Despite their major ecological role at the base of the food chain in oceanic ecosystems, the variation in phytoplankton-virus interactions as a function of abiotic factors is under studied. Indeed, efforts have focused on estimating the extraordinary interspecific diversity of these communities populating the surface of our oceans. While previous studies on phytoplankton x virus x environment interactions have analysed the effects of temperature, a key factor of climate change, there is a lack of knowledge about the effect of other abiotic factors, also impacted by global change, such as variations in nutrients and salinity. The objective of the ELVIRA project is to understand how these abiotic factors influence the phytoplankton-virus interaction quantitatively and qualitatively. This is in order to (1) characterize the phenotypic evolution, (2) identify the genomic and transcriptomic bases of these phenotypic changes and (3) establish a predictive model of the dynamics of the phytoplankton-virus system. We propose to deploy an interdisciplinary approach combining population genomics, high-throughput experimental phenotyping and modeling on an ecologically relevant phytoplankton-virus system with a worldwide distribution. In order to meet the ambitious objective of integrating the genome x phenotype x environment levels, ELVIRA brings together an international consortium composed of two French teams and a German team with complementary skills in population genomics, phytoplankton eco-physiology and theoretical biology. The ideal location of the partners in the immediate vicinity of the North, the Baltic and the Mediterranean Seas allows us to take advantage of the natural spatial variation in salinity and phosphate concentration between these sites. This lets us test whether the geographical origin impacts the genomic and phenotypic variation of microalgae isolated from these different environments, including their susceptibility to different viruses from these same sites. The project’s deliverables arise from four tasks carried out in close collaboration between the 3 teams. First, a sampling effort in the North, Baltic and Mediterranean Seas will increase the number of microalgae in the collection previously established by the consortium. This biological resource is the basis of the ELVIRA's genomic resource consisting of transcriptomes and genomes by long and short read technologies for the whole collection. Task 2 will be devoted to high-throughput phenotyping of this collection, with an exceptional exploration of the space of phytoplankton x virus x environment interactions for different conditions of salinity and phosphate concentrations. Task 3 will allow the characterization of the phenotype x genotype linkage through association analyses (GWAS) between the genomic and phenotypic resources obtained. We will first associate nucleotide variants with variations in gene expression between strains (eQTL). Then, we will associate genomic variants (nucleotide and structural) with phenotypes, with particular attention to cases of changes in phytoplankton-virus susceptibility as a function of abiotic variables. Finally, Task 4 will be devoted to the integration of the obtained phenotype x environment space into a multi-host multi-virus epidemiological model in order to predict population dynamics under current and future conditions of salinity and phosphate concentration. We believe the expected results from the analysis of these genome x phenotype x environment interactions in an original biological system will have implications for integrative biology of host-parasite interactions, population and evolutionary genomics, microbiology and integrative molecular-to-ecosystem approaches.

Environmental constraints like rising temperature and virus infections have major impacts on living organisms. Plants, as other species, develop various panels of mechanisms to cope with the effects of these stresses on growth and survival. Redox homeostasis is among the key players of cellular metabolism and cell responses to environmental constraints. It is sensitive to environmental changes and can signal these changes to response pathways, for example by modifying redox status of thiols residues in proteins. In eukaryotic cells, small RNAs (siRNA and miRNA) are major regulators for gene expression, involved in most developmental and stress response processes. The biogenesis of small RNAs is orchestrated by RNaseIII endonuclease enzymes called DICER-LIKE (DCL) and RNASE THREE-LIKE (RTL), which maturate almost all classes of double-stranded RNA precursors. Previously, we showed that the RNaseIII activity of DCL and RTL family members in Arabidopsis thaliana depends on the oxidation state of specific cysteine thiols. Recently, through whole-genome analyses, we showed that the repertoire of small RNA changes with the redox environment of the cell, suggesting that redox regulation of RNaseIII endonuclease enzymes might signal environmental changes to regulate small RNAs metabolism. Within this RoxRNase project, we will determine the thiol-redox switch mechanism of DCL and RTL, and the reduction pathways involved. The goal is to nail down the molecular bases of the thiol redox mechanisms involved in fine-tuning the small RNA biogenesis and gene expression as a response to biotic and abiotic stresses in Arabidopsis. More specifically, the project aims to elucidate how cellular redox environment regulates RNaseIII activities involved in epigenetic regulation of gene expression during plant response to high temperature and virus infection, and to identify redox regulators that control RNaseIII redox modification and activity. We propose first to identify redox post-translational modifications of Arabidopsis DCL and RTL under normal, biotic (virus infection) and abiotic (high temperature) conditions by using biochemical and mass spectrometry approaches. Then, we will examine effects of redox modifications on DCL and RTL activity, subcellular localization, and function in plant response to stress conditions by creating substitution mutations of redox-modified residues of the proteins. Next, we will study effects of redox modifications on DCL and RTL on the accumulation of small RNA and its impact on gene expression by high throughput sequencing. Finally, we will investigate the biological significance of DCL and RTL redox regulation in controlled high temperature and specific virus infection conditions and evaluate its impact on plant viability, development and fertility. Thus, this project aiming to elucidate redox-epigenetics networks in plants will deepen current understanding of how plants adapt or resist to the changing environment. We believe that the results obtained from this project will lead to establish a general link and uncover the molecular mechanisms of interplay between redox signaling, epigenetic regulation and adaptation to environmental constraints which are of major concern for all living organisms. The RoxRNase project assembles complementary expertise in the fields of redox signaling, RNA metabolism and epigenetic regulation from the three partners who have already built solid collaborations which will bring the project to success.

Developing an integrative research allowing to elucidate both the links between genotype, phenotype and fitness and the genomic bases of the variation form a major challenge in our understanding of speciation and the emergence of biodiversity. In DiversiFly, I propose to implement such approach by generating, analyzing and comparing phenotypic (morphometry, coloration, odor), genomic and ecological data on Ophrys orchids. This genus displays one of the highest rates of diversification and hybrid zones formed in the wild by some of its species makes Ophrys a promising model to better understand the causes of adaptive radiations: phenomena of intense and rapid diversification in response to ecological selection pressures. Through its approaches, DiversiFly aims at combining studies led at different evolutionary scales, working on endemic and threatened species. In a first part, the parallel study of two hybrid zones will allow us to determine what are the phenotypic traits (morphology, coloration, odor) predominantly involved in adaptation and reproductive isolation within the O. insectifera clade. Through cline analyses, outlier research and association studies, we will then confront phenotypic and genomic data (transcriptomes and GBS data) in order to look for the genomic bases of traits of interest. The links between phenotypes and individual fitness will be evaluated based on life history traits related to reproductive success and survival. As all individuals will be marked, phenotyping and fitness will be evaluated each year over the four years of the project to investigate their stability. In a second part, we will link micro and ‘macro-’ evolutionary scales by working on the whole genus Ophrys. We will use floral transcriptomes on species, representative of the diversity of the genus in order to identify genes involved in shaping floral phenotype, so particular of the Ophrys. Following a comparative approach, we will base our study on both sequence variation and gene levels of expression. Our results will have a significant impact in the field of ecology and evolutionary biology, contributing to the emergence of a new plant model, with a complex genome, to study speciation and evolutionary diversification. They will also contribute to fields such as genomics, conservation biology, systematics and taxonomy. We also believe that with its flowers mimicking insects, Ophrys forms an excellent model for scientific communication and towards general audience.

Ribosome assembly and regulation of translation activity is crucial for cell growth and response to external cues or environmental conditions in all eukaryotic organisms. Ribosomal RNA (rRNA) are the linchpins of ribosome assembly and function. The rRNAs 25S, 5.8S, 5S and 18S form, respectively, the large (LSU) and the small (SSU) subunits of the ribosome. The LSU acts as a ribozyme, catalyzing peptide bond formation, while SSU harbors the decoding center which monitors the complementarity of tRNA anticodon with mRNA codon during translation. In all eukaryotic cells, rRNA are subjects of two major types of nucleotide modifications: methylation of sugars (2’-O-ribose) and conversion of uridine to pseudouridine. Over 90% of the rRNA methylations are 2’-O-ribose methylations which are guided by antisense small nucleolar RNA (snoRNA) referred as C/D-box snoRNA. The C/D-box snoRNA associates to four nucleolar proteins called the C/D core proteins (Nop56p/p62, Nop58p, snu13/15.5K and the methyltransferase Nop1p/Fibrillarin) to form the C/D-box snoRNP. The extent and positions of rRNA 2’-O-Me fluctuate between species, however the majority of modifications occurs in functionally conserved regions. Remarkably, modulation of rRNA 2'-O-methylation affects ribosome translation fidelity and is linked to human diseases. In plants, only limited information is available on rRNA 2'-O-methylation profile, mechanism of modification and role(s) of modified nucleotides in ribosome activity. In the plant model A. thaliana, ~100 C/D-box snoRNA have been identified by computational screening of genomic sequences. Later, analysis of small RNA-seq data and targeted sequencing of nucleolar RNAs expanded the list of known C/D-box snoRNA to ~200 species. Notably, studies in Arabidopsis demonstrated that the knockout of a single C/D-box snoRNA triggers strong developmental and growth defects, and gene disruption of NUFIP, a C/D-box snoRNP assembly factor, also leads to severe developmental phenotypes and inhibited 2’-O-methylation at specific rRNA sites. We have recently performed RiboMethSeq analysis of Arabidopsis thaliana rRNA and identified both conserved and plant-specific rRNA 2’-O-Me sites. Remarkably; our data suggest that some rRNA 2’-O-Me sites do not have matching snoRNAs, suggesting 2’-O-Me through a non-canonical snoRNA and/or another guiding mechanism. Furthermore, we also observed alterations of rRNA 2’-O-Me level in both conserved and plant-specific rRNA positions in response to specific developmental conditions. Furthermore, RNA-seq analysis of Arabidopsis C/D-box snoRNPs identified a subset of non-coding RNA, other than known C/D-box snoRNA, that might be implicated in 2’-O-methylation of rRNA and /or other RNAs. In this project, we will perform deep investigation of the mechanisms controlling 2’-O-methylation of rRNA and determine connections with other rRNA modifications and translation activity of plant ribosomes. We will determine how 2’-O-ribose methylation of rRNA contributes to the production of heterogeneous ‘specialized’ ribosomes in plants and impact plant resistance and/or adaptation to heat stress conditions. The MetRibo project is intended to contribute substantially into the intense efforts currently devoted to decipher how ribosome modifications contributes to regulatory pathways and how is tuned during adaptive responses of multicellular organisms to external cues, a field to which plants have much to offer.

As the main constituent of the terrestrial plant biomass, lignocellulose represents a huge reservoir of fixed carbon and a renewable raw material that is essential for human usages. Lignocellulose biomass enclosed within secondary cell wall (SCW) is mainly composed of cellulose, hemicellulose and lignin, a complex phenolic polymer, that is deposited in specialized vascular tissues. SCW plays important roles in growth regulation, protection towards pathogens, long-distance sap transport and upright growth. Cell wall structure and composition, which is variable according to cell type, tissue and plant species, differ markedly between dicots and monocots. Even minor changes of cell wall content can trigger remarkable effects on its chemical properties and, by the way, on plant development. The synthesis and deposition of SCW polymers is controlled at the transcriptional level by a hierarchical interconnected network of transcription factors (TFs), mostly unraveled by studies in Arabidopsis. Despite a strong conservation in plant lineage, SCW regulatory network has revealed even more complex in perennials. A deeper understanding of these processes is of major interest for industry and agronomy. SCW engineering in crops or trees is a way to optimize biomass processability. Despite huge progresses, the engineering of SCW deposition has proven difficult and often impairs plant growth. While numerous works point to the role of TFs in SCW synthesis, evidence for the implication of post-transcriptional mechanisms in this process is scarce. The most convincing cases of post-transcriptional regulation of SCW synthesis involve specific microRNAs that regulate specific SCW biosynthesis genes. In this context, the coordinator/P1 and partners (P2/P3) teams have recently identified an Arabidopsis RNA-binding protein family, sharing functional similarities with the animal translation regulator Musashi/MSI, named as Musashi-like/MSIL. Results from the consortium indicate that MSILs are implicated in the cell-specific control of SCW synthesis in Arabidopsis, revealing a specific role for MSIL in the translational control of glucuronoxylan methylation and lignin deposition in the interfascicular fiber cells of the inflorescence stem. Such a translational regulation represents an additional layer of control of cell wall polymers synthesis, that opens new directions of research and alternatives for SCW engineering. MusaWall is a pioneer project that aims to study the mechanisms by which MSIL regulate SCW synthesis in Arabidopsis thaliana (P1), but also in two plant models that present SCWs with different chemical structures, Brachypodium distachyon (a grass model, P2) and Eucalyptus grandis (a model of hardwood tree, P3). In particular, 1) by using state-of-the-art genetic approaches, we will generate msil-loss-of-function lines in the grass model Brachypodium distachyon and the model of hardwood tree Eucalyptus grandis; 2) we wish to analyze the impact of MSIL in cell wall polymers synthesis in the grass and tree models and compare with Arabidopsis data; 3) in Arabidopsis more particularly, we wish to find MSIL targets that are relevant to SCW polymers synthesis and also to assess the connection existing between the MSILs and the m6A mRNA modification pathway in the SCW synthesis; 4) at last, we will analyze the MSIL-dependent regulatory network involved in SCW synthesis in interfascicular fiber cells, through the identification of actors thanks to a genetic screen in Arabidopsis . By applying a unique combination of molecular, biochemical, genetic and chemotyping approaches, MusaWall will shed light on the functional conservation of MSIL function in plants and will contribute to the identification of novel regulators with high biotechnology interest for the improvement of bioproducts conversion.