Convergent recruitment of TALE homeodomain life cycle regulators to direct sporophyte development in land plants and brown algae.

Three amino acid loop extension homeodomain transcription factors (TALE HD TFs) act as life cycle regulators in green algae and land plants. In mosses these regulators are required for the deployment of the sporophyte developmental program. We demonstrate that mutations in either of two TALE HD TF genes, OUROBOROS or SAMSARA, in the brown alga Ectocarpus result in conversion of the sporophyte generation into a gametophyte. The OUROBOROS and SAMSARA proteins heterodimerise in a similar manner to TALE HD TF life cycle regulators in the green lineage. These observations demonstrate that TALE-HD-TF-based life cycle regulation systems have an extremely ancient origin, and that these systems have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata.

Chemically-Mediated Interactions Between Macroalgae, Their Fungal Endophytes, and Protistan Pathogens.

Filamentous fungi asymptomatically colonize the inner tissues of macroalgae, yet their ecological roles remain largely underexplored. Here, we tested if metabolites produced by fungal endophytes might protect their host against a phylogenetically broad spectrum of protistan pathogens. Accordingly, the cultivable fungal endophytes of four brown algal species were isolated and identified based on LSU and SSU sequencing. The fungal metabolomes were tested for their ability to reduce the infection by protistan pathogens in the algal model Ectocarpus siliculosus. The most active metabolomes effective against the oomycetes Eurychasma dicksonii and Anisolpidium ectocarpii, and the phytomixid Maullinia ectocarpii were further characterized chemically. Several pyrenocines isolated from Phaeosphaeria sp. AN596H efficiently inhibited the infection by all abovementioned pathogens. Strikingly, these compounds also inhibited the infection of nori (Pyropia yezoensis) against its two most devastating oomycete pathogens, Olpidiopsis pyropiae, and Pythium porphyrae. We thus demonstrate that fungal endophytes associated with brown algae produce bioactive metabolites which might confer protection against pathogen infection. These results highlight the potential of metabolites to finely-tune the outcome of molecular interactions between algae, their endophytes, and protistan pathogens. This also provide proof-of-concept toward the applicability of such metabolites in marine aquaculture to control otherwise untreatable diseases.

Not in your usual Top 10: protists that infect plants and algae.

Fungi, nematodes and oomycetes belong to the most prominent eukaryotic plant pathogenic organisms. Unicellular organisms from other eukaryotic lineages, commonly addressed as protists, also infect plants. This review provides an introduction to plant pathogenic protists, including algae infecting oomycetes, and their current state of research.

The Ectocarpus IMMEDIATE UPRIGHT gene encodes a member of a novel family of cysteine-rich proteins with an unusual distribution across the eukaryotes.

The sporophyte generation of the brown alga Ectocarpus sp. exhibits an unusual pattern of development compared with the majority of brown algae. The first cell division is symmetrical and the apical-basal axis is established late in development. In the immediate upright (imm) mutant, the initial cell undergoes an asymmetric division to immediately establish the apical-basal axis. We provide evidence which suggests that this phenotype corresponds to the ancestral state of the sporophyte. The IMM gene encodes a protein of unknown function that contains a repeated motif also found in the EsV-1-7 gene of the Ectocarpus virus EsV-1. Brown algae possess large families of EsV-1-7 domain genes but these genes are rare in other stramenopiles, suggesting that the expansion of this family might have been linked with the emergence of multicellular complexity. EsV-1-7 domain genes have a patchy distribution across eukaryotic supergroups and occur in several viral genomes, suggesting possible horizontal transfer during eukaryote evolution.

A new flagellated dispersion stage in Paraphysoderma sedebokerense, a pathogen of Haematococcus pluvialis.

The blastocladialean fungus Paraphysoderma sedebokerense Boussiba, Zarka and James is a devastating pathogen of the commercially valuable green microalga Haematococcus pluvialis, a natural source of the carotenoid pigment astaxanthin. First identified in commercial Haematococcus cultivation facilities, P. sedebokerense is hypothesised to have a complex life cycle that switches between a vegetative and a resting phase depending on favourable or unfavourable growth conditions. Rather unusually for blastocladialean fungi, P. sedebokerense was described as lacking flagellated zoospores and only propagating via aplanosporic amoeboid cells. However, during repeated microscopic observation of P. sedebokerense cultivated in optimal conditions, we detected fast-swimming, transiently uniflagellated zoospores which rapidly transform into infectious amoeboid swarmers, the existence of which suggests a closer than previously thought relatedness of P. sedebokerense to its sister genera Physoderma and Urophlyctis. Additionally, we found some morphological and physiological differences between amoeboid swarmers and discuss hypotheses about their significance. These amoeboid and flagellated propagules are key to the dissemination of P. sedebokerense and are probably also the life stages most vulnerable to adverse environmental conditions. They are therefore a prime target for the development of disease management protocols in industrial cultivation facilities, a goal which requires a detailed understanding of their physiology.

Arctic marine phytobenthos of northern Baffin Island.

Global climate change is expected to alter the polar bioregions faster than any other marine environment. This study assesses the biodiversity of seaweeds and associated eukaryotic pathogens of an established study site in northern Baffin Island (72° N), providing a baseline inventory for future work assessing impacts of the currently ongoing changes in the Arctic marine environment. A total of 33 Phaeophyceae, 24 Rhodophyceae, 2 Chlorophyceae, 12 Ulvophyceae, 1 Trebouxiophyceae, and 1 Dinophyceae are reported, based on collections of an expedition to the area in 2009, complemented by unpublished records of Robert T. Wilce and the first-ever photographic documentation of the phytobenthos of the American Arctic. Molecular barcoding of isolates raised from incubated substratum samples revealed the presence of 20 species of brown seaweeds, including gametophytes of kelp and of a previously unsequenced Desmarestia closely related to D. viridis, two species of Pylaiella, the kelp endophyte Laminariocolax aecidioides and 11 previously unsequenced species of the Ectocarpales, highlighting the necessity to include molecular techniques for fully unraveling cryptic algal diversity. This study also includes the first records of Eurychasma dicksonii, a eukaryotic pathogen affecting seaweeds, from the American Arctic. Overall, this study provides both the most accurate inventory of seaweed diversity of the northern Baffin Island region to date and can be used as an important basis to understand diversity changes with climate change.

Infection of the brown alga Ectocarpus siliculosus by the oomycete Eurychasma dicksonii induces oxidative stress and halogen metabolism.

Pathogens are increasingly being recognized as key evolutionary and ecological drivers in marine ecosystems. Defence mechanisms of seaweeds, however, have mostly been investigated by mimicking infection using elicitors. We have established an experimental pathosystem between the genome brown model seaweed Ectocarpus siliculosus and the oomycete Eurychasma dicksonii as a powerful new tool to investigate algal responses to infection. Using proteomics, we identified 21 algal proteins differentially accumulated in response to Eu. dicksonii infection. These include classical algal stress response proteins such as a manganese superoxide dismutase, heat shock proteins 70 and a vanadium bromoperoxidase. Transcriptional profiling by qPCR confirmed the induction of the latter during infection. The accumulation of hydrogen peroxide was observed at different infection stages via histochemical staining. Inhibitor studies confirmed that the main source of hydrogen peroxide is superoxide converted by superoxide dismutase. Our data give an unprecedented global overview of brown algal responses to pathogen infection, and highlight the importance of oxidative stress and halogen metabolism in these interactions. This suggests overlapping defence pathways with herbivores and abiotic stresses. We also identify previously unreported actors, in particular a Rad23 and a plastid-lipid-associated protein, providing novel insights into the infection and defence processes in brown algae.

microRNAs and the evolution of complex multicellularity: identification of a large, diverse complement of microRNAs in the brown alga Ectocarpus.

There is currently convincing evidence that microRNAs have evolved independently in at least six different eukaryotic lineages: animals, land plants, chlorophyte green algae, demosponges, slime molds and brown algae. MicroRNAs from different lineages are not homologous but some structural features are strongly conserved across the eukaryotic tree allowing the application of stringent criteria to identify novel microRNA loci. A large set of 63 microRNA families was identified in the brown alga Ectocarpus based on mapping of RNA-seq data and nine microRNAs were confirmed by northern blotting. The Ectocarpus microRNAs are highly diverse at the sequence level with few multi-gene families, and do not tend to occur in clusters but exhibit some highly conserved structural features such as the presence of a uracil at the first residue. No homologues of Ectocarpus microRNAs were found in other stramenopile genomes indicating that they emerged late in stramenopile evolution and are perhaps specific to the brown algae. The large number of microRNA loci in Ectocarpus is consistent with the developmental complexity of many brown algal species and supports a proposed link between the emergence and expansion of microRNA regulatory systems and the evolution of complex multicellularity.

Saturating light and not increased carbon dioxide under ocean acidification drives photosynthesis and growth in Ulva rigida (Chlorophyta).

Carbon physiology of a genetically identified Ulva rigida was investigated under different CO2(aq) and light levels. The study was designed to answer whether (1) light or exogenous inorganic carbon (Ci) pool is driving growth; and (2) elevated CO2(aq) concentration under ocean acidification (OA) will downregulate CAext-mediated [Formula: see text] dehydration and alter the stable carbon isotope (δ (13)C) signatures toward more CO2 use to support higher growth rate. At pHT 9.0 where CO2(aq) is <1 µmol L(-1), inhibition of the known [Formula: see text] use mechanisms, that is, direct [Formula: see text] uptake through the AE port and CAext-mediated [Formula: see text] dehydration decreased net photosynthesis (NPS) by only 56-83%, leaving the carbon uptake mechanism for the remaining 17-44% of the NPS unaccounted. An in silico search for carbon-concentrating mechanism elements in expressed sequence tag libraries of Ulva found putative light-dependent [Formula: see text] transporters to which the remaining NPS can be attributed. The shift in δ (13)C signatures from -22‰ toward -10‰ under saturating light but not under elevated CO2(aq) suggest preference and substantial [Formula: see text] use to support photosynthesis and growth. U. rigida is Ci saturated, and growth was primarily controlled by light. Therefore, increased levels of CO2(aq) predicted for the future will not, in isolation, stimulate Ulva blooms.

Ligulate Desmarestia (Desmarestiales, Phaeophyceae) revisited: D. japonica sp. nov. and D. dudresnayi differ from D. ligulata.

The phylogeny of ligulate and sulfuric-acid containing species of Desmarestia, occurring worldwide from polar to temperate regions, was revised using a multigenic and polyphasic approach. Sequence data, gametophyte characteristics, and sporophyte morphology support reducing a total of 16 taxa to four different species. (1) D. herbacea, containing broad-bladed and highly branched forms, has dioecious gametophytes. The three other species have monoecious gametophytes: (2) D. ligulata which is profusely branched and, except for one subspecies, narrow-bladed, (3) Japanese ligulate Desmarestia, here described as D. japonica sp. nov., which is morphologically similar to D. ligulata but genetically distant from all other ligulate taxa. This species may have conserved the morphology of original ligulate Desmarestia. (4) D. dudresnayi, including unbranched or little branched broad-bladed taxa. A figure of the holotype of D. dudresnayi, which was lost for decades, was relocated. The taxonomy is complemented by a comparison of internal transcribed spacer and cytochrome c oxidase subunit I (cox1) as potential barcode loci, with cox1 offering good resolution, reflecting species delimitations within the genus Desmarestia.

Intracellular eukaryotic pathogens in brown macroalgae in the Eastern Mediterranean, including LSU rRNA data for the oomycete Eurychasma dicksonii .

For the Mediterranean Sea, and indeed most of the world's oceans, the biodiversity and biogeography of eukaryotic pathogens infecting marine macroalgae remains poorly known, yet their ecological impact is probably significant. Based on 2 sampling campaigns on the Greek island of Lesvos in 2009 and 1 in northern Greece in 2012, this study provides first records of 3 intracellular eukaryotic pathogens infecting filamentous brown algae at these locations: Eurychasma dicksonii, Anisolpidium sphacellarum, and A. ectocarpii. Field and microscopic observations of the 3 pathogens are complemented by the first E. dicksonii large subunit ribosomal RNA (LSU rRNA) gene sequence analyses of isolates from Lesvos and other parts of the world. The latter highlights the monophyly of E. dicksonii worldwide and confirms the basal position of this pathogen within the oomycete lineage (Peronosporomycotina). The results of this study strongly support the notion that the geographic distribution of the relatively few eukaryotic seaweed pathogens is probably much larger than previously thought and that many of the world's marine bioregions remain seriously undersampled and understudied in this respect.

Gene silencing in Fucus embryos: developmental consequences of RNAi-mediated cytoskeletal disruption.

Brown algae (Phaeophyceae) are an important algal class that play a range of key ecological roles. They are often important components of rocky shore communities. A number of members of the Fucales and Ectocarpales have provided models for the study of multicellular evolution, reproductive biology and polarized development. Indeed the fucoid algae exhibit the unusual feature of inducible embryo polarization, allowing many classical studies of polarity induction. The potential of further studies of brown algae in these important areas has been increasingly hindered by the absence of tools for manipulation of gene expression that would facilitate further mechanistic analysis and gene function studies at a molecular level. The aim of this study was to establish a method that would allow the analysis of gene function through RNAi-mediated gene knockdown. We show that injection of double-stranded RNA (dsRNA) corresponding to an α-tubulin gene into Fucus serratus Linnaeus zygotes induces the loss of a large proportion of the microtubule cytoskeleton, leading to growth arrest and disruption of cell division. Injection of dsRNA targeting ß-actin led to reduced rhizoid growth, enlarged cells and the failure to develop apical hair cells. The silencing effect on actin expression was maintained for 3 months. These results indicate that the Fucus embryo possesses a functional RNA interference system that can be exploited to investigate gene function during embryogenesis.

A molecular insight into algal-oomycete warfare: cDNA analysis of Ectocarpus siliculosus infected with the basal oomycete Eurychasma dicksonii.

Brown algae are the predominant primary producers in coastal habitats, and like land plants are subject to disease and parasitism. Eurychasma dicksonii is an abundant, and probably cosmopolitan, obligate biotrophic oomycete pathogen of marine brown algae. Oomycetes (or water moulds) are pathogenic or saprophytic non-photosynthetic Stramenopiles, mostly known for causing devastating agricultural and aquacultural diseases. Whilst molecular knowledge is restricted to crop pathogens, pathogenic oomycetes actually infect hosts from most eukaryotic lineages. Molecular evidence indicates that Eu. dicksonii belongs to the most early-branching oomycete clade known so far. Therefore Eu. dicksonii is of considerable interest due to its presumed environmental impact and phylogenetic position. Here we report the first large scale functional molecular data acquired on the most basal oomycete to date. 9873 unigenes, totalling over 3.5 Mb of sequence data, were produced from Sanger-sequenced and pyrosequenced EST libraries of infected Ectocarpus siliculosus. 6787 unigenes (70%) were of algal origin, and 3086 (30%) oomycete origin. 57% of Eu. dicksonii sequences had no similarity to published sequence data, indicating that this dataset is largely unique. We were unable to positively identify sequences belonging to the RXLR and CRN groups of oomycete effectors identified in higher oomycetes, however we uncovered other unique pathogenicity factors. These included putative algal cell wall degrading enzymes, cell surface proteins, and cyclophilin-like proteins. A first look at the host response to infection has also revealed movement of the host nucleus to the site of infection as well as expression of genes responsible for strengthening the cell wall, and secretion of proteins such as protease inhibitors. We also found evidence of transcriptional reprogramming of E. siliculosus transposable elements and of a viral gene inserted in the host genome.

Vertical distribution of epibenthic freshwater cyanobacterial Synechococcus spp. strains depends on their ability for photoprotection.

BACKGROUND: Epibenthic cyanobacteria often grow in environments where the fluctuation of light intensity and quality is extreme and frequent. Different strategies have been developed to cope with this problem depending on the distribution of cyanobacteria in the water column. PRINCIPAL FINDINGS: Here we provide an experimental proof that the light intensity plays an important role in the vertical distribution of seven, closely related, epibenthic Synechococcus spp. strains isolated from various water depths from the littoral zone of Lake Constance in Germany and cultivated under laboratory conditions. Pigment analysis revealed that the amount of chlorophyll a and total carotenoids decreased with the time of light stress exposure in three phycoerythrin-rich strains collected from 7.0 m water depth and remained low during the recovery phase. In contrast, a constant level of chlorophyll a and either constant or enhanced levels of carotenoids were assayed in phycocyanin-rich strains collected from 1.0 and 0.5 m water depths. Protein analysis revealed that while the amount of biliproteins remained constant in all strains during light stress and recovery, the amount of D1 protein from photosystem II reaction centre was strongly reduced under light stress conditions in strains from 7.0 m and 1.0 m water depth, but not in strains collected from 0.5 m depth. CONCLUSION: Based on these data we propose that light intensity, in addition to light quality, is an important selective force in the vertical distribution of Synechococcus spp. strains, depending on their genetically fixed mechanisms for photoprotection.

Algal diseases: spotlight on a black box.

Like any other living organisms, algae are plagued by diseases caused by fungi, protists, bacteria or viruses. As aquaculture continues to rise worldwide, pathogens of nori or biofuel sources are becoming a significant economic burden. Parasites are also increasingly being considered of equal importance with predators for ecosystem functioning. Altered disease patterns in disturbed environments are blamed for sudden extinctions, regime shifts, and spreading of alien species. Here we review the biodiversity and impact of pathogens and parasites of aquatic primary producers in freshwater and marine systems. We also cover recent advances on algal defence reactions, and discuss how emerging technologies can be used to reassess the profound, multi-faceted, and so far broadly-overlooked influence of algal diseases on ecosystem properties.

Detection of differential host susceptibility to the marine oomycete pathogen Eurychasma dicksonii by real-time PCR: not all algae are equal.

In the marine environment, a growing body of evidence points to parasites as key players in the control of population dynamics and overall ecosystem structure. However, their prevalence and impact on marine macroalgal communities remain virtually unknown. Indeed, infectious diseases of seaweeds are largely underdocumented, partly because of the expertise required to diagnose them with a microscope. Over the last few years, however, real-time quantitative PCR (qPCR) has emerged as a rapid and reliable alternative to visual symptom scoring for monitoring pathogens. Thus, we present here a qPCR assay suitable for the detection and quantification of the intracellular oomycete pathogen Eurychasma dicksonii in its ectocarpalean and laminarialean brown algal hosts. qPCR and microscopic observations made of laboratory-controlled cultures revealed that clonal brown algal strains exhibit different levels of resistance against Eurychasma, ranging from high susceptibility to complete absence of symptoms. This observation strongly argues for the existence of a genetic determinism for disease resistance in brown algae, which would have broad implications for the dynamics and genetic structure of natural populations. We also used qPCR for the rapid detection of Eurychasma in filamentous brown algae collected in Northern Europe and South America and found that the assay is specific, robust, and widely applicable to field samples. Hence, this study opens the perspective of combining large-scale disease monitoring in the field with laboratory-controlled experiments on the genome model seaweed Ectocarpus siliculosus to improve our understanding of brown algal diseases.

Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of photosynthesis and nitrogen fixation.

* As iron (Fe) deficiency is a main limiting factor of ocean productivity, its effects were investigated on interactions between photosynthesis and nitrogen fixation in the marine nonheterocystous diazotrophic cyanobacterium Trichodesmium IMS101. * Biophysical methods such as fluorescence kinetic microscopy, fast repetition rate (FRR) fluorimetry, and in vivo and in vitro spectroscopy of pigment composition were used, and nitrogenase activity and the abundance of key proteins were measured. * Fe limitation caused a fast down-regulation of nitrogenase activity and protein levels. By contrast, the abundance of Fe-requiring photosystem I (PSI) components remained constant. Total levels of phycobiliproteins remained unchanged according to single-cell in vivo spectra. However, the regular 16-kDa phycoerythrin band decreased and finally disappeared 16-20 d after initiation of Fe limitation, concomitant with the accumulation of a 20-kDa protein cross-reacting with the phycoerythrin antibody. Concurrently, nitrogenase expression and activity increased. Fe limitation dampened the daily cycle of photosystem II (PSII) activity characteristic of diazotrophic Trichodesmium cells. Further, it increased the number and prolonged the time period of occurrence of cells with elevated basic fluorescence (F(0)). Additionally, it increased the effective cross-section of PSII, probably as a result of enhanced coupling of phycobilisomes to PSII, and led to up-regulation of the Fe stress protein IsiA. * Trichodesmium survives short-term Fe limitation by selectively down-regulating nitrogen fixation while maintaining but re-arranging the photosynthetic apparatus.