Targeted CRISPR-Cas9-based gene knockouts in the model brown alga Ectocarpus.

Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology, but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes. Here, we report that mutations at specific target sites are generated following the introduction of CRISPR-Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method. Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE (APT) gene were isolated following treatment with 2-fluoroadenine, and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism. The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.

The microbiome of the habitat-forming brown alga Fucus vesiculosus (Phaeophyceae) has similar cross-Atlantic structure that reflects past and present drivers1.

Latitudinal diversity gradients have provided many insights into species differentiation and community processes. In the well-studied intertidal zone, however, little is known about latitudinal diversity in microbiomes associated with habitat-forming hosts. We investigated microbiomes of Fucus vesiculosus because of deep understanding of this model system and its latitudinally large, cross-Atlantic range. Given multiple effects of photoperiod, we predicted that cross-Atlantic microbiomes of the Fucus microbiome would be similar at similar latitudes and correlate with environmental factors. We found that community structure and individual amplicon sequencing variants (ASVs) showed distinctive latitudinal distributions, but alpha diversity did not. Latitudinal differentiation was mostly driven by ASVs that were more abundant in cold temperate to subarctic (e.g., Granulosicoccus_t3260, Burkholderia/Caballeronia/Paraburkholderia_t8371) or warm temperate (Pleurocapsa_t10392) latitudes. Their latitudinal distributions correlated with different humidity, tidal heights, and air/sea temperatures, but rarely with irradiance or photoperiod. Many ASVs in potentially symbiotic genera displayed novel phylogenetic biodiversity with differential distributions among tissues and regions, including closely related ASVs with differing north-south distributions that correlated with Fucus phylogeography. An apparent southern range contraction of F. vesiculosus in the NW Atlantic on the North Carolina coast mimics that recently observed in the NE Atlantic. We suggest cross-Atlantic microbial structure of F. vesiculosus is related to a combination of past (glacial-cycle) and contemporary environmental drivers.

Unusual Patterns of Mitochondrial Inheritance in the Brown Alga Ectocarpus.

Most eukaryotes inherit their mitochondria from only one of their parents. When there are different sexes, it is almost always the maternal mitochondria that are transmitted. Indeed, maternal uniparental inheritance has been reported for the brown alga Ectocarpus but we show in this study that different strains of Ectocarpus can exhibit different patterns of inheritance: Ectocarpus siliculosus strains showed maternal uniparental inheritance, as expected, but crosses using different Ectocarpus species 7 strains exhibited either paternal uniparental inheritance or an unusual pattern of transmission where progeny inherited either maternal or paternal mitochondria, but not both. A possible correlation between the pattern of mitochondrial inheritance and male gamete parthenogenesis was investigated. Moreover, in contrast to observations in the green lineage, we did not detect any change in the pattern of mitochondrial inheritance in mutant strains affected in life cycle progression. Finally, an analysis of field-isolated strains provided evidence of mitochondrial genome recombination in both Ectocarpus species.

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

A local score approach improves GWAS resolution and detects minor QTL: application to Medicago truncatula quantitative disease resistance to multiple Aphanomyces euteiches isolates.

Quantitative trait loci (QTL) with small effects, which are pervasive in quantitative phenotypic variation, are difficult to detect in genome-wide association studies (GWAS). To improve their detection, we propose to use a local score approach that accounts for the surrounding signal due to linkage disequilibrium, by accumulating association signals from contiguous single markers. Simulations revealed that, in a GWAS context with high marker density, the local score approach outperforms single SNP p-value-based tests for detecting minor QTL (heritability of 5-10%) and is competitive with regard to alternative methods, which also aggregate p-values. Using more than five million SNPs, this approach was applied to identify loci involved in Quantitative Disease Resistance (QDR) to different isolates of the plant root rot pathogen Aphanomyces euteiches, from a GWAS performed on a collection of 174 accessions of the model legume Medicago truncatula. We refined the position of a previously reported major locus, underlying MYB/NB-ARC/tyrosine kinase candidate genes conferring resistance to two closely related A. euteiches isolates belonging to pea pathotype I. We also discovered a diversity of minor resistance QTL, not detected using p-value-based tests, some of which being putatively shared in response to pea (pathotype I and III) and/or alfalfa (race 1 and 2) isolates. Candidate genes underlying these QTL suggest pathogen effector recognition and plant proteasome as key functions associated with M. truncatula resistance to A. euteiches. GWAS on any organism can benefit from the local score approach to uncover many weak-effect QTL.

Rediscovering Zygorhizidium affluens Canter: Molecular Taxonomy, Infectious Cycle, and Cryopreservation of a Chytrid Infecting the Bloom-Forming Diatom Asterionella formosa.

Parasitic Chytridiomycota (chytrids) are ecologically significant in various aquatic ecosystems, notably through their roles in controlling bloom-forming phytoplankton populations and in facilitating the transfer of nutrients from inedible algae to higher trophic levels. The diversity and study of these obligate parasites, while critical to understand the interactions between pathogens and their hosts in the environment, have been hindered by challenges inherent to their isolation and stable long-term maintenance under laboratory conditions. Here, we isolated an obligate chytrid parasite (CCAP 4086/1) on the freshwater bloom-forming diatom Asterionella formosa and characterized its infectious cycle under controlled conditions. Phylogenetic analyses based on 18S, 5.8S, and 28S ribosomal DNAs (rDNAs) revealed that this strain belongs to the recently described clade SW-I within the Lobulomycetales. All morphological features observed agree with the description of the known Asterionella parasite Zygorhizidium affluens Canter. We thus provide a phylogenetic placement for this chytrid and present a robust and simple assay that assesses both the infection success and the viability of the host. We also validate a cryopreservation method for stable and cost-effective long-term storage and demonstrate its recovery after thawing. All the above-mentioned tools establish a new gold standard for the isolation and long-term preservation of parasitic aquatic chytrids, thus opening new perspectives to investigate the diversity of these organisms and their physiology in a controlled laboratory environment.IMPORTANCE Despite their ecological relevance, parasitic aquatic chytrids are understudied, especially due to the challenges associated with their isolation and maintenance in culture. Here we isolated and established a culture of a chytrid parasite infecting the bloom-forming freshwater diatom Asterionella formosa The chytrid morphology suggests that it corresponds to the Asterionella parasite known as Zygorhizidium affluens The phylogenetic reconstruction in the present study supports the hypothesis that our Z. affluens isolate belongs to the order Lobulomycetales and clusters within the novel clade SW-I. We also validate a cryopreservation method for stable and cost-effective long-term storage of parasitic chytrids of phytoplankton. The establishment of a monoclonal pathosystem in culture and its successful cryopreservation opens the way to further investigate this ecologically relevant parasitic interaction.

High-density genome-wide association mapping implicates an F-box encoding gene in Medicago truncatula resistance to Aphanomyces euteiches.

• The use of quantitative disease resistance (QDR) is a promising strategy for promoting durable resistance to plant pathogens, but genes involved in QDR are largely unknown. To identify genetic components and accelerate improvement of QDR in legumes to the root pathogen Aphanomyces euteiches, we took advantage of both the recently generated massive genomic data for Medicago truncatula and natural variation of this model legume. • A high-density (≈5.1 million single nucleotide polymorphisms (SNPs)) genome-wide association study (GWAS) was performed with both in vitro and glasshouse phenotyping data collected for 179 lines. • GWAS identified several candidate genes and pinpointed two independent major loci on the top of chromosome 3 that were detected in both phenotyping methods. Candidate SNPs in the most significant locus (σ(A)²= 23%) were in the promoter and coding regions of an F-box protein coding gene. Subsequent qRT-PCR and bioinformatic analyses performed on 20 lines demonstrated that resistance is associated with mutations directly affecting the interaction domain of the F-box protein rather than gene expression. • These results refine the position of previously identified QTL to specific candidate genes, suggest potential molecular mechanisms, and identify new loci explaining QDR against A. euteiches.

Repeated co-option of HMG-box genes for sex determination in brown algae and animals.

In many eukaryotes, genetic sex determination is not governed by XX/XY or ZW/ZZ systems but by a specialized region on the poorly studied U (female) or V (male) sex chromosomes. Previous studies have hinted at the existence of a dominant male-sex factor on the V chromosome in brown algae, a group of multicellular eukaryotes distantly related to animals and plants. The nature of this factor has remained elusive. Here, we demonstrate that an HMG-box gene acts as the male-determining factor in brown algae, mirroring the role HMG-box genes play in sex determination in animals. Over a billion-year evolutionary timeline, these lineages have independently co-opted the HMG box for male determination, representing a paradigm for evolution's ability to recurrently use the same genetic "toolkit" to accomplish similar tasks.

Role and Evolution of the Extracellular Matrix in the Acquisition of Complex Multicellularity in Eukaryotes: A Macroalgal Perspective.

Multicellular eukaryotes are characterized by an expanded extracellular matrix (ECM) with a diversified composition. The ECM is involved in determining tissue texture, screening cells from the outside medium, development, and innate immunity, all of which are essential features in the biology of multicellular eukaryotes. This review addresses the origin and evolution of the ECM, with a focus on multicellular marine algae. We show that in these lineages the expansion of extracellular matrix played a major role in the acquisition of complex multicellularity through its capacity to connect, position, shield, and defend the cells. Multiple innovations were necessary during these evolutionary processes, leading to striking convergences in the structures and functions of the ECMs of algae, animals, and plants.

Production and Bioassay of a Diffusible Factor That Induces Gametophyte-to-Sporophyte Developmental Reprogramming in the Brown Alga Ectocarpus.

The brown alga Ectocarpus has a haploid-diploid life cycle that involves alternation between two multicellular generations, the sporophyte and the gametophyte. Life cycle generation is not determined by ploidy but by a genetic system that includes two different three amino acid loop extension homeodomain transcription factors called OUROBOROS and SAMSARA. In addition, sporophytes have been shown to secrete a diffusible factor into the medium that can induce gametophyte initial cells to switch from the gametophyte to the sporophyte developmental program. The protocol presented here describes how to produce sporophyte-conditioned medium containing the diffusible sporophyte-inducing factor and how to assay for activity of the factor using a meio-spore-based bioassay. The protocol, which describes how several steps of these procedures can be optimised, will represent a useful tool for future work aimed at characterising the diffusible factor and investigating its mode of action.

Chytrid infecting the bloom-forming marine diatom Skeletonema sp.: Morphology, phylogeny and distribution of a novel species within the Rhizophydiales.

Chytrids have long been recognised as important parasites of microalgae in freshwater systems, able to shape the dynamics of blooms, the gene pool of their host and phytoplankton succession. In the sea however, where the presence of these organisms is erratic and ephemeral, studies concerning chytrids are sparse and confined to metabarcoding surveys or microscopy observations. Despite the scarcity of data, chytrid epidemics are supposed to play an important role in marine biogeochemical cycles, being one of the drivers of phytoplankton dynamics. Here we combine microscopy observations and in silico mining of a single-cell whole genome to molecularly and morphologically characterise a novel chytrid parasite of the dominant diatom genus Skeletonema. Morphological observations highlight features of the thallus and ascertain the parasitic nature of the interaction whilst the genetic markers obtained allows for a phylogenetic reconstruction, placing the new species in the order Rhizophydiales. Thanks to the molecular data obtained we are also able to provide a first investigation of the global distribution of this organism by screening the Ocean Sampling Day (OSD) dataset, highlighting a northern transatlantic dissemination.

Novel Widespread Marine Oomycetes Parasitising Diatoms, Including the Toxic Genus Pseudo-nitzschia: Genetic, Morphological, and Ecological Characterisation.

Parasites are key drivers of phytoplankton bloom dynamics and related aquatic ecosystem processes. Yet, the dearth of morphological and molecular information hinders the assessment of their diversity and ecological role. Using single-cell techniques, we characterise morphologically and molecularly, intracellular parasitoids infecting four potentially toxin-producing Pseudo-nitzschia and one Melosira species on the North Atlantic coast. These sequences define two, morphologically indistinguishable clades within the phylum Oomycota, related to the genera of algal parasites Anisolpidium and Olpidiopsis and the diatom parasitoid species Miracula helgolandica. Our morphological data are insufficient to attribute either clade to the still unsequenced genus Ectrogella; hence it is proposed to name the clades OOM_1 and OOM_2. A screening of global databases of the barcode regions V4 and V9 of the 18S rDNA demonstrate the presence of these parasitoids beyond the North Atlantic coastal region. During a biweekly metabarcoding survey (Concarneau Bay, France), reads associated with one sequenced parasitoid coincided with the decline of Cerataulina pelagica bloom, whilst the other parasitoids co-occurred at low abundance with Pseudo-nitzschia. Our data highlight a complex and unexplored diversity of the oomycete parasitoids of diatoms and calls for the investigation of their phenology, evolution, and potential contribution in controlling their host spatial-temporal dynamics.

Transcriptome analysis highlights preformed defences and signalling pathways controlled by the prAe1 quantitative trait locus (QTL), conferring partial resistance to Aphanomyces euteiches in Medicago truncatula.

To gain an insight into the molecular mechanisms of quantitative disease resistance in Medicago truncatula to the root-infecting oomycete Aphanomyces euteiches, we selected two near-isogenic lines (NILs), NR and NS, partially resistant and susceptible, respectively, differing in the allelic state of the quantitative resistance locus (QRL) prAe1 (partially resistant to A. euteiches 1). Complementary molecular and cytological phenotyping methods showed that prAe1 alone confers quantitative resistance to A. euteiches. Root and stem tissues were colonized in NS plants and 80% of NS plants died by 21 days post-inoculation (dpi). In contrast, A. euteiches mycelium was restricted to the root cortex and the spread of symptoms was arrested in aerial parts of NR plants. A transcriptome analysis performed at 0, 1 and 6 dpi identified 1198 differentially expressed genes (DEGs) between NR and NS lines. More than 87% of the DEGs were significantly more expressed in NR. The highest number of DEGs was found in control conditions, with 723 genes over-expressed in NR versus 85 in NS. Genes belonging to secondary metabolism, pathogenesis-related (PR) proteins and kinases were significantly enriched. The significant role of the flavonoid pathway in resistance was corroborated by the detection of larger amounts of flavonoids in NR roots and the inhibition of A. euteiches zoospore germination by 2'-O-methyl-isoliquiritigenin, a compound synthesized by enzymes specifically induced in NR. Our study revealed that prAe1-dependent resistance relies mainly on the constitutive expression of defence-related pathways and signalling elements, which can be re-amplified in later time points of the infection.