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Endosymbiosis is a relationship between two organisms wherein one cell resides inside the other. This affiliation, when stable and beneficial for the ‘host’ cell, can result in massive genetic innovation with the foremost examples being the evolution of eukaryotic organelles, the mitochondria and plastids. Despite its critical evolutionary role, there is limited knowledge about how endosymbiosis is initially established and how host–endosymbiont biology is integrated. Here, we explore this issue, using as our model the rhizarian amoeba Paulinella, which represents an independent case of primary plastid origin that occurred c. 120 million yr ago. We propose the ‘chassis and engine’ model that provides a theoretical framework for understanding primary plastid endosymbiosis, potentially explaining why it is so rare.

The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles - termed chromatophores - that evolved from a cyanobacterium ∼100 million years ago, independently from plastids in plants and algae. Despite its more recent origin, at least one third of the chromatophore proteome consists of nucleus-encoded proteins that are imported by an unknown mechanism across the chromatophore double envelope membranes. Chromatophore-targeted proteins fall into two classes. Proteins exceeding 250 amino acids carry a conserved N-terminal sequence extension, termed the ‘chromatophore transit peptide’ (crTP), that is presumably involved in guiding these proteins into the chromatophore. Short imported proteins do not carry discernable targeting signals. To explore whether the import of protein is accompanied by their N-terminal processing, here we used a mass spectrometry-based approach to determine protein N-termini in Paulinella chromatophora and identified N-termini of 208 chromatophore-localized proteins. Our study revealed extensive N-terminal modifications by acetylation and proteolytic processing in both, the nucleus and chromatophore-encoded fraction of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Our results imply that the crTP mediates trafficking through the Golgi, is bipartite and surprisingly only the N-terminal third (‘part 1’) becomes cleaved upon import, whereas the rest (‘part 2’) remains at the mature proteins. In contrast, short imported proteins remain largely unprocessed. Finally, this work sheds light on N-terminal processing of proteins encoded in an evolutionary-early-stage photosynthetic organelle and suggests host-derived post-translationally acting factors involved in dynamic regulation of the chromatophore-encoded chromatophore proteome.One sentence summaryProteins targeted to the evolutionary-early-stage photosynthetic organelle of Paulinella carry a bipartite N-terminal targeting sequence that is only partially removed upon protein import.

The thecate amoeba Paulinella is a valuable model for understanding plastid organellogenesis because this lineage has independently gained plastids (termed chromatophores) of alpha-cyanobacterial provenance. Plastid primary endosymbiosis in Paulinella occurred relatively recently (90–140 million years ago, Mya), whereas the origin of the canonical Archaeplastida plastid occurred >1,500 Mya. Therefore, these two events provide independent perspectives on plastid formation on vastly different timescales. Here we generated the complete chromatophore genome sequence from P. longichromatophora (979,356 bp, GC-content = 38.8%, 915 predicted genes) and P. micropora NZ27 (977,190 bp, GC-content = 39.9%, 911 predicted genes) and compared these data to that from existing chromatophore genomes. Our analysis suggests that when a basal split occurred among photosynthetic Paulinella species ca. 60 Mya, only 35% of the ancestral orthologous gene families from the cyanobacterial endosymbiont remained in chromatophore DNA. Following major gene losses during the early stages of endosymbiosis, this process slowed down significantly, resulting in a conserved gene content across extant taxa. Chromatophore genes faced relaxed selection when compared to homologs in free-living alpha-cyanobacteria, likely reflecting the homogeneous intracellular environment of the Paulinella host. Comparison of nucleotide substitution and insertion/deletion events among different P. micropora strains demonstrates that increases in AT-content and genome reduction are ongoing and dynamic processes in chromatophore evolution.

Redox regulation in phytoplankton is critical to monitor and stabilize metabolic pathways under changing environmental conditions(1). In plastids, the thioredoxin (TRX) system is linked to photosynthetic electron transport and fine tuning of metabolic pathways to fluctuating light levels. Expansion of the number of redox signal transmitters and their protein targets, as seen in plants, is believed to increase cell robustness(2). In this study, we searched for genes related to redox regulation in the genome of the photosynthetic amoeba Paulinella micropora KR01 (hereafter, KR01). The genus Paulinella includes testate filose amoebae, in which a single clade acquired a photosynthetic organelle, the chromatophore, from an alpha cyanobacterial donor(3). This independent primary endosymbiosis occurred relatively recently (~ 124 Ma), when compared to Archaeplastida (> 1 Ga), making photosynthetic Paulinella a valuable model for studying the earlier stages of primary endosymbiosis(4). Our comparative analysis demonstrates that this lineage has evolved a thioredoxin system similar to other algae, relying however on genes with diverse phylogenetic origins (i.e., the endosymbiont, host, bacteria, red algae). One TRX of eukaryotic provenance is targeted to the chromatophore, implicating host-endosymbiont coordination of redox regulation. A chromatophore targeted glucose-6-phosphate dehydrogenase of red algal origin suggests that Paulinella exploited the existing redox regulation system in Archaeplastida to foster integration. Our study elucidates the independent evolution of the thioredoxin system in photosynthetic Paulinella, whose parts derive from the existing genetic toolkit in diverse organisms.

The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles - termed chromatophores - that evolved from a cyanobacterium, independently from plastids in plants and algae. Despite the more recent origin of the chromatophore, it shows tight integration into the host cell. It imports hundreds of nucleus-encoded proteins, and diverse metabolites are exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded transporters appears insufficient for supporting metabolic connectivity or protein import. Furthermore, chromatophore-localized biosynthetic pathways as well as multiprotein complexes include proteins of dual genetic origin, suggesting coordination of gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, nuclear factors evolved that control metabolite exchange and gene expression in the chromatophore. Here we show by mass spectrometric analyses of enriched insoluble protein fractions that, unexpectedly, nucleus-encoded transporters are not inserted into the chromatophore inner envelope membrane. Thus, despite the apparent maintenance of its barrier function, canonical metabolite transporters are missing in this membrane. Instead we identified several expanded groups of short chromatophore-targeted orphan proteins. Members of one of these groups are characterized by a single transmembrane helix, and others contain amphipathic helices. We hypothesize that these proteins are involved in modulating membrane permeability. Furthermore, we identified an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted octotrico peptide repeat expression regulators in algae and plants suggesting their convergent evolution as nuclear regulators of gene expression levels in the chromatophore.ImportanceThe endosymbiotic acquisition of mitochondria and plastids >1 billion years ago was central for the evolution of eukaryotic life. However, owing to their ancient origin, these organelles provide only limited insights into the initial stages of organellogenesis. The chromatophore in Paulinella evolved ~100 million years ago and thus, offers the possibility to gain valuable insights into early stages and common rules in organelle evolution. Critical to organellogenesis appears to be the establishment of nuclear control over metabolite exchange and gene expression in the endosymbiont. Here we show that the mechanism generating metabolic connectivity of the chromatophore fundamentally differs from the one for mitochondria and plastids, but likely rather resembles the poorly understood mechanism in various bacterial endosymbionts in plants and insects. Furthermore, we describe a novel class of chromatophore-targeted helical repeat proteins which evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function (“dark” genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion.

The uptake and conversion of a free-living cyanobacterium into a photosynthetic organelle by the single-celled Archaeplastida ancestor helped transform the biosphere from low to high oxygen. There are two documented, independent cases of plastid primary endosymbiosis. The first is the well-studied instance in Archaeplastida that occurred ca. 1.6 billion years ago, whereas the second occurred 90-140 million years ago, establishing a permanent photosynthetic compartment (the chromatophore) in amoebae in the genus Paulinella. Here, we briefly summarize knowledge about plastid origin in the Archaeplastida and then focus on Paulinella. In particular, we describe features of the Paulinella chromatophore that make it a model for examining earlier events in the evolution of photosynthetic organelles. Our review stresses recently gained insights into the evolution of chromatophore and nuclear encoded DNA sequences in Paulinella, metabolic connectivity between the endosymbiont and cytoplasm, and systems that target proteins into the chromatophore. We also describe future work with Paulinella, and the potential rewards and challenges associated with developing further this model system.

Paulinella photosynthetic species are unicellular, silica shell-forming amoebas classified into the supergroup Rhizaria. They crawl at the bottom of freshwater and brackish environments with the help of filose pseudopodia. These protists have drawn the attention of the scientific community because of two photosynthetic bodies, called chromatophores, that fill up their cells permitting fully photoautotrophic existence. Paulinella chromatophores, similarly to primary plastids of the Archaeplastida supergroup (including glaucophytes, red algae as well as green algae and land plants), evolved from free-living cyanobacteria in the process of endosymbiosis. Interestingly, these both cyanobacterial acquisitions occurred independently, thereby undermining the paradigm of the rarity of endosymbiotic events. Chromatophores were derived from α-cyanobacteria relatively recently 60-140 million years ago, whereas primary plastids originated from β-cyanobacteria more than 1.5 billion years ago. Since their acquisition, chromatophore genomes have undergone substantial reduction but not to the extent of primary plastid genomes. Consequently, they have also developed mechanisms for transport of metabolites and nuclear-encoded proteins along with appropriate targeting signals. Therefore, chromatophores of Paulinella photosynthetic species, similarly to primary plastids, are true cellular organelles. They not only show that endosymbiotic events might not be so rare but also make a perfect model for studying the process of organellogenesis. In this chapter, we summarize the current knowledge and retrace the fascinating adventure of Paulinella species on their way to become photoautotrophic organisms.

Pochłonięcie sinicy przez cudzożywne eukarionty i przekształcenie jej w plastyd na drodze endosymbiozy około 1.5 miliarda lat temu było jednym z najważniejszychwydarzeń w ewolucji życia na naszej planecie. Plastydy umożliwiły eukariontom odżywianie z wykorzystaniem światła słonecznego dając początek niezliczonej liczbie nowych gatunków roślin i glonów zaangażowanych w istotne relacje troficzne w skali całego globu. Ze względu na złożoność procesu przekształcenia endosymbionta bakteryjnego w organellum komórkowe, zjawisko endosymbiozy sinicy do niedawna uważane było za niepowtarzalne i wyjątkowe. Poglądy te podważyło odkrycie ameby Paulinella chromatophora zawierającej dwa fotosyntetyczne ciałka (chromatofory) właśnie pochodzenia sinicowego. Paulinella stanowi zatem drugi przykład endosymbiozy między sinicą a eukariontem, która miała miejsce stosunkowo niedawno, około 90-140 milionów lat temu. Niniejsza praca opisuje przemiany, jakim uległy sinicowe endosymbionty P. chromatophora, aby stać się organellami komórkowymi, w szczególności endosymbiontyczny transfer genów i ewolucję systemu importu białek do chromatoforów.

The chromatophores found in the cells of photosynthetic Paulinella species, once believed to be endosymbiotic cyanobacteria, are photosynthetic organelles that are distinct from chloroplasts. The chromatophore genome is similar to the genomes of α-cyanobacteria and encodes about 1,000 genes. Therefore, the chromatophore is an intriguing model of organelle formation. In this study, we analyzed the lipids of Paulinella micropora MYN1 to verify that this organism is a composite of cyanobacterial descendants and a heterotrophic protist. We detected glycolipids and phospholipids, as well as a betaine lipid diacylglyceryl-3-O-carboxyhydroxymethylcholine, previously detected in many marine algae. Cholesterol was the only sterol component detected, suggesting that the host cell is similar to animal cells. The glycolipids, presumably present in the chromatophores, contained mainly C16 fatty acids, whereas other classes of lipids, presumably present in the other compartments, were abundant in C20 and C22 polyunsaturated fatty acids. This suggests that chromatophores are metabolically distinct from the rest of the cell. Metabolic studies using isotopically labeled substrates showed that different fatty acids are synthesized in the chromatophore and the cytosol, which is consistent with the presence of both type I and type II fatty acid synthases, supposedly present in the cytosol and the chromatophore, respectively. Nevertheless, rapid labeling of the fatty acids in triacylglycerol and phosphatidylcholine by photosynthetically fixed carbon suggested that the chromatophores efficiently provide metabolites to the host. The metabolic and ultrastructural evidence suggests that chromatophores are tightly integrated into the whole cellular metabolism.

The conversion of free-living cyanobacteria to photosynthetic organelles of eukaryotic cells through endosymbiosis transformed the biosphere and eventually provided the basis for life on land. Despite the presumable advantage conferred by the acquisition of photoautotrophy through endosymbiosis, only two independent cases of primary endosymbiosis have been documented: one that gave rise to the Archaeplastida, and the other to photosynthetic species of the thecate, filose amoeba Paulinella. Here, we review recent genomics-informed insights into the primary endosymbiotic origins of cyanobacteria-derived organelles. Furthermore, we discuss the preconditions for the evolution of nitrogen-fixing organelles. Recent genomic data on previously undersampled cyanobacterial and protist taxa provide new clues to the origins of the host cell and endosymbiont, and proteomic approaches allow insights into the rearrangement of the endosymbiont proteome during organellogenesis. We conclude that in addition to endosymbiotic gene transfers, horizontal gene acquisitions from a broad variety of prokaryotic taxa were crucial to organelle evolution.

The thecate filose amoeba Paulinella chromatophora is a good model organism for understanding plastid organellogenesis because its chromatophore was newly derived from an alpha-cyanobacterium. Paulinella chromatophora was the only known photosynthetic Paulinella species until recent studies that suggested a species level of diversity. Here, we described a new photosynthetic species P. micropora sp. nov. based on morphological and molecular evidence from a newly established strain KR01. The chromatophore genome of P. micropora KR01 was fully determined; the genome was 976,991bp in length, the GC content was 39.9%, and 908 genes were annotated. A pairwise comparison of chromatophore genome sequences between strains KR01 and FK01, representing two different natural populations of P. micropora, showed a 99.85% similarity. Differences between the two strains included single nucleotide polymorphisms (SNPs) in CDSs, which resulted in 357 synonymous and 280 nonsynonymous changes, along with 245 SNPs in non-coding regions. Indels (37) and microinversions (14) were also detected. Species diversity for photosynthetic Paulinella was surveyed using samples collected from around the world. We compared our new species to two photosynthetic species, P. chromatophora and P. longichromatophora. Phylogenetic analyses using four gene markers revealed three distinct lineages of photosynthetic Paulinella species including P. micropora sp. nov.

Chloroplasts in photosynthetic eukaryotes originated from a cyanobacterial endosymbiosis far more than 1 billion years ago1-3. Due to this ancientness, it remains unclear how this evolutionary process proceeded. To unveil this mystery, we analysed the whole genome sequence of a photosynthetic rhizarian amoeba4, Paulinella micropora5,6, which has a chloroplast-like organelle that originated from another cyanobacterial endosymbiosis7-10 about 0.1 billion years ago11. Here we show that the predacious amoeba that engulfed cyanobacteria evolved into a photosynthetic organism very quickly in the evolutionary time scale, probably aided by the drastic genome reorganization activated by large DNA virus. In the endosymbiotic evolution of eukaryotic cells, gene transfer from the endosymbiont genome to the host nucleus is essential for the evolving host cell to control the endosymbiont-derived organelle12. In P. micropora, we found that the gene transfer from the free-living and endosymbiotic bacteria to the amoeba nucleus was rapidly activated but both simultaneously ceased within the initiation period of the endosymbiotic evolution, suggesting that the genome reorganization drastically proceeded and completed. During this period, large DNA virus appeared to have infected the amoeba, followed by the rapid amplification and diversification of virus-related genes. These findings led us to re-examine the conventional endosymbiotic evolutionary scenario that exclusively deals with the host and the symbiont, and to extend it by incorporating a third critical player, large DNA virus, which activates the drastic gene transfer and genome reorganization between them. This Paulinella version of the evolutionary hypothesis deserves further testing of its generality in evolutionary systems and could shed light on the unknown roles of large DNA viruses13 in the evolution of terrestrial life.

The widespread algal and plant (Archaeplastida) plastid originated >1 billion years ago, therefore relatively little can be learned about plastid integration during the initial stages of primary endosymbiosis by studying these highly derived species. Here we focused on a unique model for endosymbiosis research, the photosynthetic amoeba Paulinella micropora KR01 (Rhizaria) that underwent a more recent independent primary endosymbiosis about 124 Mya. A total of 149 Gbp of PacBio and 19 Gbp of Illumina data were used to generate the draft assembly that comprises 7,048 contigs with N50=143,028 bp and a total length of 707 Mbp. Genome GC-content was 44% with 76% repetitive sequences. We predicted 32,358 genes that contain 73% of the complete, conserved genes in the BUSCO database. The mean intron length was 882 bp, which is significantly greater than in other Rhizaria (86∼184 bp). Symbiotic bacteria from the culture were isolated and completed genomes were generated from three species (Mesorhizobium amorphae Pch-S, Methylibium petroeiphilum Pch-M, Polaromonas sp. Pch-P) with one draft genome (Pimelobacter simplex Pch-N). Our holobiont data establish P. micropora KR01 as a model for studying plastid integration and the role of bacterial symbionts in Paulinella biology.

Plastids evolved from a cyanobacterium that was engulfed by a heterotrophic eukaryotic host and became a stable organelle. Some of the resulting eukaryotic algae entered into a number of secondary endosymbioses with diverse eukaryotic hosts. These events had major consequences on the evolution and diversification of life on Earth. Although almost all plastid diversity derives from a single endosymbiotic event, analysis of nuclear genomes of plastid-bearing lineages has revealed a mosaic origin of plastid-related genes. In addition to cyanobacterial genes, plastids recruited for their functioning eukaryotic proteins encoded by the host nucleus and also bacterial proteins of non-cyanobacterial origin. Thus, plastid proteins and plastid-localized metabolic pathways evolved by tinkering using the gene toolkits provided by the cyanobacterial endosymbiont, its eukaryotic host, and other bacteria. This mixed heritage seems especially complex in secondary algae containing green plastids, the acquisition of which appears to have been facilitated by many previous acquisitions of red algal genes (the “red carpet hypothesis”).

Summary An amoeba named Paulinella harbours ‘chromatophores', cyanobacterium-derived photosynthetic bodies that evolved independent of plastids. Proteomics has shown that hundreds of nucleus-encoded proteins are targeted to the chromatophore, revealing the host cell's contributions to its recently established organelle.

Symbioses between phototrophs and heterotrophs (a.k.a 'photosymbioses') are extremely common, and range from loose and temporary associations to obligate and highly specialized forms. In the history of life, the most transformative was the 'primary endosymbiosis,' wherein a cyanobacterium was engulfed by a eukaryote and became genetically integrated as a heritable photosynthetic organelle, or plastid. By allowing the rise of algae and plants, this event dramatically altered the biosphere, but its remote origin over one billion years ago has obscured the sequence of events leading to its establishment. Here, we review the genetic, physiological and developmental hurdles involved in early primary endosymbiosis. Since we cannot travel back in time to witness these evolutionary junctures, we will draw on examples of unicellular eukaryotes (protists) spanning diverse modes of photosymbiosis. We also review experimental approaches that could be used to recreate aspects of early primary endosymbiosis on a human timescale.

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5′ end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5′ end sequence of 28–33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution.

© 2017, National Academy of Sciences. All rights reserved. The early evolutionary history of the chloroplast lineage remains an open question. It is widely accepted that the endosymbiosis that established the chloroplast lineage in eukaryotes can be traced back to a single event, in which a cyanobacterium was incorporated into a protistan host. It is still unclear, however, which Cyanobacteria are most closely related to the chloroplast, when the plastid lineage first evolved, and in what habitats this endosymbiotic event occurred. We present phylogenomic and molecular clock analyses, including data from cyanobacterial and chloroplast genomes using a Bayesian approach, with the aim of estimating the age for the primary endosymbiotic event, the ages of crown groups for photosynthetic eukaryotes, and the independent incorporation of a cyanobacterial endosymbiont by Paulinella. Our analyses include both broad taxon sampling (119 taxa) and 18 fossil calibrations across all Cyanobacteria and photosynthetic eukaryotes. Phylogenomic analyses support the hypothesis that the chloroplast lineage diverged from its closet relative Gloeomargarita, a basal cyanobacterial lineage, ~2.1 billion y ago (Bya). Our analyses suggest that the Archaeplastida, consisting of glaucophytes, red algae, green algae, and land plants, share a common ancestor that lived ~1.9 Bya. Whereas crown group Rhodophyta evolved in the Mesoproterozoic Era (1,600–1,000 Mya), crown groups Chlorophyta and Streptophyta began to radiate early in the Neoproterozoic (1,000–542 Mya). Stochastic mapping analyses indicate that the first endosymbiotic event occurred in low-salinity environments. Both red and green algae colonized marine environments early in their histories, with prasinophyte green phytoplankton diversifying 850–650 Mya.

The endosymbiotic acquisition of mitochondria and plastids more than 1 Ga ago profoundly impacted eukaryote evolution. At the heart of understanding organelle evolution is the re-arrangement of the endosymbiont proteome into a host-controlled organellar proteome. However, early stages in this process as well as the timing of events that underlie organelle integration remain poorly understood. The amoeba Paulinella chromatophora contains cyanobacterium-derived photosynthetic organelles, termed "chromatophores," that were acquired more recently (around 100 Ma ago). To explore the re-arrangement of an organellar proteome during its integration into a eukaryotic host cell, here we characterized the chromatophore proteome by protein mass spectrometry. Apparently, genetic control over the chromatophore has shifted substantially to the nucleus. Two classes of nuclear-encoded proteins-which differ in protein length-are imported into the chromatophore, most likely through independent pathways. Long imported proteins carry a putative, conserved N-terminal targeting signal, and many specifically fill gaps in chromatophore-encoded metabolic pathways or processes. Surprisingly, upon heterologous expression in a plant cell, the putative chromatophore targeting signal conferred chloroplast localization. This finding suggests common features in the protein import pathways of chromatophores and plastids, two organelles that evolved independently and more than 1 Ga apart from each other. By combining experimental data with in silico predictions, we provide a comprehensive catalog of almost 450 nuclear-encoded, chromatophore-targeted proteins. Interestingly, most imported proteins seem to derive from ancestral host genes, suggesting that the re-targeting of nuclear-encoded proteins that resulted from endosymbiotic gene transfers plays only a minor role at the onset of chromatophore integration.

The study of diseased human cells and of cells isolated from the natural environment will likely be revolutionized by single cell genomics (SCG). Here, we used protein similarity networks to explore within- and between-cell DNA differences from SCG data derived from six individual rhizarian cells related to Paulinella ovalis and proteins from the complete genome of another rhizarian, Bigelowiella natans. We identified shared and distinct DNA components within our SCG data and between P. ovalis and B. natans. We show that network properties such as assortativity and degree effectively discriminate genome features between SCG assemblies and that SCG data follow the power law with a small number of protein families dominating networks.

First-generation Sanger DNA sequencing revolutionized science over the past three decades and the current next-generation sequencing (NGS) technology has opened the doors to the next phase in the sequencing revolution. Using NGS, scientists are able to sequence entire genomes and to generate extensive transcriptome data from diverse photosynthetic eukaryotes in a timely and cost-effective manner. Genome data in particular shed light on the complicated evolutionary history of algae that form the basis of the food chain in many environments. In the Eukaryotic Tree of Life, the fact that photosynthetic lineages are positioned in four supergroups has important evolutionary consequences. We now know that the story of eukaryotic photosynthesis unfolds with a primary endosymbiosis between an ancestral heterotrophic protist and a captured cyanobacterium that gave rise to the glaucophytes, red algae and Viridiplantae (green algae and land plants). These primary plastids were then transferred to other eukaryotic groups through secondary endosymbiosis. A red alga was captured by the ancestor(s) of the stramenopiles, alveolates (dinoflagellates, apicomplexa, chromeridae), cryptophytes and haptophytes, whereas green algae were captured independently by the common ancestors of the euglenophytes and chlorarachniophytes. A separate case of primary endosymbiosis is found in the filose amoeba Paulinella chromatophora, which has at least nine heterotrophic sister species. Paulinella genome data provide detailed insights into the early stages of plastid establishment. Therefore, genome data produced by NGS have provided many novel insights into the taxonomy, phylogeny and evolutionary history of photosynthetic eukaryotes.

Eukaryotes co-opted photosynthetic carbon fixation from prokaryotes by engulfing a cyanobacterium and stably integrating it as a photosynthetic organelle (plastid) in a process known as primary endosymbiosis. The sheer complexity of interactions between a plastid and the surrounding cell that started to evolve over 1 billion years ago, make it challenging to reconstruct intermediate steps in organelle evolution by studying extant plastids. Recently, the photosynthetic amoeba Paulinella chromatophora was identified as a much sought-after intermediate stage in the evolution of a photosynthetic organelle. This article reviews the current knowledge on this unique organism. In particular it describes how the interplay of reductive genome evolution, gene transfers, and trafficking of host-encoded proteins into the cyanobacterial endosymbiont contributed to transform the symbiont into a nascent photosynthetic organelle. Together with recent results from various other endosymbiotic associations a picture emerges that lets the targeting of host-encoded proteins into bacterial endosymbionts appear as an early step in the establishment of an endosymbiotic relationship that enables the host to gain control over the endosymbiont.

The rhizarian amoeba Paulinella chromatophora harbors two photosynthetic cyanobacterial endosymbionts (chromatophores), acquired independently of primary plastids of glaucophytes, red algae and green plants. These endosymbionts have lost many essential genes, and transferred substantial number of genes to the host nuclear genome via endosymbiotic gene transfer (EGT), including those involved in photosynthesis. This indicates that, similar to primary plastids, Paulinella endosymbionts must have evolved a transport system to import their EGT-derived proteins. This system involves vesicular trafficking to the outer chromatophore membrane and presumably a simplified Tic-like complex at the inner chromatophore membrane. Since both sequenced Paulinella strains have been shown to undergo differential plastid gene losses, they do not have to possess the same set of Toc and Tic homologs. We searched the genome of Paulinella FK01 strain for potential Toc and Tic homologs, and compared the results with the data obtained for Paulinella CCAC 0185 strain, and 72 cyanobacteria, eight Archaeplastida as well as some other bacteria. Our studies revealed that chromatophore genomes from both Paulinella strains encode the same set of translocons that could potentially create a simplified but fully-functional Tic-like complex at the inner chromatophore membranes. The common maintenance of the same set of translocon proteins in two Paulinella strains suggests a similar import mechanism and/or supports the proposed model of protein import. Moreover, we have discovered a new putative Tic component, Tic62, a redox sensor protein not identified in previous comparative studies of Paulinella translocons.