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The PhD thesis is composed of three publications on genomic, metabolic, and cellular integration between the host and its symbionts in the tripartite nested mealybug system. The articles revealed a path to an intimate endosymbiosis that can be compared to what we think happened before (and to some extent after) bacterial ancestors of key eukaryotic organelles, mitochondria and plastids, became highly integrated into their host cells. I argue that these much younger symbioses may tell us something about how the mitochondria and plastids came to be, at the very least by revealing what types of evolutionary events are possible as stable intracellular relationships proceed along the path of integration.

This thesis is devoted to the evolution of the heme biosynthetic pathway in eukaryotic phototrophs with particular emphasis on algae possessing secondary and tertiary red and green derived plastids. Based on molecular biology and bioinformatics approaches it explores the diversity and similarities in heme biosynthesis among different algae. The core study of this thesis describes the heme biosynthesis in Bigelowiella natans and Guillardia theta, algae containing a remnant endosymbiont nucleus within their plastids, in dinoflagellates containing tertiary endosymbionts derived from diatoms called dinotoms, and in Lepidodinium chlorophorum, a dinoflagellate containing a secondary green plastid. The thesis further focusses on new insights in the heme biosynthetic pathway and general origin of the genes in chromerids the group of free-living algae closely related to apicomplexan parasites.

Coral reefs form marine-biodiversity hotspots of enormous ecological, economic, and aesthetic importance that rely energetically on a functional symbiosis between the coral animal and a photosynthetic alga. The ongoing decline of corals worldwide due to anthropogenic influences heightens the need for an experimentally tractable model system to elucidate the molecular and cellular biology underlying the symbiosis and its susceptibility or resilience to stress. The small sea anemone Aiptasia is such a model organism and the main aims of this dissertation were 1) to assemble and analyze its genome as a foundational resource for research in this area and 2) to investigate the role of miRNAs in modulating gene expression during the onset and maintenance of symbiosis. The genome analysis has revealed numerous features of interest in relation to the symbiotic lifestyle, including the evolution of transposable elements and taxonomically restricted genes, linkage of host and symbiont metabolism pathways, a novel family of putative pattern-recognition receptors that might function in host-microbe interactions and evidence for horizontal gene transfer within the animal-alga pair as well as with the associated prokaryotic microbiome. The new genomic resource was used to annotate the Aiptasia miRNA repertoire to illuminate the role of post-transcriptional regulatory mechanisms in regulating endosymbiosis. Aiptasia encodes a majority of species-specific miRNAs and first evidence is presented that even evolutionary conserved miRNAs are undergoing recent differentiations within the Aiptasia genome. The analysis of miRNA expression between different states of Symbiodinium infection further revealed that species-specific and conserved miRNAs are symbiotically regulated. In order to detect functional miRNA-mRNA interactions and to investigate the downstream effects of such miRNA action, a protocol for cross-linking immunoprecipitations of Argonaute, the central protein of the miRNA-induced silencing complex, was developed. This method identified binding sites of miRNAs on a transcriptome-wide scale and revealed target genes of symbiotically regulated miRNAs that were identified previously to be involved in the symbiosis. In summary, this dissertation provides novel insights into miRNA-mediated post-transcriptional modulation of the host transcriptome and by presenting a critically needed genomic resource, lays the foundation for the continued development of Aiptasia as a model for coral symbiosis.

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Chemotaxis is the process by which motile bacteria navigate chemical gradients in order to position themselves in optimum environments for growth and metabolism. Sensory input from both the external environment and the internal cellular environment are sensed by chemotaxis transducers and transduced to a two-component system whose output interacts with the flagellum thereby regulating motility. Chemotaxis has been implicated in establishing the endosymbiotic relationship between the motile alpha-proteobacterium Rhizobium leguminosarum biovar viciae and its host Pisum sativa, the pea plant. An approach combing bioinformatical sequence analysis, molecular genetics, and behavioral analysis was used to characterize the chemotaxis system of R. leguminosarum and determine its contribution to this bacterium s lifestyle. A genome search revealed the presence of two chemotaxis gene clusters, che1 and che2. Homologs of each che cluster are major chemotaxis operons controlling flagellar motility in other bacterial species. For this reason we sought to determine the contribution of each che cluster to chemotaxis in R. leguminosarum. We found that while both che clusters contribute to the regulation of motility, che1 is the major che cluster controlling chemotaxis. Using competitive nodulation assays we determined that che1, but not che2, is essential for competitive nodulation. The major che cluster, che1, encodes a chemotaxis transducer, IcpA-Rl, with a globin coupled sensor domain. Chemotaxis transducers with a globin coupled sensor domain comprise a large class of proteins found in bacteria and archaea. These proteins have been shown to bind heme and sense oxygen and are therefore termed HemATs for heme-binding aerotaxis transducers. However, sequence analysis of IcpA-Rl reveals that it lacks the requisite amino acid residues for heme-binding and is therefore unlikely to sense oxygen. We present evidence that IcpA-Rl is likely an energy transducer and represents a novel function of the globin coupled sensor domain in sensing energy related parameters.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Sequenced diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum belong to the chromist algae harboring secondary plastids, which display distinct evolutionary history when compared to photosynthetic organelles from rhodophytes, green algae and plants. Via secondary endosymbiosis, heterotrophic eukaryotic ancestor of diatoms engulfed red alga, and in addition to the new organelle, it obtained fitness increasing peculiarities in the chimerical cell metabolism and lifestyle. We examined phylogeny and in silico localization of the nuclear-encoded but plastid located enzymes of tryptophan biosynthesis. We suggest that the diatom tryptophan pathway represents an extreme in the trend of plastid (cyanobacterial) enzymes to be replaced by eukaryotic isoforms. In addition, the gene napped during the endosymbiotic gene transfer from the diatom plastid genome to the diatom nucleus (psb28) was described.

For millions of years, the nutrient exchange symbiosis between corals and their endosymbiotic algae has formed the foundation of the ecological success of coral reefs. Yet, in recent decades anthropogenic climate change is increasingly destabilizing this symbiosis, and thus the reefs that rely on it. High-temperature anomalies have caused mass mortality of corals due to repeated coral bleaching, the expulsion or digestion of symbionts by the host during stress. Hence, in-depth knowledge of the cellular processes of bleaching is required to conceive strategies to maintain the ecological functioning of coral reefs. In this thesis, we investigated the role of symbiotic nutrient cycling in the bleaching response of corals. For this, we examined the mechanisms that underlie the functioning of the symbiosis in a stable state and how heat stress affects these metabolic interactions during coral bleaching. Our findings reveal that the functioning of the coral – algae symbiosis depends on the resource competition between host and symbionts. In a stable state, symbiotic competition for ammonium limits nitrogen availability for the algal symbiont, thereby ensuring symbiotic carbon translocation and recycling. During heat stress, however, increased metabolic energy demand shifts host metabolism from amino acid synthesis to degradation. The resulting net release of ammonium by the host, coupled with the stimulated activity of associated nitrogen-fixing microbes, substantially increases nitrogen availability for algal symbionts. Subsequently, stimulated algal growth causes selfish retention of carbon, thereby further reducing energy availability for the host. This positive feedback loop disturbs symbiotic nutrient recycling, eventually causing the collapse of carbon translocation by the symbiont. Hence, heat stress causes shifts in metabolic interactions, which directly and indirectly destabilizes the symbiosis, and ultimately undermines the ecological benefits of hosting algal symbionts for corals. In summary, this thesis shows that integrating symbiotic nutrient cycling into our conceptual understanding of coral bleaching is likely to improve our ability to predict coral bleaching in light of environmental conditions and may ultimately help to conceive new strategies to preserve coral reef functioning.

Les plastes primaires proviennent d'une cyanobactérie qui a établi une relationendosymbiotique avec un hôte eucaryote. Cet événement a donné naissance au super-groupeArchaeplastida qui inclut les Viridiplantae (algues vertes et plantes terrestres), les Rhodophyta (alguesrouges) et les Glaucophyta. Suite à l'endosymbiose primaire, les algues rouges et vertes ont étendu lacapacité de photosynthèse à d'autres lignées eucaryotes via des endosymbioses secondaires. Bien quedes progrès considérables aient été réalisés dans la compréhension de l'évolution des eucaryotesphotosynthétiques, d'importantes questions sont restées ouvertes, telles que l’identité de la lignéecyanobactérienne la plus proche des plastes primaires ainsi que le nombre et l'identité des partenairesdans les endosymbioses secondaires.Ma thèse a consisté à étudier l'origine et l'évolution précoce des eucaryotes photosynthétiques enutilisant des approches phylogénétiques et phylogénomiques. Je montre par mon travail que les plastesprimaires ont évolué à partir d'un symbiote phylogénétiquement proche de Gloeomargarita lithophora,une cyanobactérie représentant un clade s’étant diversifié précocement et qui a été détectéeuniquement dans les milieux terrestres. Ce résultat fournit des pistes nouvelles sur le contexteécologique dans lequel l'endosymbiose primaire a probablement eu lieu. En ce qui concerne l'évolutiondes lignées eucaryotes avec des plastes secondaires, je montre que les génomes nucléaires deschlorarachniophytes et des euglénophytes, deux lignées photosynthétiques avec des plastes dérivésd'algues vertes, encodent un grand nombre de gènes acquis par transferts depuis des algues rouges.Enfin, je mets en évidence que SELMA, la machinerie de translocation des protéines à travers laseconde membrane externe des plastes rouges secondaires à quatre membranes, a une histoireétonnamment compliquée aux implications évolutives importantes : les cryptophytes ont recruté unensemble de composants de SELMA différent de ceux des haptophytes, straménopiles et alvéolés.Ainsi, ma thèse a permis d’identifier pour la première fois la lignée cyanobactérienne la plus proche desplastes primaires et apporte de nouvelles pistes pour éclaircir les événements complexes qui ontjalonné l’évolution des eucaryotes photosynthétiques secondaires. / Primary plastids derive from a cyanobacterium that entered into an endosymbioticrelationship with a eukaryotic host. This event gave rise to the supergroup Archaeplastida whichcomprises Viridiplantae (green algae and land plants), Rhodophyta (red algae) and Glaucophyta. Afterprimary endosymbiosis, red and green algae spread the ability to photosynthesize to other eukaryoticlineages via secondary endosymbioses. Although considerable progress has been made in theunderstanding of the evolution of photosynthetic eukaryotes, important questions remained debatedsuch as the present-day closest cyanobacterial lineage to primary plastids as well as the number andidentity of partners in secondary endosymbioses.The main objectives of my PhD were to study the origin and evolution of plastid-bearing eukaryotesusing phylogenetic and phylogenomic approaches to shed some light on how primary and secondaryendosymbioses occurred. In this work, I show that primary plastids evolved from a close relative ofGloeomargarita lithophora, a recently sequenced early-branching cyanobacterium that has been onlydetected in terrestrial environments. This result provide interesting hints on the ecological setting whereprimary endosymbiosis likely took place. Regarding the evolution of eukaryotic lineages with secondaryplastids, I show that the nuclear genomes of chlorarachniophytes and euglenids, two photosyntheticlineages with green alga-derived plastids, encode for a large number of genes acquired by transfersfrom red algae. Finally, I highlight that SELMA, the translocation machinery putatively used to importproteins across the second outermost membrane of secondary red plastids with four membranes, has asurprisingly complex history with strong evolutionary implications: cryptophytes have recruited a set ofSELMA components different from those present in haptophytes, stramenopiles and alveolates.In conclusion, during my PhD I identified for the first time the closest living cyanobacterium to primaryplastids and provided new insights on the complex evolution that have undergone secondary plastid-bearing eukaryotes