PLASTID-TARGETED PROTEINS ARE ABSENT FROM THE PROTEOMES OF ACHLYA HYPOGYNA AND THRAUSTOTHECA CLAVATA (OOMYCOTA, STRAMENOPILA): IMPLICATIONS FOR THE ORIGIN OF CHROMALVEOLATE PLASTIDS AND THE ‘GREEN GENE’ HYPOTHESIS Lindsay Rukenbrod A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Center for Marine Science University of North Carolina Wilmington 2012 Approved by Advisory Committee D. Wilson Freshwater Jeremy Morgan Allison Taylor J. Craig Bailey Chair Accepted by Dean, Graduate School This thesis has been prepared in the style and format consistent with the Journal of Eukaryotic Microbiology. ii TABLE OF CONTENTS ABSTRACT .....................................................................................................................iv ACKNOWLEDGMENTS ..................................................................................................vi DEDICATION ................................................................................................................. vii LIST OF TABLES .......................................................................................................... viii LIST OF FIGURES ..........................................................................................................ix CHAPTER 1: Implications for the origin of chromalveolate plastids ............................... X INTRODUCTION .................................................................................................. 1 METHODS............................................................................................................ 3 RESULTS AND DISCUSSION ............................................................................. 4 Revised Hypotheses for the Evolution of Chromalveolate Plastids ............ 6 CHAPTER 2: Do chromalveolate genomes encode ‘green genes’? ............................ 15 INTRODUCTION ................................................................................................ 16 METHODS.......................................................................................................... 18 RESULTS AND DISCUSSION ........................................................................... 19 Green Genes in Oomycetes and Other Chromalveolates? ...................... 22 SUPPLEMENTAL INFORMATION................................................................................ 32 LITERATURE CITED .................................................................................................... 41 iii ABSTRACT Chapter 1 The chromalveolate hypothesis predicts that extant nonphotosynthetic stramenopiles are secondarily nonphotosynthetic and derived from ancestors bearing a secondary red- type plastid. To test this hypothesis, proteomes of the oomycetes Achlya hypogyna and Thraustotheca clavata were canvassed for plastid-targeted genes. Proteins for each species encoding putative plastid-targeting signal peptides were identified, annotated, and assigned to protein families if possible. Forty-six candidate proteins were culled from the two genomes. Bioinformatic analyses revealed that the proteomes of Achlya and Thraustotheca do not encode plastid-targeted genes acquired by endosymbiotic gene transfer. All proteins possessing non-mitochondrial-targeting signal peptides identified were judged to belong to the secretome (i.e, extracellularly secreted proteins). These results indicate that oomycetes are ancestrally aplastidic stramenopiles and do not support the chromalveolate theory of plastid evolution. Revised hypotheses for the origin of plastids characterized by chlorophylls a and c and fucoxanthin are presented. It is concluded that alveolate and stramenopile plastids are likely tertiary or higher order plastids, not secondary plastids. Chapter 2 The hypothesis that a green algal symbiosis preceded the red algal symbiont that gave rise to red-type plastids in the ancestors of the chromalveolates is reexamined. A network approach was used to detect nuclear encoded proteins from the genomes of Achlya hypogyna, Thraustotheca clavata, other oomycetes, and other chromalveolates iv that cluster with green algal genes. Twelve oomycete proteins clustering with green algal genes at high stringency were annotated and selected for further analyses. Representative homologs from all other eukaryotic taxa available were aligned to sequences comprising each network and maximum likelihood trees were constructed from these alignments. Protein trees derived from these data exhibited obvious errors resulting from taxon biases and heterotachy. These results argue that ‘green genes’ detected in phylogenomics studies are artifactual and not indicative of endosymbiotic gene transfer. v ACKNOWLEDGMENTS My thanks go to my advisor, Dr. J. Craig Bailey, whose enthusiasm about molecular protistology caught my interest in the very beginning of my scientific education. His continuous encouragement, wit, and sense of humor made this journey an enjoyable one. Ian Misner and Dr. Chris Lane of the University of Rhode Island have also been instrumental in my education, providing feedback and technical support in my research. I’d also like to thank my committee members, Dr. D. Wilson Freshwater, Dr. Jeremy Morgan, and Dr. Allison Taylor, for their encouragement and flexibility throughout this process. My lab mates past and present, particularly Cory Dashiell, Erika Shwarz, Ashley Hayes, and Allison Martin, helped me maintain my focus over the years throughout failed DNA extractions, computer malfunctions, approaching deadlines, and many other graduate school related challenges. The Department of Biology and Marine Biology, the Center for Marine Science and the National Science Foundation provided financial support for my education and research. Finally, I’d like to thank my parents and my husband for supporting me every step of the way. vi DEDICATION I’d like to dedicate this to my mother, whose endless patience has allowed me to explore life with few restrictions and overwhelming love and support. vii LIST OF TABLES Table Page Chapter 1 1. Protein IDs for 46 hypothetical proteins detected in the genomes of Thraustotheca and/or Achlya.. ............................................................................ 9 2. Protein ID numbers, annotations and protein family designations.. ................... 11 3. Proteins sorted into one of 14 unique protein families.. ..................................... 13 4. List of seven proteins from the Achlya and Thraustotheca and putative homologs found in the Arabidopsis thaliana plastid proteome.. ........................ 14 Chapter 2 1. List of 12 annotated proteins from the Achlya and/or Thraustotheca proteomes or other oomycetes found in EGNs . ........................................................................ 24 viii LIST OF FIGURES Figure Page Chapter 1 1. Hypotheses for the origin of complex, higher order chlorophyll a+c-containing plastids in chromalveolates. ....................................... 8 Chapter 2 1. Three examples of putative green genes in oomycete genomes based on EGN analysis. .................................................................... 25 2. DEXDc ML tree ................................................................................................... 26 3. RPB ML tree ....................................................................................................... 27 4. ALDH ML tree ..................................................................................................... 28 5. TOR-containing kinase ML tree .......................................................................... 29 6. YAK1 ML tree: .................................................................................................... 30 7. ALS ML tree....................................................................................................... 31 ix CHAPTER 1: Implications for the origin of chromalveolate plastids. x INTRODUCTION The evolutionary origin and subsequent movement of secondary and higher order plastids among photosynthetic eukaryotes is the subject of intense debate. The principal key to unraveling the evolutionary history of plastids is an accurate understanding of the relationships among both host and plastid lineages (Archibald 2009; Green 2011). This goal is hampered by the mosaic nature of eukaryotic genomes comprised of lineage- specific genes inherited vertically, thousands of genes acquired by endosymbiotic gene transfer (EGT), and genes obtained via lateral gene transfer (LGT) (Archibald 2008; Green 2011; Keeling 2009; Larkum 2007). The chromalveolate hypothesis posits that the alveolates, cryptomonads, haptophytes and stramenopiles are monophyletic and that the last common ancestor of these lineages was a photosynthetic alga bearing a red-type plastid (Cavalier-Smith 1999; 2003). This notion is supported, in the first instance, by the fact that photosynthetic members of these chlorophyll a+c-containing groups all possess red- type plastids surrounded by three or four unit membranes [the so-called chloroplast- endoplasmic reticulum, or CER], a feature indicative of secondary endosymbiosis (Dodge 1975; Foth and McFadden 2003; Guillot and Gibbs 1980a, b; Gibbs 1981a, b; Köhler et al. 1997). Second, nuclear-encoded plastid-targeted proteins in these algae are characterized by the presence of a 5’ bipartite signal sequence that directs gene products to the plastid and across the outer- and inner-pair of plastid membranes