Bacteria, including the human pathogen Vibrio cholerae, can enter a temporary non-proliferating state, also known as dormant, in response to a variety of environmental stresses. In the dormant state the cellular metabolic activity is absent or reduced to a maintenance level. Cells resuscitate and revert to proliferation when conditions for growth are restored. For their ability to survive host-imposed and environmental stressful conditions, cells in a dormant state have been associated to antimicrobial tolerance, chronic infections and environmental dispersion. In the SurVi project, we will investigate V. cholerae dormant state on all fronts - cell physiology, genome integrity, de novo cell morphology reshaping - by a multi-disciplinary integrated approach. The genetic circuits, molecular mechanisms, morphological changes and metabolism throughout the dormancy cell program (entry, maintenance, exit) will be characterised in minute details. We present a straightforward way to induce the formation of V. cholerae dormant cells by the simple addition of L-Arabinose to the growth medium. This permits to trigger formation and resuscitation of quantities of non-proliferating dormant cells in a short time window. Beside L-Arabinose, dormancy will be induced by at least two other means, cold exposure and treatment with cell wall targeting antibiotics. Comparing different stresses will permit to obtain a comprehensive view of the dormant state and pinpoint the dormant formation and reversion core cellular program. A better knowledge of the physiology of dormant cells is necessary to engineer new molecules to treat bacteria in a non-growth state and eradicate the environmental reservoirs of dormant bacteria. In addition, we will study the mechanism of action of L-Arabinose, because of its potential interest as a novel prophylactic agent against cholera and possibly other bacterial infections or diseases.

Plants constantly adjust their development in response to variations of environmental parameters, both above and below ground. How do plants sense and integrate various concomitant external signals? How does each signal influence the response to another? These questions are at the heart of the NitReST project, in which I will address the interplay between plant nutrition and growth responses to light or temperature. Nitrogen is one of the most important nutrients for plant growth and development. In temperate aerobic soils, nitrate ions (NO3-) are the main source of nitrogen for many plant species and roots are exposed to large variations of nitrate availability. To ensure high crop yields, nitrate-based fertilizers are supplied in large excess in agricultural soils. However, such excess is transient due to depletion by roots and leakage, which has potentially harmful consequences for the environment. It is crucial to understand how plants deal with nitrate supply and how it affects their development, especially in constraining environments. A good example of such challenging situations is the shade of competitive neighbours. In some species like Arabidopsis thaliana, unfavourable light conditions lead to elongation of vegetative aerial organs like hypocotyls but a restriction of root growth. This response, known as the shade-avoidance syndrome (SAS), helps plants overtop competitors and get better access to sunlight. Similarly, a slight elevation of ambient temperature like those associated with the current climate change also triggers a developmental response called thermomorphogenesis that includes hypocotyl elongation. The phenotypic consequences as well as the underlying signalling pathways are highly similar to SAS. Nonetheless, contrary to SAS, higher temperatures favour root elongation. This difference may have an impact on how plants can take up available nutrients from soil in both contexts. The NitReST project aims at deciphering how plants integrate nitrate nutrition and growth responses to shade or elevated ambient temperature. The main hypothesis is that nitrate fluxes are regulated in seedlings upon perception of a shade signal or high temperature and a sufficient soil nitrate concentration is required to ensure hypocotyl and root elongation in these contexts. NitReST will tackle this issue by answering two complementary questions. First, I will address how light or temperature conditions affect nitrate uptake, transport and assimilation in young seedlings. To do so, I will combine metabolomics experiments with a reverse genetics approach to (1) understand how the different steps of nitrate homeostasis are regulated in SAS and thermomorphogenesis and (2) identify key players of the nitrate pathway involved in environment-driven growth responses. Second, I will determine how nitrate availability impacts hypocotyl and root elongation in young seedlings in response to shade or elevated temperature. The thorough phenotyping with combined treatments will be followed by two complementary approaches (GWAS and comparative transcriptomics) to describe the natural variation underlying the interactions between shade/temperature and nitrate supply and identify regulators of this interplay. One originality of the approach will be to combine the use of Arabidopsis thaliana as the main model species to new emerging models from the Brassicaceae family: Brassica rapa and Cardamine hirsuta. Both are close relatives to Arabidopsis, with their genome sequenced, the possibility to get mutant by CRISPR technology, and they display contrasted responses to shade or temperature. This will allow me to understand how [NO3-] affects species with different strategies towards shade or high temperature. The outcomes of the NitReST project will have a strong impact for both fundamental and applied research as they will bring new insights to how to optimize the use of nitrate fertilizers in the context of dense environments and climate change.

Plants must constantly respond to a wide range of signals, including stresses, in order to coordinate their development and survival within a dynamic environment. One way in which this is achieved is through chemical modifications of proteins, allowing flexible and rapid changes of the proteome to alter cellular and physiological outputs. Protein acetylation is one such modification, which occurs on the N-termini (Nt) and internal lysines (K) of many proteins. Despite its prevalence, and in contrast to other wellstudied modifications (e.g., phosphorylation), our knowledge of: (i) the regulation, specificity and plasticity of protein acetylation, and (ii) its downstream functional consequences on protein activity and physiology are severely lacking, especially in plants. It is therefore extremely timely to elucidate the multifaceted functions of protein acetylation and open up this new area of plant molecular biology, in which Europe has the capacity to take a world lead through strategic ERA-CAPS funding. The overarching aim of the KatNat project is to provide a mechanistic understanding of protein acetylation in plants, with a particular focus on investigating the enzymes that catalyze this modification (Nt- and K-acetyltransferases) and the resultant effects on proteostasis, photosynthesis, and metabolism. Crucially, this work will be carried out within the context of agronomically relevant stresses. KatNat consists of four interrelated objectives that will answer broad questions: (1) How does abiotic stress regulate the global Nt- and K-acetylome?; (2) What are the specificities, targets and stress-responsive dynamics of the acetyltransferases?; (3) How does protein acetylation impact protein stability and turnover?; (4) How does protein acetylation in plastids regulate photosynthesis and metabolism? By answering these connected questions, KatNat will not only shift the forefront of the field but will provide regulatory mechanisms and fundamental new insight into how plants sense and respond to environmental changes, which may become entered into textbooks. Last but not least, the obtained information will identify key new targets for the future development of superior crops. The KatNat consortium brings together five European groups who all have a significant, demonstrable interest in the study of protein acetylation, and who have the highly complementary expertise in mass spectrometry, protein biochemistry and molecular plant biology required to carry out this original and competitive research at the highest international level. Consortium members already have a world lead in this field, and several members currently collaborate informally. The synergistic value of our collaboration will be the development and exploitation of an understudied area of in plant science, with key importance to agriculture. The proposed research is highly innovative, aligns closely with ERA-CAPS priority themes and has measurable and impactful outcomes that will shed light onto this emerging, exciting and important new area of plant molecular biology.

According to the World Health Organization, antibiotic resistance is among the most serious threats to global health, food security and development. It affects humans and animals in every country and the gravity of infections is increasing due to antibiotic treatment failure. Staphylococcus aureus is among the most commonly reported antibiotic-resistant bacteria. Despite recent efficient prophylactic measures, it remains a leading cause of community- and healthcare-associated diseases ranging from minor skin infection to life threatening bacteremia. Its “success” as a pathogen is dues to the expression of numerous virulence factors, a high capacity for adaptation to biotopes, and to the emergence of multidrug-resistant strains. Tight control of transcription and post-transcriptional events have established roles in S. aureus pathogenicity and factors, including regulatory proteins, metabolites and regulatory RNAs contributes to a rapid adaptation to adverse environments. Regulatory RNAs are now demonstrated to be ubiquitous regulators that use a wide variety of mechanism to carry out their activities. They sense metabolites, interacts with proteins, modulate the RNA polymerase, release stalled ribosomes and in numerous documented cases base pair with mRNAs to modulate their expression. Consequently, they have emerged as key modulators of pathogenicity, antibiotic resistance and metabolic adaptation. Using bioinformatics and experimental approaches, we identified numerous small putative regulatory RNAs (sRNA) in S. aureus. However, the function and targets of most of them are unknown. This is partly due to technological bottleneck impeding phenotype and target discoveries. We recently developed an approach to determine phenotypes associated with sRNA gene deletions in S. aureus. The strategy allows testing simultaneously many mutants and the identification of weak phenotypes. We applied it in the context of antibiotic resistance. A first screen permitted the identification of sRNA gene deletions conferring an altered bacterial fitness in the presence of low concentrations of antibiotics. With the RRARE project, we will identify sRNA mutants conferring an altered fitness in different growth conditions including the presence of antibiotics using the experimental setup we developed. Our first results demonstrate the efficiency of the method. We wish to test more antibiotics with a recently improved mutant library. We will then focus on the characterization of sRNA mutants with relevant phenotypes. For that, we first need to identify the sRNA targets. This is a limiting step in deciphering sRNA function as none of the technics are fully satisfactory. We recently developed a ribosome profiling (Ribo-Seq) method with S. aureus and demonstrated that it is a highly efficient method. We proposed to use to this technic to find the targets of sRNAs uncovered by our experiments. One of our collaborator recently created an algorithm to model the complete set of mRNA-miRNA interaction in a eukaryotic cell. We propose to create a bacterial version of this algorithm. The availability of global data from Ribo-seq on RNA abundance and translation will be a unique opportunity to model system-wide sRNA-mRNA interactions in S. aureus. RRARE will contribute to understand RNA-RNA regulations and antibiotic susceptibility associated with sRNAs. Antagonizing directly antibiotic resistances is a proposed strategy to improve antibiotic efficiencies and therefore molecules interfering with specific sRNAs or their targets could increase antibiotic efficiencies. Many antibiotics used clinically target RNAs and more generally, molecules targeting RNAs are under development. Consequently, a long-term application of the proposed work would the identification and development of molecules that interfere with regulatory RNAs that we will characterize.

Meiotic crossovers allow for the shuffling of alleles between homologous chromosomes. The frequency and distribution of meiotic crossovers determine which traits are inherited together and which ones are reassorted to produce new combinations on which selection can act. The distribution of crossovers along the chromosomes is neither uniform nor random, but the mechanisms and evolutionary forces that impose crossover patterning are poorly understood. Major questions are still standing: Why do crossovers form preferentially in some regions of the genome? How does crossover patterning affect allele segregation and population dynamics? Further, crossover mispositioning can cause chromosome segregation defects, leading to aneuploid, largely unviable, gametes and embryos. Elucidating where and how much genomes recombine is therefore key to understand causes of infertility, to reveal the forces contributing to genome evolution, and to provide breeders with better tools to create new crops. My work has contributed to identify factors limiting crossover formation during meiosis in Arabidopsis thaliana and Caenorhabditis elegans. In Arabidopsis, mutation of such anti-crossover factors can lead to an unprecedented 7.8-fold increase in the number of crossovers formed. A very intriguing observation is that, in either wild type or in these hyper-recombinogenic mutants, presence of polymorphisms can impact crossover distribution, either attracting and repelling them. Similar observations have been made in yeast. Two classes of crossovers exist, which rely on different recombination proteins and DNA intermediates. Preliminary observations suggest that, in Arabidopsis, class I crossovers would tend to preferentially form in polymorphic regions while class II crossovers would avoid them. Presence of polymorphisms is therefore emerging as a major force contributing to crossover patterning, but data are still scarce to describe and understand this phenomenon. With a combination of genetics, genomics and cytology approaches, the POLYREC project aims at deciphering how the distribution of crossovers is impacted by polymorphism density across the genome and what the underlying mechanisms are, in Arabidopsis. We will produce the first genome-wide and high-resolution map of crossover events in regard to polymorphism using both already available and newly built genetic resources. This approach is now made possible thanks to the development of massive parallel sequencing of long fragments of DNA, allowing for cheap and efficient identification of thousands of crossover events. Further, we will establish how each class of crossovers behave in response to variation of polymorphism density along chromosomes, using a combination of genetics approach with available mutants as well as further developing ChIP-seq assays on meiotic tissues in Arabidopsis. Finally, we will proceed to a genetic screen to identify how the homologous recombination machinery could both sense and react to the presence of polymorphisms. The POLYREC project will also lead to the production of new genetic resources that will be made available to the Arabidopsis community and beyond, which could be used for mapping of diverse traits and QTLs, e.g. of agronomical interest. Understanding how polymorphism can affect recombination has great potential not only to decipher the forces contributing to genome evolution, but also to improve plant breeding programs through knowledge transfer. As a young researcher, the POLYREC project will allow me to further develop my independence. My publication track record, the fellowships I have obtained, the projects I have designed and the network I have built demonstrate that I am well positioned to make a significant contribution to the field.