Pesticides are of limited use against bacterial diseases in crops due to a lack of effective and non-toxic molecules. Thus, genetic selection of resistant crops remains the most effective approach to control bacterial pathogens. Resistance breeding requires a conceptual jump to efficiently design significant and durable resistance to a large variety of pathogens in a large number of crops simultaneously. The CROpTAL project aims at identifying plant susceptibility hubs in major crops (cereals, citrus, legumes and brassicaceae) targeted by Xanthomonas virulence-promoting TAL (Transcription Activator-Like) type III effectors. These conserved susceptibility targets could then be used for marker-assisted breeding of loss-of-susceptibility by selection of inactive variants of those hubs. These results will contribute to the development of durable resistance to a broad range of bacterial pathogens in the selected crops.

In the context of climate change, it appears essential to unravel the mechanisms governing abiotic stress tolerance in higher plants, in order to build predictive models and use this knowledge to assist selection and design of stress tolerant crops. We have previously uncovered remarkable adaptations in seed mitochondria, which because of the ability of seeds to survive desiccation, display impressive tolerance to abiotic stress. In particular, seed mitochondria accumulate high levels of small heat shock proteins (sHSP) and late embryogenesis abundant proteins (LEA). The sHSP are the most widespread but less conserved HSP. They contribute to the molecular chaperone network that assists protein biogenesis and homeostasis under stress conditions (sHSPs are stress inducible). In eukaryotes, mitochondrial sHSP (M-sHSP) have only been identified in plants and insects. LEA proteins are highly hydrophilic proteins, generally intrinsically disordered, which accumulate in desiccation tolerant organisms, and whose functions still remain largely enigmatic. The MITOZEN project aims at deciphering the molecular function and physiological role of the mitochondrial sHSP and LEA proteins (M-sHSP and M-LEA) in the model plant Arabidopsis thaliana. The genome of Arabidopsis harbors 17 sHSP genes (including 3 M-sHSP) and more that 50 LEA genes, among which we have recently identified 5 M-LEA genes. The molecular functions of the M-sHSP and M-LEA will be explored using biochemical and biophysical approaches to study recombinant proteins produced in Escherichia coli. Their structural features and protective activities (oligomerisation, secondary structure, chaperone activities, membrane protection) will be examined in the context of temperature stress and dehydration using a large panel of techniques and in vitro assays. The goal is to determine the potential molecular functions of the different M-sHSP and M-LEA in the context of stress tolerance (desiccation in seeds, high temperature in seeds and plants). A reverse genetics approach will be developed in Arabidopsis to explore the role of M-M-sHSPs and M-LEAs in the physiology and development of plants. Single and multiple knock-out mutant lines will be constructed, as well as overexpressors using an inducible system. Their phenotypic characterization will focus on seed development and abiotic stress tolerance of plants, including mitochondrial function. The integration of data provided by these multidisciplinary approaches (bioinformatics, biochemistry and biophysics, genetics, physiology) will shed light on the function and importance of the different M-sHSP and M-LEA in the development and stress tolerance of plants. It will also increase knowledge about molecular chaperones and in particular with respect to their yet unexplored role in the context of dehydration, and will shed novel light on the function of LEA proteins.

The objectives of the FLUOPATH project are 1) to find new biomarkers (promoeters that induce the expression of genes of interest) coupled to a fluorescent biosensor allowing to acquire new knowledge in bacterial cell physiology related to the impact of technological disturbances inducing stresses. The study will be focused on the growth, survival and virulence of two pathogens in dairy products (milk, diluted cheese and if possible solid cheese) and 2) to use this knowledge combined with the knowledge present in the scientific literature to improve the models for predicting the microbiological risk in dairy products. The pathogens considered will be L. monocytogenes and B. cereus. The matrices considered will be liquid milk as well as model diluted and undiluted cheese (gelified matrix). New models based on the use at the scale of the single cell and the whole population of biomarkers will be developed to predict growth, resistance and virulence (invasive capacity for L. monocytogenes and entry into sporulation and toxin production for B. cereus) to stress while taking into account cellular variability. A precise quantification of the impact of stressful industrial conditions will be carried out on bacterial responses at the cellular level: probability of single cell / whole poulation growth, survival, toxin production, spore formation, invasive capacity. The challenge is to design and validate in food matrices bacterial biomarkers of phenotypes of interest in risk assessment in order to incorporate the intensity of biomarker response into exposure assessment models.

Plant responses to biotic aggressions involve a great diversity of molecules including regulatory proteins and hormones. Among these actors, small secreted peptides, also named peptide phytohormones or phytocytokines, may directly interact with pathogens or act in signalling and cell-to-cell communication. They are produced from non-functional precursors through a maturation process, making characterization difficult only on the basis of their gene sequences. Only a small fraction of the genes liable to encode these secreted peptides has been described and their impact and diversity appears to be seriously underestimated. The main goal of STRESS-PEPT is to better understand the plant responses to biotic stress at the peptidome level and to characterize new molecular actors involved in defence mechanisms. Based on our experience and previous results, we plan to develop and apply a multidisciplinary genome-wide approach combining bioinformatics, differential transcriptomics and peptidomics to identify new secreted peptides in Arabidopsis and to describe their contribution to plant responses to different representative biotrophic and necrotrophic pathogens (oomycete, fungus and bacterial elicitor). The proposed methodology is organized in three complementary tasks: (i) An original bioinformatics pipeline will be used to screen the Arabidopsis thaliana genome in order to identify genes encoding precursors of secreted peptides, to cluster and classify them in gene families by homology and phylogenetic profiling and to predict the putative mature peptides through a sensitive conserved motif searching method ; these predictions will be integrated to RNAseq transcriptomics analyses applied on Arabidopsis in presence or absence of the different pathogens in order to tag the fraction of the secreted peptide precursor genes that are transcriptionally regulated by pathogen aggression(s). (ii) The same biological samples will be used to prepare extracts of peptides, from apoplastic fluids and total extracts, through an original protocol optimized for efficient mass spectrometry (MS) analysis: a LC-MS/MS based peptidomics approach will be applied on the different peptide extracts for the identification and the differential quantification of the secreted peptides and to characterize their post-translational modifications. (iii) A selection of promising peptides, based on transcriptomics and peptidomics data, will be made for functional analyses including assays on knock-out mutants and overexpressing transgenic lines. Synthetic peptide treatments will be performed in order to understand and validate their role in plant-pathogen interactions. The STRESS-PEPT consortium gathers bioinformaticians, molecular biologists, biochemists and plant pathologists expert of each studied pathosystem as well as a proteomics platform. The already established collaborations between these partners will ensure close cooperation and synergy throughout the 4 year project, and ensure obtaining results of high interest for the plant biology community. All the data generated will be stored and organized for efficient querying in a relational database publicly available at the end of the project. STRESS-PEPT will lead to significant advances in the discovery of new secreted peptides which are key players in danger sensing and the modulation of immune responses. According to their conservation among plant species, these peptides might be used in innovative strategies aiming at jointly optimizing plant quality and resistance, and should open new opportunities for sustainable crop management.

Human and wildlife animals are exposed to multiple sources of environmental stressors including chemicals such as persistent organic pollutants (POPs) and endocrine disrupting compounds (EDCs). In addition to the important public health issues related to such exposures, EDCs are suspected to elicit ecosystems toxicity with an impact on the food chain and biodiversity and a significant economic burden linked to the increase of metabolic and neurodevelopmental disorders. In this complex and multifactorial context, new and innovative approaches are warranted to address potential linkages between such environmental exposure and health outcomes. Whereas exposure models in toxicology and ecotoxicology traditionally link a given external exposure source with a target organism, the vision of CREATIvE is to consider the organism as both an internal exposure source and a target. Specifically, its ambition is to assess potential health consequences from the release of POP mixtures from an internal storage site (the source) by understanding their complex biological modes of action (MoAs) on the target tissues of the same organism. It is well known that POPs bio-accumulate in living organisms and are stored in specific tissues e.g. adipose tissue (AT) brain, and liver, for long periods of time. Therefore, these tissues represent internal chronic sources of pollutants possibly leading to various disorders including metabolic and neurodegenerative diseases. Such “internal” exposures are not satisfactorily captured by current methods based on investigating different types of external POPs exposure via gavage, injection or acute inhalation. The proposed protocol will not replace the existing ones but will be complementary, taking into account for the first time internal sources of exposure. The aim of CREATIvE is to develop a novel strategy exploring the effects of an internal exposure from grafted contaminated AT. The kinetics and consequences from a redistribution of POPs and their metabolites from grafted contaminated AT on several tissues and organs, e.g. liver, brain and host AT will be studied. The proposed integrated approach is a combination of experimental studies (chemical quantitative measurements in tissues, metabolomics, transcriptomics) and computational modeling (PBPK and systems biology approaches). The advantage of developing such integrated approaches is the possibility to identify the systemic effects of internal mixture exposure at different biological levels, by mimicking the reality of human and animal exposure. As results, new biomarkers will be characterized, and novel complementary models will be proposed which will help at increasing Agregated Exposure Pathways (AEPs) information. To our knowledge such a strategy is clearly innovative and different from existing studies. In a recent preliminary study, an allograft model was developed at Paris Descartes, consisting in a mouse graft of contaminated AT to a non-contaminated mouse. We demonstrated that four weeks after transplantation, the grafts are vascularized and functional. In those initial studies, donor AT was contaminated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and we showed that this contaminant was indeed redistributed to different tissues with different kinetics. Based on this acquired proof of concept, CREATIvE will explore the kinetics of a low dose POP mixture release from an internal source of exposure, and most importantly will assess the toxic effects of such mixtures on other tissues and organs. After improvement of the experimental model, a mixture of twelve environmentally relevant POPs will be studied at low doses with the aim to better understand the consequences of POP mixture release from a unique internal source of exposure.