AN ABSTRACT OF THE DISSERTATION OF Kyle R. Pomraning for the degree of Doctor of Philosophy in Molecular and Cellular Biology presented on November 20, 2012 Title: Characterization of Neurospora crassa and Fusarium graminearum Mutants Defective in Repeat-induced Point Mutation Abstract approved: Michael Freitag Mutation of repetitive DNA by repeat-induced point mutation (RIP) is a process that occurs in many filamentous fungi of the Ascomycota during the sexual cycle. Concurrently, direct DNA repeats are often deleted by homologous recombination at high frequency during the sexual cycle. Thus, the processes of RIP and deletion compete to either mutate or remove repetitive DNA from the genome of filamentous fungi during sexual cycles. Both processes contribute to genome streamlining by controlling proliferation of transposable elements and by limiting expansion of gene families. While the genetic requirements for deletion by homologous recombination are well known, the mechanism behind the specific detection and mutation of repetitive DNA by RIP has yet to be elucidated as only a single gene essential for RIP, rid, has been identified. We have developed Fusarium graminearum as a model organism for the study of RIP by showing that it mutates repetitive DNA frequently during the sexual cycle and that the mutations due to RIP are dependent on rid. Further, we have sequenced a genetic mapping strain of F. graminearum (00-676-2) and identified 62,310 single nucleotide polymorphisms (SNPs) compared to the reference strain (PH-1). The SNP map will be useful for quickly mapping new mutants by bulk segregant analysis and high-throughput sequencing for which bioinformatic tools were specifically developed. The groundwork has thus been laid for identification of novel RIP mutants in F. graminearum, which being homothallic has a major advantage for identification of recessive mutations. We used a forward genetics approach to shed light on the mechanism of RIP in Neurospora crassa. Two rrr mutants that dominantly reduce RIP and recombination were characterized and identified as different mutated alleles of the same gene, rrr-1L496P and rrr- 1G325N by bulk segregant analysis and high-throughput sequencing. Bioinformatic characterization suggests RRR-1 belongs to a previously uncharacterized group of dynamin- like proteins, which are generally involved in membrane fission and fusion. RRR-1-GFP localizes to the nuclear membrane, but not DNA, suggesting it affects RIP and recombination frequency indirectly by altering nuclear membrane dynamics during sexual development and thereby altering temporal aspects of RIP and recombination. We used a reverse genetics approach to determine whether high frequency RIP and homologous recombination of repetitive DNA during the sexual cycle are linked mechanistically or spatio-temporally. We tested strains where genes important for deletion by homologous recombination were knocked out and found all to be completely RIP competent except mre11, which, while sterile in homozygous deletion crosses, displayed lower RIP frequency in heterozygous crosses. This suggests that mre11 has roles in homologous recombination as well as non- homologous end joining may be important for RIP. Collectively, this work developed methods for efficiently mapping mutations and identified a novel protein that reduces RIP and recombination frequency but did not identify any mechanistic link between the two processes. ©Copyright by Kyle R. Pomraning November 20, 2012 All Rights Reserved Characterization of Neurospora crassa and Fusarium graminearum Mutants Defective in Repeat-induced Point Mutation by Kyle R. Pomraning A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented November 20, 2012 Commencement June 2013 Doctor of Philosophy dissertation of Kyle R. Pomraning presented on November 20, 2012. APPROVED: Major Professor, representing Molecular and Cellular Biology Director of the Molecular and Cellular Biology Program Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Kyle R. Pomraning, Author ACKNOWLEDGEMENTS I would like to express my tremendous gratitude to everyone who has supported me throughout graduate school. My advisor, Michael Freitag, brought me into the light of fungal research and provided the scaffolding for scientific success, without which I could not have completed this thesis. To my friends and colleagues, Erin Bredeweg and Brett Mellbye, I am grateful for constant moral support and enthusiastic scientific discussion. I would also like to express my thanks to all who have toiled beside me at the bench including Lanelle Connolly, Pallavi Phatale, Kristina Smith, Joe Whalen, Jon Galazka, Steve Friedman, Greg Ekberg, Kayly Lembke, Phuong Pham, Mu Feng and the many students from Oregon and abroad who have come through Michael’s lab. To Jason Cumbie and Henry Priest I offer my appreciation for assistance with all things computational. My thanks also go out to my graduate committee, Kerry McPhail, Todd Mockler, Steve Strauss and Tom Wolpert for providing a positive environment during the most critical junctures of the graduate experience. To Kelly Vining, Snezhka Oliferendo, Scott Baker, Kevin Mc Cluskey, Monika Schmoll, Fred Bowring, David Catcheside, Eric Selker, Georgi Mitev, Bruce Geller and Buck Wilcox I offer my thanks for fruitful collaborations. To my family, Alex, Don and Mona Pomraning I am grateful for the December beach extravaganza each of the last five years. Finally, I thank Bubbles for lowering my blood pressure. CONTRIBUTION OF AUTHORS Chapter 1: Kyle R. Pomraning wrote the chapter. Chapter 2: Joseph P. Whalen, Lanelle R. Connolly, Kristina M. Smith, Kyle R. Pomraning and Michael Freitag performed the experiments and data analysis. Kyle R. Pomraning and Michael Freitag wrote the manuscript. Chapter 3: Sequencing of the Mauriceville strain was done by Kristina M. Smith and Michael Freitag. Fred Bowring isolated the octad strains in David Catcheside’s lab at Flinders University, Australia. The strains were sequenced by Michael Freitag and analyzed by Kyle R. Pomraning. All other experiments and analyses were performed by Kyle R. Pomraning. Kyle R. Pomraning and Michael Freitag wrote the manuscript. Chapter 4: Ann T. Kobsa (Hageman) performed the mutagenesis and identified mutants in Eric Selker’s lab at the University of Oregon, Eugene. All other experiments and analyses were performed by Kyle R. Pomraning. Kyle R. Pomraning wrote the chapter. Chapter 5: Kyle R. Pomraning performed the experiments and wrote the chapter. Chapter 6: Kyle R. Pomraning performed the experiments and wrote the chapter. TABLE OF CONTENTS Page Introduction: repeat-induced gene inactivation in fungi ........................................................... 1 Genome defense in filamentous fungi ................................................................................... 2 RIP and MIP are fungal specific processes .............................................................................. 2 Discovery of repeat-induced gene inactivation in filamentous fungi..................................... 4 Duplicated DNA and nearby single copy sequences are targeted by MIP and RIP ................ 6 Use of RIP for molecular biology ............................................................................................ 8 Length constraints for targeting of duplicated DNA by RIP and MIP ..................................... 8 Divergence constraints for targeting of duplicated DNA by RIP and MIP ............................ 11 Characterization of the effects of RIP ................................................................................... 12 RIP acts on paired sequences but mutates single strands of DNA independently ............... 13 RIP and MIP most likely act on cytosine ............................................................................... 14 Mutations in a group of orthologous fungal-specific cytosine methyltransferase-like genes result in enigmatic phenotypes ............................................................................................ 16 RIP is not dependent on the homologous recombination machinery ................................. 18 Effects of mating type on RIP frequency .............................................................................. 20 Effects of translesion polymerases on RIP frequency .......................................................... 21 Severity of RIP in heterozygous crosses with wild N. crassa isolates ................................... 21 Distribution and severity of RIP in the fungi: experimental data ......................................... 22 Distribution and severity of RIP in the fungi: computational prediction of past RIP ........... 23 Evolutionary effects of RIP.................................................................................................... 25 Evolutionary effects of RIP beyond transposons and rDNA ................................................. 28 Unanswered questions about RIP ........................................................................................ 29 Characterization of RIP in Fusarium graminearum .................................................................. 33 Abstract ................................................................................................................................