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Viral Dsrna and Killer System in Saccharomyces Paradoxus

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Viral Dsrna and Killer System in Saccharomyces Paradoxus Viral dsRNA and Killer System in Saccharomyces Paradoxus Mahsan Nematbakhsh A thesis submitted for the degree of Doctor of Philosophy of Imperial College London Department of Life Sciences Faculty of Natural Sciences Imperial College London June 2016 1 DECLARATION I certify that this thesis represents my own work, unless otherwise stated. All external contributions and any information derived from other sources have been acknowledged accordingly. MAHSAN NEMATBAKHSH The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 2 In loving memory of my late grandfathers, Mirza Mahmoud Amin and Fatolah Nematbakhsh… Dedicated to my loving husband Meysam, my wonderful son Ali and my darling daughter Layli, who are all so full of life; without them my PhD journey would not have been possible. Dedicated to my mom & dad Sima and Masoud, the most precious gift in my life: for their endless support, love and encouragement throughout my life. 3 ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest gratitude to Professor Austin Burt for accepting me onto his research team and for all his patience and valuable inputs to successfully drive this project forward. I would like also to thank my co-supervisor, Professor Tony Cass and Dr. Vassiliki Koufopanou for their continued support and guidance throughout the project. Thank you to colleagues, past and present, particularly Dr. Muthu and Ms. Susan Lomas for their continued support and encouragement throughout my tenure. Finally, a very special thank you to my family members, relatives and friends for being there for me. Last but not least, to the Almighty God for giving me the strength and perseverance to complete my studies within the stipulated time frame and for keeping me surrounded by these wonderful people. 4 Abstract Some yeast strains (killer strains) release killer toxins which kill other strains of the same or different species of yeast but not the strain producing the toxin itself. In Saccharomyces cerevisiae, killer toxins are encoded by either dsRNA viruses or nuclear genes. This study aims to characterise the genetic basis of the killer toxin synthesised in Saccharomyces paradoxus, a wild non-domesticated relative of S. cerevisiae. One hundred and nine stains of S. paradoxus gathered from Silwood Park, Continental Europe, Far East, and North America were screened for the killer-immune phenotype against killer and sensitive yeast strains. In order to find whether the killer toxin is encoded by dsRNA or a nuclear gene, the killer strains were treated by cyclohexamide that removes the dsRNA which carries the killer toxin gene. The strains were also screened for presence and absence of dsRNA by directly visualising the dsRNA on the agarose gel and also by Next Generation Sequencing (NGS). Several strains were found to have the killer phenotype (30% of the screened strains). In the majority of killer strains the toxins were encoded by dsRNA except the strains from Canada and one strain from continental Europe, which seems to have other genetic bases. Sixteen full sequences of large dsRNA variants (L-A; with the length of about 4.5 kb) composed of L-A-Q, L-A-D1, L-A-C, L-A- P1.1, L-A-P1.2, L-A-P1.3, L-A-P1.4, L-A-P1.5, L-A-P1.6, L-A-P1.7, L-A-P2.1, L-A-P2.2, L-A-P2.3, L-A-P2.4, L-A-P2.5, and L-A-P2.6, and seven new types of medium size dsRNA (M; with the length between 0.8 and 2kb) composed of MQ, MC, M-P1G1, M-P1G2, M-P1G3-1, M-P1G3-2, M-P1G5, and M-P1SG, have been identified in this study. M28 was the only M dsRNA in S. paradoxus which was nearly identical to the M28 from S. cerevisiae; therefore likely to have been transferred between the two species. To test whether the killer toxins were encoded from the new types of M dsRNAs, the MQ ORF sequence from S. paradoxus was cloned and expressed into S. cerevisiae and then tested for the killer phenotype. The S. paradoxus MQ protein was successfully expressed in the S. cerevisiae strain and a positive killer response in the killer assay confirmed its function as a killer toxin. 5 List of Tables Table 1-1. Characterisation of dsRNA mycovirus families (Fauquet et al., 2005) 17 Table 1-2. The characterisation of the species in Totivirus genus (Dinman et al., 1991, Kang et al., 2001, Kondo et al., 2016, Fauquet et al., 2005, Li et al., 2011). 18 Table 1-3. The genetic basis of the killer toxins in S. cerevisiae (Goto et al., 1991, Goto et al., 1990a, Rodríguez-Cousiño et al., 2011, SCHMITT and TIPPER, 1995, Magliani et al., 1997b) 23 Table 1-4. The genetic basis of killer phenotype in yeast species 24 Table 1-5. Reported dsRNA and killer activity in Saccharomyces species (Ivannikova et al., 2007, Naumov et al., 2005, Naumov et al., 2009) 25 Table 2-1. The killer-immune phenotype of yeast strains against the S. cerevisiae sensitive strain, M894, S. cerevisiae killer strains, K1 (K1 617), K2 (K2 618), and K28 (MS 300), and the S. paradoxus non-killer strains, CBS 8437, T18.2, A33. SDT and DDT represent Same-Day-Test and Different-Day-Test spraying of the immune tester and K, PK, VPK and (-) represent Killer, Poor Killer, Very poor and non-killer, respectively. 39 Table 2-2. The effect of ethanol on the killer activity of the three S. paradoxus strains, O62.5, T21.4, and Y8.5, and the concentrated medium that contains Q62.5 killer toxin; K (Killer), PK (Poor Killer), VPK (Very Poor Killer), VVPK (Very Very Poor Killer) and (-) non-killer. 42 Table 3-1. Summary of the results of killer assay, dsRNA extraction of S. paradoxus strains and S1 nuclease treatment of the dsRNA. ”K” is the abbreviation of killer and “-“ means it is not killer. 57 Table 3-2. The distribution of dsRNAs and killer phenotypes in S. paradoxus strains from four different regions. The columns show: the total number of strains, dsRNA⁺; the number of strains containing dsRNA, dsRNA⁺ K⁺; the number of strains carrying dsRNA and showing the killer phenotype, dsRNA⁺ K⁻ the number of strains carrying dsRNA and not showing the killer phenotype and dsRNA⁻ K⁺ the number of strains that do not have dsRNA but show the killer phenotype. 61 Table 3-3. The cyclohexamide treatment of S.paradoxus killer strains. Both the Same-Day-Test (SDT) and Different-Day-Test (DDT)were performed to test the killer phenotype of the strains before and after the cycloheximide treatment. The presence of L and M dsRNA checked by dsRNA extraction and running on the gel. C1, C2, and C3 are the three clones from each cyclohexamide treated strain which their killer phenotype was tested. Only CECT10176 strain, which does not carry any dsRNA, did not lose its killer phenotype, after cycloheximide treatment. In the rest of the killer strains, minimum one clone became non – Killer after cyclohexamide treatment. dsRNA extraction of the non – killer clones indicated that, M dsRNA is removed from all the non-killer clones. K, NK, PK and VPK are the abbreviations of Killer, Non-killer, Poor Killer and Very poor respectively. “+” and “-“indicate the presence and absent of the mentioned dsRNA in each column in each strain. 62 Table 4-1. The results of killer assay, the gel electrophoresis of dsRNA extraction and dsRNA sequencing of Q62.5, DBVPG4650 and CBS8441. 74 Table 4-2. The viral dsRNAs reported in S. cerevisiae (Icho and Wickner, 1989b, Rodríguez-Cousiño et al., 2013, Russell et al., 1997, Meskauskas, 1990, SCHMITT and TIPPER, 1995, Rodríguez-Cousiño et al., 2011, PARK et al., 1996, Esteban et al., 1992b, Rodriguez-Cousiño et al., 1991) and S. paradoxus (Chang et al., 2015), and those found in S. paradoxus in this study. (C) is the abbreviation of Coding and (NC) is the abbreviation of Non-Coding parts of M. In terms of L, all the L-A found in this study contain full sequences of L-A except L-A-P1-g1 and L-A-P1-p1, which contained only gag and pol genes. 75 Table 4-3. The nucleic acid and amino identities between the different types of L-A dsRNA in S. cerevisiae and S. paradoxus. The numbers on the top that are marked with the blue lines are nucleic acid identities and the numbers at the bottom marked with green lines are amino acid identities. Based on the identities between different L-A and their phylogeny tree, there are three groups of dsRNA in the S. paradoxus strains. 77 Table 4-4. The result of the prediction of the structure of the new M dsRNAs and their preprotoxins 83 6 Table 8-1. The killer phenotype of S. paradoxus strains. 119 Table 8-2. Media (1L): 121 Table 8-3. The list of the strains in each library of NGS. 122 Table 8-4. The insertion size, number of reads after pairing the reads in each library and the percentage of reads mapped to nuclear and mitochondrial genome with and without considering MMQ200. 124 Table 8-5.
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