A FORWARD GENETIC SCREEN FOR ISOLATION OF FUSION DEFECTIVE MATING TYPE MINUS MUTANTS IN CHLAMYDOMONAS REINHARDTII

Year 2021, , 321 - 332, 17.05.2021

Münevver Aksoy , Tammy La , Nuenyan Lam , Charlene Forest

https://doi.org/10.15671/hjbc.689397

Cited By: 1

Abstract

Many biological processes require cell fusion, therefore defects in cell fusion result in many diseases. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study cell-cell fusion. The objective of this study was to identify genes that are involved in C. reinhardtii mating type minus (MT_) gamete fusion. A forward genetics approach was taken in our work. We created several MT_ fusion defective mutants using DNA insertional mutagenesis. These mutants were normal in the early stages of mating; they agglutinated with mating type plus (MT+), removed their walls, adhered to their mating partner through their mating structures, but the cells did not fuse, indicating that the DNA insertional mutants were defective in the latest stages of fusion. The number of insertions was confirmed by Southern blots. Mutant J1 had one insertion and the flanking genomic DNA was cloned by TAIL-PCR and RESDA-PCR. The insertion is in a gene predicted to be involved in 5-deoxystrigol biosynthesis.

Keywords

Chlamydomonas reinhardtii, mating type minus, fusion defective mutant

References

• 1. E. H. Chen, E. N. Olson, Unveiling the mechanisms of cell-cell fusion, Science, 308 (2005) 369–373.
• 2. G. B. Harris, H.E., Stern, D.B., Witman, Ed., The Chlamydomonas Sourcebook, 2nd Ed. Academic Press, 2009.
• 3. S. S. Merchant, S. E. Prochnik, O. Vallon, E. H. Harris, S. J. Karpowicz, G. B. Witman, A. Terry, A. Salamov, L. K. Fritz-Laylin, L. Maréchal-Drouard, W. F. Marshall, L. H. Qu, D. R. Nelson, A. A. Sanderfoot, M. H. Spalding, V. V. Kapitonov, Q. Ren, P. Ferris, E. Lindquist, H. Shapiro, S. M. Lucas, J. Grimwood, J. Schmutz, I. V. Grigoriev, D. S. Rokhsar, A. R. Grossman, P. Cardol, H. Cerutti, G. Chanfreau, C. L. Chen, V. Cognat, M. T. Croft, R. Dent, S. Dutcher, E. Fernández, H. Fukuzawa, D. González-Ballester, D. González-Halphen, A. Hallmann, M. Hanikenne, M. Hippler, W. Inwood, K. Jabbari, M. Kalanon, R. Kuras, P. A. Lefebvre, S. D. Lemaire, A. V. Lobanov, M. Lohr, A. Manuell, I. Meier, L. Mets, M. Mittag, T. Mittelmeier, J. V. Moroney, J. Moseley, C. Napoli, A. M. Nedelcu, K. Niyogi, S. V. Novoselov, I. T. Paulsen, G. Pazour, S. Purton, J. P. Ral, D. M. Riaño-Pachón, W. Riekhof, L. Rymarquis, M. Schroda, D. Stern, J. Umen, R. Willows, N. Wilson, S. L. Zimmer, J. Allmer, J. Balk, K. Bisova, C. J. Chen, M. Elias, K. Gendler, C. Hauser, M. R. Lamb, H. Ledford, J. C. Long, J. Minagawa, M. D. Page, J. Pan, W. Pootakham, S. Roje, A. Rose, E. Stahlberg, A. M. Terauchi, P. Yang, S. Ball, C. Bowler, C. L. Dieckmann, V. N. Gladyshev, P. Green, R. Jorgensen, S. Mayfield, B. Mueller-Roeber, S. Rajamani, R. T. Sayre, P. Brokstein, I. Dubchak, D. Goodstein, L. Hornick, Y. W. Huang, J. Jhaveri, Y. Luo, D. Martínez, W. C. A. Ngau, B. Otillar, A. Poliakov, A. Porter, L. Szajkowski, G. Werner, K. Zhou, The Chlamydomonas genome reveals the evolution of key animal and plant functions, Science, 318 (2007) 245–250.
• 4. R. Sager, S. Granick, Nutritional control of sexuality in Chlamydomonas reinhardi, J. Gen. Physiol., (1954) 729–742.
• 5. W. S. Adair, C. Hwang, U. W. Goodenough, Identification and visualization of the sexual agglutinin from the mating-type plus flagellar membrane of Chlamydomonas, Cell, 33-1 (1983) 183–193.
• 6. P. J. Ferris, E. Virginia Armbrust, U. W. Goodenough, Genetic structure of the mating-type locus of Chlamydomonas reinbardtii, Genetics, 160-1 (2002) 181–200.
• 7. P. J. Ferris, S. Waffenschmidt, J. G. Umen, H. Lin, J. H. Lee, K. Ishida, T. Kubo, J. Lau, U. W. Goodenough, Plus and minus sexual agglutinins from Chlamydomonas reinhardtii, Plant Cell, 17-2 (2005) 597–615.
• 8. K. Bergman, U. W. Goodenough, D. A. Goodenough, J. Jawitz, H. Martin, Gametic differentiation in Chlamydomonas Reinhardtii: II. flagellar membranes and the agglutination reaction, J. Cell Biol., 67 (1975) 606–622.
• 9. C. L. Forest, D. A. Goodenough, U. W. Goodenough, Flagellar membrane agglutination and sexual signaling in the conditional Gam-1 mutant of Chlamydomonas, J. Cell Biol., 79 (1978), 74–84.
• 10. W. I. Snell, M. Buchanan, A. Clausell, Lidocaine reversibly inhibits fertilization in Chlamydomonas: A possible role for calcium in sexual signalling, J. Cell Biol., 94-3 (1982) 607–612.
• 11. S. M. Pasquale, U. W. Goodenough, Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii, J. Cell Biol., 105-5 (1987) 2279–2292.
• 12. M. J. Buchanan, S. H. Imam, W. A. Eskue, W. J. Snell, Activation of the cell wall degrading protease, lysin, during sexual signalling in Chlamydomonas: the enzyme is stored as an inactive, higher relative molecular mass precursor in the periplasm, J. Cell Biol., 108 (1989) 199–207.
• 13. W. J. Snell, W. A. Eskue, M. J. Buchanan, Regulated secretion of a serine protease that activates an extracellular matrix-degrading metalloprotease during fertilization in Chlamydomonas, J. Cell Biol., 109 (1989) 1689–1694.
• 14. Y. Matsuda, T. Saito, T. Yamaguchi, H. Kawase, Cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii is a metalloprotease and digests the sodium perchlorate-insoluble component of cell wall, J. Biol. Chem., 260 (1985) 6373–6377.
• 15. U. W. Goodenough, R. L. Weiss, Gametic differentiation in Chlamydomonas reinhardtii: III. cell wall lysis and microfilament-associated mating structure activation in wild-type and mutant strains, J. Cell Biol., 67-3 (1975) 623–637.
• 16. U. W. Goodenough, P. A. Detmers, C. Hwang, Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants, J. Cell Biol., 92 (1982) 378–386.
• 17. C. L. Forest, Specific contact between mating structure membranes observed in conditional fusion-defective Chlamydomonas mutants, Exp. Cell Res., 148 (1983) 143–154.
• 18. R. L. Weiss, D. A. Goodenough, U. W. Goodenough, Membrane particle arrays associated with the basal body and with contractile vacuole secretion in Chlamydomonas, J. Cell Biol., 71-1 (1977) 133–143.
• 19. C. L. Forest, Genetic control of plasma membrane adhesion and fusion in Chlamydomonas gametes, J. Cell Sci., 88-5 (1987) 613–621.
• 20. P. J. Ferris, J. P. Woessner, U. W. Goodenough, A sex recognition glycoprotein is encoded by the plus mating-type gene fus1 of Chlamydomonas reinhardtii, Mol. Biol. Cell, 7-8 (1996) 1235–1248.
• 21. M. J. Misamore, S. Gupta, W. J. Snell, The Chlamydomonas Fus1 protein is present on the mating type plus fusion organelle and required for a critical membrane adhesion event during fusion with minus gametes, Mol. Biol. Cell, 14 (2003) 2530–2542.
• 22. T. Mori, H. Kuroiwa, T. Higashiyama, T. Kuroiwa, GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization, Nat. Cell Biol., 8 (2006) 64–71.
• 23. Y. Liu, R. Tewari, J. Ning, A. M. Blagborough, S. Garbom, J. Pei, N. V. Grishin, R. E. Steele, R. E. Sinden, W. J. Snell, O. Billker, The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes, Genes Dev., 22 (2008) 1051–1068.
• 24. L. H. Friedman, L., Colwin, A.L., Colwin, Fine structural aspects of fertilization in Chlamydomonas reinhardi, J. Cell Sci., 3 (1968) 115–128.
• 25. C. L. Forest, R. K. Togasaki, Selection for conditional gametogenesis in Chlamydomonas reinhardi, Proc. Natl. Acad. Sci. U. S. A., 72 (1975) 3652–3655.
• 26. K. L. Kindle, High-frequency nuclear transformation of Chlamydomonas reinhardtii, Proc. Natl. Acad. Sci. USA, 87-3 (1990) 1228–1232.
• 27. X. Li, R. Zhang, W. Patena, S. S. Gang, S. R. Blum, N. Ivanova, R. Yue, J. M. Robertson, P. A. Lefebvre, S. T. Fitz-Gibbon, A. R. Grossman, M. C. Jonikas, An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii, Plant Cell, 28 (2016) 367–387.
• 28. S. Lumbreras, V., Stevens, D.R., Purton, Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron, Plant J., 14 (1998) 441–447.
• 29. D. R. Stevens, J. D. Rochaix, S. Purton, The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas, Mol. Gen. Genet., 251-1 (1996) 23–30.
• 30. R. Sager, Genetic systems in Chlamydomonas., Science, 132-3438 (1960) 1459–1465.
• 31. C. L. Forest, Mutational disruption of the 9 + 2 structure of the axoneme of Chlamydomonas flagella, J. Cell Sci., 61 (1983) 423–436.
• 32. F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. a Smith, K. Struhl, C. J. Wiley, R. D. Allison, M. Bittner, S. Blackshaw, Current Protocols in Molecular Biology (2003).
• 33. R. M. Dent, C. M. Haglund, B. L. Chin, M. C. Kobayashi, K. K. Niyogi, Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii, Plant Physiol., 137 (2005) 545–556.
• 34. D. González-Ballester, A. De Montaigu, A. Galván, E. Fernández, Restriction enzyme site-directed amplification PCR: A tool to identify regions flanking a marker DNA, Anal. Biochem., 340 (2005) 330–335.
• 35. B. Cenkci, J. L. Petersen, G. D. Small, REX1 a novel gene required for DNA repair, J. Biol. Chem., 278-25 (2003) 22574–22577. 36. M. Aksoy, W. Pootakham, A. R. Grossman, Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation, Plant Cell, 26-10 (2014) 4214–4229.
• 37. M. Iseki, K. Shida, K. Kuwabara, T. Wakabayashi, M. Mizutani, H. Takikawa, Y. Sugimoto, Evidence for species-dependent biosynthetic pathways for converting carlactone to strigolactones in plants, J. Exp. Bot., 69-9 (2018) 2305–2518.
• 38. D. Gobena, M. Shimels, P. J. Rich, C. Ruyter-Spira, H. Bouwmeester, S. Kanuganti, T. Mengiste, G. Ejeta, Mutation in sorghum LOW GERMINATION STIMULANT 1 alters strigolactones and causes Striga resistance, Proc. Natl. Acad. Sci. U. S. A., 114-17 (2017) 4471–4476.