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Molecular Mechanisms of Salt and Osmotic Stress Tolerance in Frankia Strains Isolated from Casuarina Trees

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Molecular Mechanisms of Salt and Osmotic Stress Tolerance in Frankia Strains Isolated from Casuarina Trees University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Spring 2017 Molecular mechanisms of salt and osmotic stress tolerance in Frankia strains isolated from Casuarina trees REDIET OSHONE University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation OSHONE, REDIET, "Molecular mechanisms of salt and osmotic stress tolerance in Frankia strains isolated from Casuarina trees" (2017). Doctoral Dissertations. 161. https://scholars.unh.edu/dissertation/161 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. Molecular Mechanisms of Salt and Osmotic Stress Tolerance in Frankia Strains Isolated from Casuarina trees BY Rediet Tamire Oshone B.Sc., Mekelle University, 2005 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy In Genetics May, 2017 ii This dissertation has been examined and approved in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics by: Dissertation Director, Louis S. Tisa, Professor of Microbiology and Genetics Estelle Hrabak, Associate Professor of Genetics and Molecular Biology W. Kelly Thomas, Professor of Genetics Feixia Chu, Associate Professor of Biochemistry, Molecular, and Cellular Biology Antony Champion, Plant Molecular Biology, Institut de recherche pour le développement (IRD), France On April 03, 2017 Original approval signatures are on file with the University of New Hampshire Graduate School. iii ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Louis Tisa, for providing me with the resources and funding required for the completion of my research. The continued financial support by Dr. Tisa obviated the need to financially support myself and allowed me to solely focus on my research. Dr. Tisa provided me with invaluable guidance and advice and was actively engaged in the entire process of my research- thank you Dr. Tisa! I would also like to extend my deepest gratitude to my dissertation committee members, Dr. Estelle Hrabak, Dr. Feixia Chu, Dr. W. Kelly Thomas, and Dr. Antony Champion, for the valuable comments, suggestions, and guidance they provided. The UNH graduate school, which awarded me a generous merit-based graduate fellowship when I first joined the graduate school in August 2011, deserves my heartfelt appreciation and gratitude. The generous award made my settlement in the USA smooth and easier. Last but not least, I would like to thank my family for always being there for me and for giving me unconditional support. The selfless devotion and sacrifice of my mother to ensure a better future for me and my siblings deserves the highest appreciation. I’m also grateful to my father for instilling in me the love of academia and science. His passion for education and the extraordinary commitment he showed to make environment conducive for my academic progress was undoubtedly a key positive force in my academic journey. I was raised to be competitive from an early age and settling for second best was never an option. Although it seemed a little strenuous, or even iv unnecessary at times, the high expectations my parents placed on me molded my work ethic, discipline, and outlook on life in general; I’m forever grateful for that. The following funding sources supported the research in this dissertation: New Hampshire Agricultural Experimental Station (NHAES) National Institute of Food and Agriculture - USDA Academy of Applied Sciences – REAP USAID Borlaug Fellowship Hatch NH637 Summer TA fellowship NH Graduate School Travel Grants MCBS Travel Grants Zsigray Travel Grants v Contents ACKNOWLEDGEMENTS ................................................................................................................................ iii LIST OF FIGURES ......................................................................................................................................... viii LIST OF TABLES ............................................................................................................................................. ix Abstract ......................................................................................................................................................... x Introduction .................................................................................................................................................. 1 Soil salinization.......................................................................................................................................... 1 The actinorhizal symbiosis ........................................................................................................................ 2 Status of Frankia genomes ....................................................................................................................... 5 Actinorhizal symbiosis and environmental stress ................................................................................... 11 The Casuarina – Frankia association and salt tolerance ......................................................................... 12 Salt and osmotic stress tolerance of mechanisms of microorganisms ................................................... 14 Research background and objectives ..................................................................................................... 17 Materials and Methods ............................................................................................................................... 19 Frankia Growth Media and Culture Conditions ...................................................................................... 19 Plant growth media ................................................................................................................................. 23 Salt sensitivity assay for Frankia ............................................................................................................. 24 Protein content and dry weight determination .................................................................................. 24 Vesicle induction and nitrogenase activity ............................................................................................. 25 Effect of salt treatment on the nodulation of Casuarina cunninghamiana ........................................... 25 The effect of symbiosis with Frankia on the salt tolerance of Casuarina cunninghamiana ................... 26 Genome sequencing of Frankia strains .................................................................................................. 27 Accession numbers ................................................................................................................................. 30 Pan genome analysis ............................................................................................................................... 30 Average nucleotide identity, average amino acid identity and average genomic distance ................... 31 Phylogenetic Analysis .............................................................................................................................. 31 RNA-Seq sample preparation and data analysis ..................................................................................... 32 Proteome analysis of salt-stressed Frankia ............................................................................................ 34 Quantitative reverse transcription PCR (qRT-PCR) ................................................................................. 38 Amino Acid analysis ................................................................................................................................ 40 Cloning and expression of salt tolerance genes in strain CcI3 ................................................................ 41 Results ......................................................................................................................................................... 44 Comparative Genomic analysis of salt tolerance in Casuarina isolates ................................................. 44 vi Casuarina isolates manifest diverse salt tolerance levels ................................................................... 44 Salt tolerance is highly dependent on the external supply of nitrogen.............................................. 44 In vitro level of salt tolerance was not correlated with symbiotic performance under salt stress .... 48 Genomic characteristics of Frankia strains isolated from Casuarina trees ........................................ 52 Average nucleotide identity, average amino acid identity, and genome to genome distance .......... 52 Pan-genome analysis reveals a high abundance of singletons among all of the strains .................... 55 The two salt-tolerant strains contain many hypothetical proteins absent in the other strains ......... 57 Tolerant and sensitive strains contain the same set of classical salt-tolerance genes ....................... 60 Transcriptome analysis of salt stress tolerance in Casuarina isolates ...................................................
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