Into the genome: advances in the world of algal genomics

The widespread photosynthetic organelle (plastid) originated ca. 1.6 billion years ago in a heterotrophic protist via primary endosymbiosis involving a beta-cyanobacterium. Evidence will be presented that generally supports the single origin of this plastid in the common ancestor of the Archaeplastida, with some recent insights into the putative branching order of the three phyla in this kingdom. The only other known case of primary plastid origin is in the clade comprised of photosynthetic rhizarian amoebae, Paulinella. Members of this genus contain a photosynthetic organelle, referred to as a chromatophore that is derived from an alpha-cyanobacterial endosymbiont. I will present the results of our analysis of Paulinella cultures and of significant genome and transcriptome data that was generated from Paulinella chromatophora and other species in this genus. Our goal was to gain insights into the process of primary plastid establishment. We find that P. chromatophora is light sensitive and has undergone large-scale duplication of EGT-derived HLI genes that are involved in adaptation to high light. Phylogenomic analysis uncovered extensive recruitment of bacterium-derived genes that appear to counteract gene losses from the chromatophore due to Muller’s ratchet. Because nearly all of the genes still encoded on the chromatophore genome are of cyanobacterial affiliation, we postulate that HGTs from non-endosymbiont sources was key to chromatophore survival. This suggests that phagotrophy was likely maintained after plastid origin to facilitate HGT from prey or other symbiotic bacteria. How these insights inform us about the process of organellogenesis will be discussed.

Red algae (Rhodophyta) include more than 7,000 species thrive in marine and freshwater habitats. Red algae play a critical role in the eukaryote tree of life as the donor through secondary endosymbiosis of the plastid that subsequently gave rise to chlorophyll-c containing groups such as diatoms, dinoflagellates, haptophytes, and cryptophytes. However, only limited genome data are available from economically important red algal species. Here, we report the complete nuclear genome of the agarophyte Gracilariopsis chorda and present genome comparisons with other algal species. We also assembled the plastid and mitochondrial genomes from the largest dataset including 45 from the red algae (17 novel plastid genomes) to elucidate the evolution of these organelles. We find extreme conservation of plastid and mitochondrial genome structures in the major lineages of Florideophyceae. Only three minor structural types were detected in this group that are explained by recombination events of the duplicated rRNA operons. A similar high level of structural conservation (however, with different gene content) was found in seed plants. Three major plastid genome structures were identified from representatives of 46 orders of angiosperms and 3 orders of the gymnosperms. Our results provide a comprehensive accounting of plastid gene loss and genome rearrangement events within Archaeplastida and lead to one major conclusion. From a pool of highly rearranged plastid genomes in red and green algae, the aquatic (Florideophyceae) and terrestrial (seed plants) multicellular lineages display extreme conservation in plastid genome architecture. We speculate that the origin of complex reproductive tissue types in red seaweeds and in seed plants likely set in place developmental pathways linking the nuclear and organelle genomes that constrained evolution of the photosynthetic compartment.

Chloroplast genomes, relics of an ancient endosymbiotic cyanobacterial genome, are typically circular, double-stranded DNA molecules. Their size usually ranges between 100-200 kb and they encode 100-200 genes. While multipartite architectures of mitochondrial genomes evolved several times independently during the evolution of eukaryotes, fragmented plastid genomes are only known in dinoflagellates. Here we report on the chloroplast structure of the Cladophorales, an order of green seaweeds. Based on a combination of short NGS reads, long sequencing reads and RNA-seq, we conclude that they do not possess a single circular chloroplast genome but a genome fragmented into multiple molecules, 2-3 kb in size, each containing one protein-coding gene. In that aspect, the chloroplast genome of the Cladophorales somewhat resembles dinoflagellates chloroplast minicircles. However, in Cladophorales the molecules are linear single-stranded molecules that fold onto themselves in a hairpin-like structure due to the presence of extensive inverted repeats. So far we assembled 13 hairpins, each harbouring a distinct full-length chloroplast gene (e.g. atpA, atpB, atpH, atpI, petA, petB, psaA, psaC, psbA, psbB, psbC, psbD, rbcL). RNA-seq analysis confirmed that these genes are transcribed and produce functional transcripts. Phylogenetic analyses indicate that the genes are more rapidly evolving compared to chloroplast genes of other green algae. As far as known such a chloroplast structure is unique among eukaryotes.

Dinoflagellates are an important group of primary producers and grazers, predominantly found in the marine environment. Almost all dinoflagellates are free-living (including species that cause harmful algal blooms); Symbiodinium, the only symbiotic lineage, is associated with cnidarian hosts (e.g. corals and jellyfish) and diverse coral reef animals. The origin of plastid in dinoflagellates can be traced back to multiple, serial events of endosymbiosis, during which genetic material could have been transferred from the engulfed endosymbiont (of prokaryote and eukaryote origins) to the host nucleus. The frequency and functional impact of exogenous gene recruitment in dinoflagellates are known to be as great as in prokaryotes, impacting some of the most fundamental traits. Due to its remarkable complexity, evolution of dinoflagellate genomes is sometimes considered as the “worst-case” scenario in microbial eukaryotes. Furthermore, dinoflagellate genomes are huge — often estimated 20–230 Gbp — and they exhibit anomalous characteristics e.g. non‐canonical nucleotides and unusual intron‐exon splice signals; sequencing and assembly of these genomes remain a challenge. Symbiodinium genomes are orders of magnitude smaller (estimated 1.2–3Gbp) than their free-living counterparts, thus providing a practical, initial analysis platform for dinoflagellate genomics. Two genomes of Symbiodinium were recently published, with more dinoflagellate genomes expected to become available. Here I will present our recent findings from the analysis of three Symbiodinium genomes and their biological implications for our understanding of dinoflagellate (and algal) evolution. Technical challenges of sequencing dinoflagellate genomes and future perspectives will also be discussed.

Chlamydiales were recently proposed to have sheltered the future cyanobacterial ancestor of plastids in a common inclusion. The intracellular pathogens are thought to have donated those critical transporters that triggered the efflux of photosynthetic carbon and the consequent onset of symbiosis. Chlamydiales are also suspected to have encoded glycogen metabolism TTS (Type Three Secretion) effectors responsible for photosynthetic carbon assimilation in the eukaryotic cytosol.

We now review the reasons underlying other chlamydial lateral gene transfers evidenced in the descendants of plastid endosymbiosis. In particular, we show that half of the genes encoding enzymes of tryptophan synthesis in Archaeplastida are of chlamydial origin. Tryptophan is known to define an essential cue triggering two alternative modes of replication in Chlamydiales. In addition, sophisticated tryptophan starvation mechanisms are known to have been implemented as antibacterial defences by their eukaryotic hosts. We propose that Chlamydiales have donated their tryptophan operon to the emerging plastid to ensure increased synthesis of tryptophan by the plastid ancestor. This would have allowed massive expression of the tryptophan rich chlamydial transporters responsible for symbiosis. It would also have allowed possible export of this valuable amino-acid in the inclusion of the tryptophan hungry pathogens. Free-living single cell cyanobacteria are devoid of proteins able to transport this amino-acid. We therefore investigated the phylogeny of the E.coli Tyr/Trp transporters and found yet another LGT from Chlamydiales to Archaeplastida thereby considerably strengthening our proposal.

Using powerful bioinformatics tools and comparative genomics is allowing us to identify novel components of photosynthesis. This information will help elucidate new photosynthetic functions and provide opportunities for engineering plants and algae for efficient solar energy utilization, increased agricultural outputs and improved resiliency to changing global environments.  The GreenCut represents an informatics assemblage of nuclear-encoded proteins that are conserved among photosynthetic organisms of the green lineage (Viridiplantae), but that are either not present, or poorly conserved in heterotrophic (nonphotosynthetic) organisms. Many uncharacterized GreenCut proteins (unknown specific functions) appear to have regulatory functions or roles in the biogenesis of the photosynthetic apparatus based on specific domains in their protein sequences. Other GreenCut proteins have no domains that are currently informative with respect to function. Generating insertional mutants of genes encoding GreenCut proteins in Chlamydomonas reinhardtii has allowed for the characterization of mutant phenotypes, suggesting roles for a number of these proteins with respect to photosynthetic activities. One GreenCut protein was recently shown to be involved in stabilizing the assembly of photosystem I (PSI) under oxic conditions, and is potentially involved in protecting oxygen sensitive PSI cofactors (e.g. Fe-S clusters) during the assembly process; as the Earth became oxygenated mechanisms must have evolved that protect existing oxygen sensitive cofactors from disruption. Other novel proteins of the GreenCut appear to be important for the biogenesis/stability of the cytochrome b6f complex. I will discuss the GreenCut and the ways in which it is being used to examine photosynthetic function and evolution.

Marine macroalgae are host to a wide range of prokaryotic and eukaryotic life, creating a dynamic and complex community of specialists or generalists that can be beneficial (mutualistic), neutral (commensal) or harmful (parasitic) organisms. Bacteria are the dominant active group that make up the microbiome, and macroalgal-bacterial studies suggest that there is a core microbiome at the phylum level consisting of the Gammaproteobacteria, Bacteroidetes, Alphaproteobacteria, Firmicutes and Actinobacteria.  Until relatively recently, studies of bacteria associated with various macroalgal hosts were undertaken using cultures and electron microscopy. Next generation sequencing (NGS) is now revolutionising the subject and revealing the extent of bacterial diversity in these microbiomes. This talk will review what is known about microbiomes of marine macroalgae with a particular focus on the red algae.  It will explore the notion of a core microbiome for different host groups and also the spatial and temporal ecological impact on host photosynthesis of different types of epiphytes.  Focussing in particular on the prokaryote component of the microbiome, the nature of the relationships between the bacteria and host and the implications for ecosystem function and environmental change including ocean acidification and increasing sea surface temperatures will be explored.  Drawing upon our results of the microbiome of Corallina officinalis – the first for a geniculate coralline alga – differences in microbiome composition will be compared both within and between fleshy and calcified species. Evidence that the epiphytic bacteria provide important services to hosts that are vital to their health, performance and resilience will be discussed. Ways forward to identify prokaryote and eukaryote diversity, to understand their roles in productivity, and the overall nature of these relationships will be explored.

Marine viruses that infect marine microorganisms are recognized as major ecological and evolutionary driving forces, shaping community structure and controlling cycling of nutrient energy in the marine environment. A major challenge in our current understanding of host-virus interactions in the marine environment is to decode the wealth of genomic and metagenomic data and translate it into cellular mechanisms that mediate host susceptibility and resistance to viral infection. Nevertheless, the cellular mechanisms that govern these host-virus dynamics are largely underexplored. Recent reports highlighted a novel genomic inventory found in marine viruses which can encode for auxiliary metabolic genes previously thought to be restricted to their host genomes. Thus, these genes can expand the metabolic capabilities of the infected host cell (Virocell) and the flux of the nutrients and metabolites between the cell and its micro-environment.

Emiliania huxleyi is a globally important coccolithophore forming massive algal blooms in the North Atlantic Ocean that are routinely infected and terminated by large DNA viruses (EhVs) belong to the coccolithoviruses. We explore the molecular and metabolic basis for these host-virus dynamics and the signal transduction pathways that mediate host-virus interactions. By combining genome-enabled technologies, analytical chemistry and advance cell imaging approaches, we were able to identify several fundamental metabolic pathways that mediate these host-virus interactions. We revealed the role of viral-encoded sphingolipid biosynthesis, redox and DMS metabolism and their function in determining host cell fate (e.g. PCD and autophagy) and viral replication strategies.

Pythium porphyrae is responsible for devastating outbreaks in seaweed farms of Pyropia, the most valuable seaweed worldwide. While the genus Pythium contains many well studied plant and animal pathogens, the infection strategies and genome content of P. porphyrae remains to be elucidated.  Recent reports also indicated the ability of P.porphyrae to infect, colonize and reproduce on a great variety of land plants, begging the question of its potential land origin, as well as the molecular mechanisms underpinning its host specificity. Here, we used RNA sequencing to provide the first description of P. porphyrae gene repertoire and assess its similarity to fully sequenced Pythiums. Using ab-initio detection strategies, similarity based and manual annotation, we found that the P.porphyrae gene repertoire is strikingly similar to the ones described for classical phytopathogenic Pythium species, including Crinklers, elicitins, cellulases, CBEL-like proteins and a total absence of RxLR effectors. Comparative genomics revealed that 1507 genes, including CBEL-like proteins and elicitins, have orthologs in some plant infecting Pythiums but not in the animal pathogen P.insidiosum. Despite 34% of the P.porphyrae proteome having no orthologs in other sequenced Pythiums, we could not identify any enzyme involved in the degradation of red algal-specific cell wall components. Complementary infection experiments indicated that P.porphyrae is specific of Bangiales, contrasting with the general broad host range observed on land plants, perhaps suggesting a recent adaptation linked with the development of Pyropia cultivation.

Fouling is a paramount phenomenon in the marine environment. However, surfaces of certain seaweeds like the brown alga Fucus vesciculosus, remain relatively free from heavy fouling and are covered by a thin film of epibiotic microorganisms. We found that Fucus in a given habitat harbors a distinct epibacterial community. Fucus also produces defence compounds like fucoxanthin, DMSP reducing bacterial settlement. These compounds have also been found to be strain specific in their action, thus probably assisting Fucus in ‘gardening’ a distinct bacterial community on its surface. This idea was further supported by a study that correlated the surface concentration of the defence compounds with the presence or absence of different bacterial clades. Several bacterial groups were found to be positively or negatively affected by the compounds present on surfaces of Fucus individuals. Seaweeds also account for a substantial proportion of all introduced species. The East Asian red macroalga Gracilaria vermiculophylla has successfully invaded several temperate areas of the Northern hemisphere. Although foulers have the potential to determine invasion success or failure of invasive seaweeds, this perspective has been ignored so far. We tested whether the impressive invasion success of Gracilaria may be enhanced by a rapid adaptation of defence against potentially facultative new target microfoulers in the invaded range. Native and invasive Gracilaria populations were equally well defended against presently co-occurring bacterial foulers. However, native populations were weakly defended against bacteria from the invaded range, while invasive populations were weakly defended against bacteria from the native range. Thus, the invasive populations exhibited an adaptation of their defence capacity to cope with the new foes, but have lost capacity to fend off old foes. These results provide the first evidence that confrontation by new foulers can trigger a rapid defence adaptation of aquatic weeds, which could be necessary for algal invasiveness.