirnov, A., et al. (2019) Revisions to the classification, nomenclature, and diversity of eukaryotes. J Eukaryot Microbiol 66: 4-119.
Arras, S.D.M., and Fraser, J.A. (2016) Chemical inhibitors of non-homologous end joining increase targeted construct integration in Cryptococcus neoformans. PLoS One 11: e0163049.
Barnes, R.L., and McCulloch, R. (2007) Trypanosoma brucei homologous recombination is dependent on substrate length and homology, though displays a differential dependence on mismatch repair as substrate length decreases. Nucleic Acids Res 35: 3478-3493.
Benz, C., and Urbaniak, M.D. (2019) Organising the cell cycle in the absence of transcriptional control: dynamic phosphorylation co-ordinates the Trypanosoma brucei cell cycle post-transcriptionally. PLoS Pathog 15: e1008129.
Byrum, J., Jordan, S., Safrany, S.T., and Rodgers, W. (2004) Visualization of inositol phosphate-dependent mobility of Ku: depletion of the DNA-PK cofactor InsP6 inhibits Ku mobility. Nucleic Acids Res 32: 2776-2784.
Carradec, Q., Pelletier, E., Da Silva, C., Alberti, A., Seeleuthner, Y., Blanc-Mathieu, R., et al. (2018) A global atlas of eukaryotic genes. Nat Commun 9: 373.
Clayton, C.E. (2016) Gene expression in Kinetoplastids. Curr Opin Microbiol 32: 46-51.
Dean, S., Sunter, J.D., and Wheeler, R.J. (2017) TrypTag.org: a trypanosome genome-wide protein localisation resource. Trends Parasitol 33: 80-82.
Dean, S., Sunter, J., Wheeler, R.J., Hodkinson, I., Gluenz, E., and Gull, K. (2015) A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol 5: 140197.
Eichinger, L., Lee, S.S., and Schleicher, M. (1999) Dictyostelium as model system for studies of the actin cytoskeleton by molecular genetics. Microsc Res Tech 47: 124-134.
Faktorová, D., Bär, A., Hashimi, H., McKenney, K., Horák, A., Schnaufer, A., et al. (2018) TbUTP10, a protein involved in early stages of pre-18S rRNA processing in Trypanosoma brucei. Mol Biochem Parasitol 225: 84-93.
Faktorová, D., Nisbet, R.E.R., Fernández Robledo, J.A., Casacuberta, E., Sudek, L., Allen, A.E., et al. (2020) Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nat Methods 17: 481-494. https://doi.org/10.1038/s41592-020-0796-x.
Faktorová, D., Valach, M., Kaur, B., Burger, G., and Lukeš, J. (2018) Mitochondrial RNA editing and processing in diplonemid protists. In RNA Metabolism in Mitochondria, Gray, M.W., and Cruz-Reyes, J. Cham, Switzerland: (eds): Springer International Publishing AG, pp. 145-176.
Flegontova, O., Flegontov, P., Malviya, S., Audic, S., Wincker, P., de Vargas, C., et al. (2016) Extreme diversity of diplonemid eukaryotes in the ocean. Curr Biol 26: 3060-3065.
Gawryluk, R.M.R., del Campo, J., Okamoto, N., Strassert, J.F.H., Lukeš, J., Richards, T.A., et al. (2016) Morphological identification and single-cell genomics of marine diplonemids. Curr Biol 26: 3053-3059.
George, E., Husnik, P.F., Tashyreva, D., Prokopchuk, G., Horák, A., Kwong, W.K., et al. (2020) Highly reduced genomes of protist endosymbionts show evolutionary convergence. Curr Biol 30: 925-933.
Goins, C.L., Gerik, K.J., and Lodge, J.K. (2006) Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes. Fung Genet Biol 43: 531-544.
Goos, C., Dejung, M., Janzen, C.J., Butter, F., and Kramer, S. (2017) The nuclear proteome of Trypanosoma brucei. PLoS One 12: e0181884.
Hegemann, J.H., Heick, S.B., Pöhlmann, J., Langen, M.M., and Fleig, U. (2014) Targeted gene deletion in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Methods Mol Biol 1163: 45-73.
von der Heyden, S., Chao, E.E., Vickerman, K., and Cavalier-Smith, T. (2004) Ribosomal RNA phylogeny of bodonid and diplonemid flagellates and the evolution of euglenozoa. J Eukaryot Microbiol 51: 402-416.
Jackson, A.P., Vaughan, S., and Gull, K. (2006) Evolution of tubulin gene arrays in trypanosomatid parasites: genomic restructuring in Leishmania. BMC Genomics 18: 261.
Kaur, B., Valach, M., Peña-Diaz, P., Moreira, S., Keeling, P.J., Burger, G., et al. (2018) Transformation of Diplonema papillatum, the type species of the highly diverse and abundant marine microeukaryotes Diplonemida (Euglenozoa). Environ Microbiol 20: 1030-1040.
Kaur, B., Záhonová, K., Valach, M., Faktorová, D., Prokopchuk, G., Burger, G., and Lukeš, J. (2020) Gene fragmentation and RNA editing without borders: eccentric mitochondrial genomes of diplonemids. Nucleic Acids Res 48: 2694-2708.
Kiethega, G.N., Yan, Y., Turcotte, M., and Burger, G. (2013) RNA-level unscrambling of fragmented genes in Diplonema mitochondria. RNA Biol 10: 301-313.
Krejci, L., Altmannova, V., Spirek, M., and Zhao, X. (2012) Homologous recombination and its regulation. Nucleic Acids Res 40: 5795-5818.
Lam, S.S., Martell, J.D., Kamer, K.J., Deerinck, T.J., Ellisma n, M.H., Mootha, V.K., and Ting, A.Y. (2015) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods 12: 51-54.
López-García, P., Vereshchaka, A., and Moreira, D. (2007) Eukaryotic diversity associated with carbonates and fluid sea water interface in lost city hydrothermal field. Environ Microbiol 9: 546-554.
Lukeš, J., Flegontova, O., and Horák, A. (2015) Diplonemids. Curr Biol 25: R702-704.
Lukeš, J., Wheeler, R., Jirsová, D., David, V., and Archibald, J.M. (2018) Massive mitochondrial DNA content in diplonemid and kinetoplastid protists. IUBMB Life 70: 1267-1274.
Malkova, A., and Haber, J.E. (2012) Mutations arising during repair of chromosome breaks. Annu Rev Genet 46: 455-473.
Marande, W., and Burger, G. (2007) Mitochondrial DNA as a genomic jigsaw puzzle. Science 318: 415.
Marande, W., Lukeš, J., and Burger, G. (2005) Unique mitochondrial genome structure in diplonemids, the sister group of kinetoplastids. Eukaryot Cell 4: 1137-1146.
Massana, R. (2011) Eukaryotic picoplankton in surface oceans. Annu Rev Microbiol 65: 91-110.
Matthews, K.R., McCulloch, R., and Morrison, L.J. (2015) The within-host dynamics of African trypanosome 726 infections. Philos Trans R Soc Lond B Biol Sci 370: 20140288.
McKean, P.G., Vaughan, S., and Gull, K. (2001) The extended tubulin superfamily. J Cell Sci 114: 2723-2733.
Moreira, S., Valach, M., Aoulad-Aissa, M., Otto, C., and Burger, G. (2016) Novel modes of RNA editing in mitochondria. Nucleic Acids Res 44: 4907-4919.
Mukherjee, I., Hodoki, Y., Okazaki, Y., Fujinaga, S., Ohbayashi, K., and Nakano, S.I. (2019) Widespread dominance of kinetoplastids and unexpected presence of diplonemids in deep freshwater lakes. Front Microbiol 10: 2375.
Nayak, T., Szewczyk, E., Oakley, C.E., Osmani, A., Ukil, L., Murray, S.L., et al. (2006) A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics 172: 1557-1566.
Nenarokova, A., Záhonová, K., Krasilnikova, M., Gahura, O., McCulloch, R., Zíková, A., et al. (2019) Causes and effects of loss of classical nonhomologous end joining pathway in parasitic eukaryotes. mBio 10: e01541-19.
Ninomiya, Y., Suzuki, K., Ishii, C., and Inoue, H. (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci USA 101: 12248-12253.
Okamoto, N., Gawryluk, R.M.R., Campo, J., Strassert, J.F.H., Lukeš, J., Richards, T.A., et al. (2019) A revised taxonomy of diplonemids including the Eupelagonemidae n. fam. and a type species, Eupelagonema oceanica n. gen. & sp. J Eukaryot Microbiol 66: 519-524.
Peña-Diaz, P., Mach, J., Kriegová, E., Poliak, P., Tachezy, J., and Lukeš, J. (2018) Trypanosomal mitochondrial intermediate peptidase does not behave as a classical mitochondrial processing peptidase. PLoS One 13: e0196474.
Prokopchuk, G., Tashyreva, D., Yabuki, A., Horák, A., Masařová, P., and Lukeš, J. (2019) Morphological, ultrastructural, motility and evolutionary characterization of two new Hemistasiidae species. Protist 170: 259-282.
Rodgers, K., and McVey, M. (2016) Error-prone repair of DNA double-strand breaks. J Cell Physiol 231: 15-24.
Rodríguez-Ezpeleta, N., Teijeiro, S., Forget, L., Burger, G., and Lang, B.F. (2009) Construction of cDNA libraries: focus on protists and fungi. In Methods in Molecular Biology, Vol. 533, Parkinson, J. (ed) . Totowa, NJ: Humana Press, pp. 33-47.
Roy, J., Faktorová, D., Benada, O., Lukeš, J., and Burger, G. (2007) Description of Rhynchopus euleeides n. sp. (Diplonemea), a free-living marine euglenozoan. J Eukaryot Microbiol l54: 137-145.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory.
Simpson, A.G.B. (1997) The identity and composition of the Euglenozoa. Archiv für Protistenkunde 148: 318-328.
Son, M.Y., and Hasty, P. (2019) Homologous recombination defects and how they affect replication fork maintenance. AIMS Genet 5: 192-211.
Sunter, J.D., Yanase, R., Wang, Z., Catta-Preta, C.M.C., Moreira-Leite, F., Myskova, J., et al. (2019) Leishmania flagellum attachment zone is critical for flagellar pocket shape, development in the sand fly, and pathogenicity in the host. Proc Natl Acad Sci U S A 116: 6351-6360.
Tashyreva, D., Prokopchuk, G., Votýpka, J., Yabuki, A., Horák, A., and Lukeš, J. (2018a) Life cycle, ultrastructure, and phylogeny of new diplonemids and their endosymbiotic bacteria. mBio 9: e02447-17.
Tashyreva, D., Prokopchuk, G., Yabuki, A., Kaur, B., Faktorová, D., Votýpka, J., et al. (2018b) Phylogeny and morphology of new diplonemids from Japan. Protist 169: 158-179.
Trahan, C., Aguilar, L.-C., and Oeffinger, M. (2016) Single-step affinity purification (ssAP) and mass spectrometry of macromolecular complexes in the yeast S. cerevisiae. Methods Mol Biol 1361: 265-287.
Valach, M., Léveillé-Kunst, A., Gray, M.W., and Burger, G. (2018) Respiratory chain complex I of unparalleled divergence in diplonemids. J Biol Chem 293: 16043-16056.
Valach, M., Moreira, S., Faktorová, D., Lukeš, J., and Burger, G. (2016) Post-transcriptional mending of gene sequences: looking under the hood of mitochondrial gene expression in diplonemids. RNA Biol 13: 1204-1211.
de Vargas, C., Audic, S., Henry, N., Decelle, J., Mahe, F., Logares, R., et al. (2015) Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean. Science 348: 1261605.
Vickerman, K. (2000) Diplonemids (class: Diplonemea Cavalier Smith, 1993). In An Illustrated Guide to the Protozoa, Vol. 2, 2nd edn, Lee, J.J., Leedale, G.F., and Bradbury, P. (eds). Lawrence, Kansas, USA: Society of Protozoologists, pp. 1157-1159.
Waters, C.A., Strande, N.T., Wyatt, D.W., Pryor, J.M., and Ramsden, D.A. (2014) Nonhomologous end joining: a good solution for bad ends. DNA Repair 17: 39-51.
Yi, Z., Berney, C., Hartikainen, H., Mahamdallie, S., Gardner, M., Boenigk, J., et al. (2017) High-throughput sequencing of microbial eukaryotes in Lake Baikal reveals ecologically differentiated communities and novel evolutionary radiations. FEMS Microbiol Eco 93. https://doi.org/10.1093/femsec/fix073.
Zhao, Z., Liu, H., Luo, Y., Zhou, S., An, L., Wang, C., et al. (2014) Molecular evolution and functional divergence of tubulin superfamily in the fungal tree of life. Sci Rep 23: 6746.
On the possibility of yet a third kinetochore system in the protist phylum Euglenozoa
Functional differentiation of Sec13 paralogues in the euglenozoan protists
Highly flexible metabolism of the marine euglenozoan protist Diplonema papillatum
Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses