Comparative analysis of nodulation-related small secreted peptides across legume species A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Diana Trujillo IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Nevin Young November 2017 © Diana Trujillo 2017 Acknowledgments I would like to thank all those who, one way or another, have made this work possible. First and foremost, thanks go to my advisor, Nevin Young, who helped shape my vision for this project and was always available to give me support or the necessary guidance in the right direction. I would also like to thank Kevin Silverstein who played a large role during the development of my LSE pipeline, and was a helpful mentor in bioinformatic matters. Thanks to the other members of my advisory committee, Peter Morrell, Michael Sadowsky, and Robert Stupar, who provided a fresh outlook and valuable advice that helped to improve this study. I would like to thank Joseph Guhlin and Peng Zhou who were always one step away when I had Unix concerns, and Shaun Curtin and Roxanne Denny who guided me through the technical aspects of growing or transforming Medicago. I am grateful that I had a strong network of friends and colleagues to discuss biology, coding, and life. To Allison Haaning, Beth Fallon, Christina Smith, Leland Werden, Derek Nedveck, and Eli Krumholz: thanks for sharing the journey. I would like to thank my mother, María Cecilia, who instilled my love of reading, truth-seeking, and plants. To my husband, Dylan Huss, thank you for always calling to see if I already ate (including as I typed these words). i Table of Contents Acknowledgments................................................................................................................ i Table of Contents ................................................................................................................ ii List of Tables ..................................................................................................................... iv List of Figures ..................................................................................................................... v List of Abbreviations ......................................................................................................... vi Chapter 1: Introduction ....................................................................................................... 1 1.1 Legumes form a unique relationship with nitrogen-fixing rhizobia ......................... 1 1.2 Rhizobial fate is determined by legume hosts ........................................................... 2 1.3 The role of small-secreted peptides in nodulation remains largely unexplored ........ 4 1.4 Lineage-specific expansions can lead to phylogeny-restricted traits ........................ 6 1.5 Project overview ........................................................................................................ 7 Chapter 2: Cross-species examination of legume signaling peptides reveals that nodule- specific PLAT domain proteins are required for nodulation .............................................. 9 2.1 Summary ................................................................................................................... 9 2.2 Keywords ................................................................................................................ 10 2.3 Introduction ............................................................................................................. 10 2.4 Materials and Methods ............................................................................................ 13 2.4.1 Legume genomic and RNA-seq data ................................................................ 13 2.4.2 Identification of nodulation-related LSEs in small secreted peptide families .. 13 2.4.3 Confirmation of candidate LSEs ...................................................................... 15 2.4.4 Synteny analyses of NPDs and Phaseoleae-specific nodulins ......................... 16 2.4.5 CRISPR/Cas9 mediated knockout of NPD genes ............................................ 16 2.4.6 Nodulation Phenotyping of NPD mutants ........................................................ 17 2.4.7 RNA-seq differential expression analyses ........................................................ 18 2.4.8 Microscopy and nodule staining ....................................................................... 19 2.4.9 Accession Numbers .......................................................................................... 19 2.5 Results ..................................................................................................................... 20 2.5.1 Overview of identified LSEs ............................................................................ 20 2.5.2 A nodule-specific subset of the PLAT domain protein family expanded exclusively in the Medicago lineage ......................................................................... 22 2.5.3 Multi-gene NPD knockouts lines show differences in nodulation phenotypes 23 2.5.4 Rhizobia lose N2-fixation capacity when all five NPD genes are knocked out 26 2.5.5 Transcriptome analysis of nodules from two- and three-gene knockout lines . 27 2.6 Discussion ............................................................................................................... 28 2.6.1 Types of LSEs found in this study.................................................................... 28 2.6.2 Phaseoleae-specific nodulins have expanded independently in Glycine and Phaseolus legumes .................................................................................................... 31 2.6.3 The NPDs are a previously unexplored family related to nodulation .............. 32 2.6.4 Utility of LSE studies ....................................................................................... 35 2.7 Acknowledgements ................................................................................................. 36 2.8 Tables ...................................................................................................................... 37 ii 2.9 Figures ..................................................................................................................... 40 Chapter 3: Genomic characterization of the LEED..PEEDs, a gene family unique to the Medicago lineage .............................................................................................................. 54 3.1 Summary ................................................................................................................. 54 3.2 Keywords ................................................................................................................ 55 3.3 Introduction ............................................................................................................. 55 3.4 Material and methods .............................................................................................. 57 3.4.1 Plant genome and transcriptome sequences ..................................................... 57 3.4.2 Detection of LPs in plant genomes and transcriptomes ................................... 57 3.4.3 Trifolium pratense DNA-seq and Pisum sativum RNA-seq analysis ............... 58 3.4.4 Transcript levels of LPs in M. truncatula ......................................................... 59 3.4.5 Synteny and collinearity comparisons .............................................................. 60 3.4.6 Phylogenetic analysis of LP sequences ............................................................ 60 3.4.7 Accession Numbers .......................................................................................... 61 3.5 Results ..................................................................................................................... 61 3.5.1 The LP gene family is specific to the Medicago lineage .................................. 61 3.5.2 Genomic architecture of the LP gene family .................................................... 63 3.5.3 Phylogenetic relationship between LP genes ................................................... 64 3.5.4 The LEED..PEED family is nodule-specific in M. truncatula ......................... 65 3.6 Discussion ............................................................................................................... 66 3.7 Acknowledgements ................................................................................................. 70 3.8 Tables ...................................................................................................................... 71 3.9 Figures ..................................................................................................................... 74 4 Conclusions .................................................................................................................... 80 5 Bibliography .................................................................................................................. 84 6 Appendix ........................................................................................................................ 96 iii List of Tables Table 2.1 Overview of genomic and RNA-seq data sources used in the LSE discovery pipeline .............................................................................................................................. 37 Table 2.2 Overview of identified LSEs and magnitude of expansion .............................