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Phytoplankton-virus interactions are major determinants of geochemical cycles in the oceans. Viruses are responsible for the redirection of carbon and nutrients away from larger organisms back towards microorganisms via the lysis of microalgae in a process coined the "viral shunt". Virus-host interactions are generally expected to follow "boom and bust" dynamics, whereby a numerically dominant strain is lysed and replaced by a virus resistant strain. Here, we isolated a microalga and its infective nucleo-cytoplasmic large DNA virus (NCLDV) concomitantly from the environment in the surface NW Mediterranean Sea, Ostreococcus mediterraneus, and show continuous growth in culture of both the microalga and the virus. Evolution experiments through single cell bottlenecks demonstrate that, in the absence of the virus, susceptible cells evolve from one ancestral resistant single cell, and vice-versa; that is that resistant cells evolve from one ancestral susceptible cell. This provides evidence that the observed sustained viral production is the consequence of a minority of virus-susceptible cells. The emergence of these cells is explained by low-level phase switching between virus-resistant and virus-susceptible phenotypes, akin to a bet hedging strategy. Whole genome sequencing and analysis of the ~14 Mb microalga and the ~200 kb virus points towards ancient speciation of the microalga within the Ostreococcus species complex and frequent gene exchanges between prasinoviruses infecting Ostreococcus species. Re-sequencing of one susceptible strain demonstrated that the phase switch involved a large 60 Kb deletion of one chromosome. This chromosome is an outlier chromosome compared to the streamlined, gene dense, GC-rich standard chromosomes, as it contains many repeats and few orthologous genes. While this chromosome has been described in three different genera, its size increments have been previously associated to antiviral immunity and resistance in another species from the same genus. Mathematical modelling of this mechanism predicts microalga-virus population dynamics consistent with the observation of continuous growth of both virus and microalga. Altogether, our results suggest a previously overlooked strategy in phytoplankton-virus interactions.

Understanding factors that generate, maintain, and constrain host-parasite associations is of major interest to biologists. Although little studied, many extremely virulent micro-eukaryotic parasites infecting microalgae have been reported in the marine plankton. This is the case for Amoebophrya, a diverse and highly widespread group of Syndiniales infecting and potentially controlling dinoflagellate populations. Here, we analyzed the time-scale gene expression of a complete infection cycle of two Amoebophrya strains infecting the same host (the dinoflagellate Scrippsiella acuminata), but diverging by their host range (one infecting a single host, the other infecting more than one species). Over two-thirds of genes showed two-fold differences in expression between at least two sampled stages of the Amoebophrya life cycle. Genes related to carbohydrate metabolism as well as signaling pathways involving proteases and transporters were overexpressed during the free-living stage of the parasitoid. Once inside the host, all genes related to transcription and translation pathways were actively expressed, suggesting the rapid and extensive protein translation needed following host-cell invasion. Finally, genes related to cellular division and components of the flagellum organization were overexpressed during the sporont stage. In order to gain a deeper understanding of the biological basis of the host-parasitoid interaction, we screened proteins involved in host-cell recognition, invasion, and protection against host-defense identified in model apicomplexan parasites. Very few of the genes encoding critical components of the parasitic lifestyle of apicomplexans could be unambiguously identified as highly expressed in Amoebophrya. Genes related to the oxidative stress response were identified as highly expressed in both parasitoid strains. Among them, the correlated expression of superoxide dismutase/ascorbate peroxidase in the specialist parasite was consistent with previous studies on Perkinsus marinus defense. However, this defense process could not be identified in the generalist Amoebophrya strain, suggesting the establishment of different strategies for parasite protection related to host specificity.

Background Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired-end reads from Ostreococcus tauri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture. Results The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq Illumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate-receptor like gene. Conclusion High coverage (>80 fold) paired-end Illumina sequencing enables a high quality 95% complete genome assembly of a compact ~13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1103) contains supplementary material, which is available to authorized users.

International audience; Background: Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired end reads from Ustreococcus touri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture. Results: The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq II lumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate receptor like gene. Conclusion: High coverage (>80 fold) paired end Illumina sequencing enables a high quality 95% complete genome assembly of a compact 13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture.

The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation.

Cell division depends on the correct localization of the cyclin-dependent kinases that are regulated by phosphorylation, cyclin proteolysis, and protein-protein interactions. Although immunological assays can define cell cycle protein abundance and localization, they are not suitable for detecting the dynamic rearrangements of molecular components during cell division. Here, we applied an in vivo approach to trace the subcellular localization of 60 Arabidopsis (Arabidopsis thaliana) core cell cycle proteins fused to green fluorescent proteins during cell division in tobacco (Nicotiana tabacum) and Arabidopsis. Several cell cycle proteins showed a dynamic association with mitotic structures, such as condensed chromosomes and the preprophase band in both species, suggesting a strong conservation of targeting mechanisms. Furthermore, colocalized proteins were shown to bind in vivo, strengthening their localization-function connection. Thus, we identified unknown spatiotemporal territories where functional cell cycle protein interactions are most likely to occur.

Protein ubiquitination is a posttranslational regulatory process essential for plant growth and interaction with the environment. E3 ligases, to which the seven in absentia (SINA) proteins belong, determine the specificity by selecting the target proteins for ubiquitination. SINA proteins are found in animals as well as in plants, and a small gene family with highly related members has been identified in the genome of rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), Medicago truncatula, and poplar (Populus trichocarpa). To acquire insight into the function of SINA proteins in nodulation, a dominant negative form of the Arabidopsis SINAT5 was ectopically expressed in the model legume M. truncatula. After rhizobial inoculation of the 35S:SINAT5DN transgenic plants, fewer nodules were formed than in control plants, and most nodules remained small and white, a sign of impaired symbiosis. Defects in rhizobial infection and symbiosome formation were observed by extensive microscopic analysis. Besides the nodulation phenotype, transgenic plants were affected in shoot growth, leaf size, and lateral root number. This work illustrates a function for SINA E3 ligases in a broad spectrum of plant developmental processes, including nodulation.

We report the draft genome sequence of the model moss Physcomitrella patens and compare its features with those of flowering plants, from which it is separated by more than 400 million years, and unicellular aquatic algae. This comparison reveals genomic changes concomitant with the evolutionary movement to land, including a general increase in gene family complexity; loss of genes associated with aquatic environments (e.g., flagellar arms); acquisition of genes for tolerating terrestrial stresses (e.g., variation in temperature and water availability); and the development of the auxin and abscisic acid signaling pathways for coordinating multicellular growth and dehydration response. The Physcomitrella genome provides a resource for phylogenetic inferences about gene function and for experimental analysis of plant processes through this plant's unique facility for reverse genetics.

Aquatic nodulation on the tropical legume Sesbania rostrata occurs at lateral root bases via intercellular crack-entry invasion. A gene was identified (Srprx1) that is transiently up-regulated during the nodulation process and codes for a functional class III plant peroxidase. The expression strictly depended on bacterial nodulation factors (NFs) and could be modulated by hydrogen peroxide, a downstream signal for crack-entry invasion. Expression was not induced after wounding or pathogen attack, indicating that the peroxidase is a symbiosis-specific isoform. In situ hybridization showed Srprx1 transcripts around bacterial infection pockets and infection threads until they reached the central tissue of the nodule. A root nodule extensin (SrRNE1) colocalized with Srprx1 both in time and space and had the same NF requirement, suggesting a function in a similar process. Finally, in mixed inoculation nodules that were invaded by NF-deficient bacteria and differed in infection thread progression, infection-associated peroxidase transcripts were not observed. Lack of Srprx1 gene expression could be one of the causes for the aberrant structure of the infection threads.

The identification of promoters and their regulatory elements is one of the major challenges in bioinformatics and integrates comparative, structural, and functional genomics. Many different approaches have been developed to detect conserved motifs in a set of genes that are either coregulated or orthologous. However, although recent approaches seem promising, in general, unambiguous identification of regulatory elements is not straightforward. The delineation of promoters is even harder, due to its complex nature, and in silico promoter prediction is still in its infancy. Here, we review the different approaches that have been developed for identifying promoters and their regulatory elements. We discuss the detection of cis-acting regulatory elements using word-counting or probabilistic methods (so-called “search by signal” methods) and the delineation of promoters by considering both sequence content and structural features (“search by content” methods). As an example of search by content, we explored in greater detail the association of promoters with CpG islands. However, due to differences in sequence content, the parameters used to detect CpG islands in humans and other vertebrates cannot be used for plants. Therefore, a preliminary attempt was made to define parameters that could possibly define CpG and CpNpG islands in Arabidopsis, by exploring the compositional landscape around the transcriptional start site. To this end, a data set of more than 5,000 gene sequences was built, including the promoter region, the 5′-untranslated region, and the first introns and coding exons. Preliminary analysis shows that promoter location based on the detection of potential CpG/CpNpG islands in the Arabidopsis genome is not straightforward. Nevertheless, because the landscape of CpG/CpNpG islands differs considerably between promoters and introns on the one side and exons (whether coding or not) on the other, more sophisticated approaches can probably be developed for the successful detection of “putative” CpG and CpNpG islands in plants.

While the molecular events involved in cell responses to heat stress have been extensively studied, our understanding of the genetic basis of basal thermotolerance, and particularly its evolution within the green lineage, remains limited. Here, we present the 13.3-Mb haploid genome and transcriptomes of a halotolerant and thermotolerant unicellular green alga, Picochlorum costavermella (Trebouxiophyceae) to investigate the evolution of the genomic basis of thermotolerance. Differential gene expression at high and standard temperatures revealed that more of the gene families containing up-regulated genes at high temperature were recently evolved, and less originated at the ancestor of green plants. Inversely, there was an excess of ancient gene families containing transcriptionally repressed genes. Interestingly, there is a striking overlap between the thermotolerance and halotolerance transcriptional rewiring, as more than one-third of the gene families up-regulated at 35 degrees C were also up-regulated under variable salt concentrations in Picochlorum SE3. Moreover, phylogenetic analysis of the 9,304 protein coding genes revealed 26 genes of horizontally transferred origin in P. costavermella, of which five were differentially expressed at higher temperature. Altogether, these results provide new insights about how the genomic basis of adaptation to halo- and thermotolerance evolved in the green lineage.

As part of the European Scientists Sequencing Arabidopsis program, a contiguous region (396 607 bp) located on chromosome 4 around the APETALA2 gene was sequenced. Analysis of the sequence and comparison to public databases predicts 103 genes in this area, which represents a gene density of one gene per 3.85 kb. Almost half of the genes show no significant homology to known database entries. In addition, the first 45 kb of the contig, which covers 11 genes, is similar to a region on chromosome 2, as far as coding sequences are concerned. This observation indicates that ancient duplications of large pieces of DNA have occurred in Arabidopsis.

Background: The moss Physcomitrella patens as a model species provides an important reference for early-diverging lineages of plants and the release of the genome in 2008 opened the doors to genome-wide studies. The usability of a reference genome greatly depends on the quality of the annotation and the availability of centralized community resources. Therefore, in the light of accumulating evidence for missing genes, fragmentary gene structures, false annotations and a low rate of functional annotations on the original release, we decided to improve the moss genome annotation. Results: Here, we report the complete moss genome re-annotation (designated V1.6) incorporating the increased transcript availability from a multitude of developmental stages and tissue types. We demonstrate the utility of the improved P. patens genome annotation for comparative genomics and new extensions to the cosmoss.org resource as a central repository for this plant "flagship" genome. The structural annotation of 32,275 protein-coding genes results in 8387 additional loci including 1456 loci with known protein domains or homologs in Plantae. This is the first release to include information on transcript isoforms, suggesting alternative splicing events for at least 10.8% of the loci. Furthermore, this release now also provides information on non-protein-coding loci. Functional annotations were improved regarding quality and coverage, resulting in 58% annotated loci (previously: 41%) that comprise also 7200 additional loci with GO annotations. Access and manual curation of the functional and structural genome annotation is provided via the www.cosmoss.org model organism database. Conclusions: Comparative analysis of gene structure evolution along the green plant lineage provides novel insights, such as a comparatively high number of loci with 5'-UTR introns in the moss. Comparative analysis of functional annotations reveals expansions of moss house-keeping and metabolic genes and further possibly adaptive, lineage-specific expansions and gains including at least 13% orphan genes.