With 429 000 deaths per year, malaria remains the most devastating parasitic disease for humans. It is caused by Plasmodium parasites and transmitted by Anopheles mosquitoes. The efficiency of artemisinin-based combination therapies (ACTs), the spearhead of malarial treatments, is now threatened by the appearance and spreading of artemisinin-resistant parasites. Moreover, the development of control strategies to block parasite transmission is a priority of WHO. In this project, we propose to study the mode of action of two antimalarial drugs, primaquine (PQ) and plasmodione (PD) that are active against gametocytes, the parasite stages responsible for human to mosquito transmission. PQ is the only available antimalarial medicine with established activity against mature gametocytes and is currently under intense clinical validation for widespread use in combination with ACTs. PD and derivatives are new early leads displaying fast-acting antimalarial activity and potent transmission-blocking properties. These drugs kill parasites most likely through pleiotropic redox-mediated mechanisms that remain poorly understood. While having distinct bioactivations, some of their modes of action seem to share common features. Indeed, while PQ is transformed by human cytochromes cytP450 into presumably highly active hydroxylated metabolites, the antimalarial activity of PD comes largely from its specific bioactivation within the infected erythrocytes and subsequent redox cycling properties. The existence of putative targets proteins for these compounds also remains an opened question. To decipher their complex mode of action, we propose to set up a multidisciplinary approach combining different expertise in organic synthesis, chemical proteomics, biochemistry, cell biology, and genetics, and to use the yeast as a model in parallel to Plasmodium studies. Our project has three main objectives: 1) to monitor the oxidative damages caused by PQ/PD, 2) to identify target proteins and redox enzymes controlling drug sensitivity and resistance in yeast and parasites, 3) to confirm the contribution of these candidate genes to drug sensitivity and resistance in Plasmodium. We expect that this proposal will allow us to shed light and understand the biochemical pathways and genes implicated in the modes of action of these antimalarials, and will allow their drug targeting as essential components in asexual and sexual parasites, responsible for malaria physiopathology and transmission, respectively.

Cyanobacteria are present in a large range of habitats and climates, including extreme conditions going from deserts to arctic lakes. They are equipped with all the physiological mechanisms needed to survive to extreme and fluctuating environments allowing them to be the predominant species in ecosystems under specific environmental conditions. Under certain conditions, a sudden and rapid growth of one or some cyanobacterial species is induced leading to blooms. There is a direct relationship between the frequency of cyanobacterial blooms and the augmentation of available nutritive resources caused by human activities. Climate changes also favor the appearance of cyanobacteria blooms suggesting that this phenomenon will be amplified in the future. The cyanobacterial blooms disrupt the functioning of the continental aquatic ecosystems with serious consequences for the production of drinking water, or recreational aquatic activities. These disturbances are largely connected to the capacity of some cyanobacteria to synthesize toxins hazardous for human and animal health. Among them, the microcystins (MCs) are the most common as they are produced by the most common bloom-forming species Microcystis and Planktothrix (cHAB: cyanobacterial Harmful Algal Bloom). The concentration of toxins present in each bloom varies. Indeed, for a toxic species, only some strains present in the bloom synthesize toxins, and in addition, in these toxic cells, toxin biosynthesis depends on environmental conditions. Thus, despite that the survey of cyanobacterial cells and toxins is one request of safety agencies in Europe and France, it is impossible to predict the concentration of toxins only from biomass data. This impossibility of prediction represents big costs for the water end-users and a health risk for populations. For this reason, it becomes urgent to understand the determinism of the toxin production during the blooms and one way to achieve this goal is to found what are the factors regulating toxin synthesis and to elucidate the functional role of toxins. Several stress conditions increases cyanotoxin synthesis but it is not clear if there is a direct effect of stress factors or an indirect effect via photosynthesis and/or photoprotection. It is also not clear, why cyanotoxin producers are more resistant to stress. Is it a direct effect of the toxin or a photoprotective mechanism up-regulated by their presence? Thus, it is crucial for the understanding of cHAB to examine the fluctuation of toxic cyanobacterial sub-populations and toxin synthesis in relationship with the rates of photosynthesis and protection mechanisms in strains isolated from natural environment and usually present in blooms. This is the main objective of CYPHER. We will study the connection between microcystin synthesis and the cellular redox changes generated by variations in photosynthesis and in photoprotective mechanisms in MCs producer Planktothrix presenting different phenotypes and living in different ecosystems. We will also study the possible role of toxins on photosynthesis and photoprotective mechanisms under stress conditions. The originality of our proposition is to put together experts in: ecology, toxin synthesis, toxin modification and transcriptomics (partner 1), comparative genomics and secondary metabolites (partners 2) and cellular photosynthesis and photoprotective mechanisms (partner 3) to do a study that should help further predict the concentration of cyanotoxin producing strains in blooms generated by different environmental conditions, especially associated with a changing climate and a more polluted world. Thus, the expected fundamental knowledge will help ecological scientists or end-users in the large application on quality and surveys of water plans.

Streptomyces are major filamentous soil-dwelling bacteria found in a large diversity of natural habitats and particularly in the rhizosphere, the immediate zone around the plant root system. These bacteria are characterized by their ability to produce a wide range of specialized metabolites, notably anti-fungal and anti-bacterial compounds. Consequently, most than two-thirds of the antibiotics used in medicine derived from Streptomyces species. Recent studies showed that the microbial composition of the soil, and particularly the presence of certain Streptomyces strains, plays a major role in plant health, notably by competing with phytopathogenic microorganisms. In this context, identifying and understanding the role of beneficial Streptomyces can have a profound impact on the strategies developed to improve plant health and to reduce the use of chemical pesticides. Despite their impressive ability to produce bioactive compounds, only a few Streptomyces strains have been developed in commercial plant protection products suggesting that a better knowledge of the behavior of these bacteria in the environment is needed to improve their agricultural use. The STREPTOCONTROL project aims to understand the biological activity and to optimize the use of the AGN23 Streptomyces strain, selected following a screening strategy to identify bacteria able to induce the plant immune system and to produce antifungal metabolites. Spraying Steptomyces spores or mycelium on aerial parts of the plants protects them from fungal infection suggesting that the strain could be used in formulations to protect crops. However, the mode of action of this strain is not known, as well as its behavior in natural environments. The first objective of the STREPTOCONTROL project is to identify the compounds involved in the antifungal activity and the induction of plant defense responses by using a combination of genomic and biochemical approaches. To reach this goal, a high quality genome sequence of AGN23 will be produced and annotated. This will allow predicting specialized gene clusters and regulatory factors involved in the expression of these clusters and will help the biochemical identification of active metabolites. High-throughput screening tools will be used to identify and purify active fractions and structural approaches (MS/MS and RMN) will allow defining the chemical structures of active metabolites. The second major objective is to engage genetic and molecular studies to confirm the identification of gene clusters involved in the prediction of active metabolite. A collection of UV-mutant has been recently prepared and will be screened for the loss or overexpression of antifungal activity. Re-sequencing of the selected mutants will allow the identification of candidate genes. Complementary approaches will use the recently develop CRISPR/CAS9 strategy to delete each of the specialized metabolite gene cluster and to evaluate the phenotype of the genome edited strains. The third objective is to study the behavior of the strain in controlled environmental conditions and in presence of various types of plant and microbial species. The construction of modified reporter strains will allow monitoring the expression of gene clusters involved in the production of active compounds, to study the impact of rhizosphere colonization of various plant species and of microbial composition. Together, the STREPTOCONTROL project will bring new fundamental knowledge on the activity of Streptomyces, essential microbial components of the plant root microbiome and will serve as a basis to improve the development of these microorganisms as new environmentally friendly products for plant protection.

Potato, the 4th worldwide food crop culture, represents a major economic stake both in developed and developing countries. Its culture is recommended by Food and Agriculture Organization of the United Nations. A limiting factor in the expansion of potato culture is its sensitivity to a wide diversity of pathogens. As a consequence potato culture exhibits a high level of treatment frequency. Since the increasing interest of European and National governments for limiting chemical inputs, biocontrol appears as one of the alternative tools instead of or in complement to chemical treatments. The National plan Ecophyto claims to reduce by half the chemical treatments -if possible-, hence stimulates the development and use of all novel practices including biocontrol. In addition to the endemic Pectobacterium populations (P. atrosepticum, P. carotovorum, P. wasabiae), two Dickeya species (D. dianthicola and D. solani) emerged successively and recently in potato cultures in Europe though Dickeya populations were, initially, considered to be restricted to tropical and subtropical plant hosts and areas. The P. atrosepticum, P. carotovorum, P. wasabiae, D. dianthicola and D. solani species co-exist in a same contaminated field or in symptomatic tissues. Hence, treatment against blackleg and soft rot diseases should take into account the natural and complex diversity of the pathogen populations and its dynamics. COMBICONTROL project aims at evaluating, combining and accommodating biocontrol strategies against the highly diverse and dynamic populations of the Pectobacterium and Dickeya bacterial pathogens which induce blackleg and soft rot diseases on potato plants and tubers. Hence, COMBICONTROL will produce basic knowledge (1) in the dynamics and function (protection efficiency) of biocontrol agents when they colonize the potato rhizosphere; (2) in the structure and dynamics (including the characterization of novel taxons) of Pectobacterium and Dickeya populations pathogens which will be annually collected from fields, as well as in their sensitivity to the studied biocontrol agents; (3) in the genetic and functional characterization of the potential emergence traits (species-specific and virulence traits) of Dickeya solani and Dickeya dianthicola pathogens. COMBICONTROL will also strengthen a dialog between academic researchers and potato seed producers about Pectobacterium and Dickeya disease and control. To achieve these objectives, COMBICONTROL associate 3 academic partners and 2 private partners. The 3 academic partners are teams belonging to the Integrative Biology of the Cell (I2BC)-Saclay Plant sciences (at Gif-sur-Yvette), Institut d’Ecologie et des Sciences de l’Environnement de Paris (IEES at Paris) and Laboratory Microbiology, Adaptation & Pathogenesis (MAP at Villeurbanne). The private partners SIPRE and RD3PT are R&D sectors of the CNPPT and FN3PT organizations, respectively. They are the main national actors in the production and certification of potato seeds. Though COMBICONTROL focuses on biocontrol approach, it will be conducted in a tight coordination with other emerging projects of SIPRE and RD3PT about pathogen survey and development of other plant protection strategies. All together, the partners are already updated in the use of omics, and combine expertise in plant pathology, biocontrol, ecology, epidemiology, taxonomy, structural and functional genomics, genetics and agronomy. COMBICONTROL is the first ANR-network on Pectobacterium and Dickeya potato pathogens that associate academic and private partners. This consortium will accelerate knowledge transfer from research to applied fields (and reciprocally) and will produce data of national and international interests. Because Pectobacterium and Dickeya diseases are present in Europe and world wide, this consortium will be a solid interlocutor for developing further research programs in collaboration with laboratories from other countries.

Members of the ATP-dependent chromatin remodeling complexes (remodelers), including the BAF complex, are recurrently mutated in human cancers. We recently reported a genome-wide investigation of remodeler function in embryonic stem (ES) cells (De Dieuleveult et al., Nature 2016). We detected extensive binding of remodelers to promoters and enhancer elements, and unexpectedly, to CTCF-occupied elements. CTCF is a DNA-binding protein that divides the genome into separated chromatin domains called ‘Regulatory Neighborhoods’. This 3D organization is thought to minimize illegitimate contacts between (onco-)genes and nearby unrelated enhancers, with recent data confirming that mutations in CTCF-sites are recurrent in human cancers. By combining the expertise of the three partners in the project, we will investigate the function of chromatin remodelers at CTCF-sites and determine how they contribute to organize 3D genome structure. Our 3D-reMODEL project has four objectives that will systematically elucidate the functional link between chromatin remodelers and CTCF in the structuring of mammalian 3D DNA domains and how perturbed collaboration between remodelers and CTCF may contribute to the cancerous phenotype: 1. We will describe how CTCF co-localizes with nine different remodelers in an orientation dependent manner at different types of DNA domains (TADs and various types of sub-TADs) in mouse ES cells. 2. Using ChIP-seq, we will dissect how the nine remodelers contribute to CTCF binding and Cohesin stabilization or vice-versa. 3. We will unravel the nature of CTCF-remodeler co-occupancy by testing for (direct) interactions between CTCF and remodelers. We will also characterize protein-protein interaction (PPI) networks of CTCF/Cohesin using co-immunoprecipitation and mass spectrometry approaches. 4. Using a combination of high resolution Hi-C and HiChIP approaches, we will analyze how loss-of-function of each remodeler affects the formation of different types of 3D DNA domains. In particular, we will monitor the consequence of Brg1 loss of function to determine if its function in regulating 3D DNA domains can predict part of its tumor suppressor function. We have performed preliminary experiments in which we mapped the ChIP-seq distribution of CTCF in ES cells depleted of either Chd4, Brg1 or Ep400 remodeler. We found that loss of each these remodeler alters CTCF binding with a specific pattern, suggesting a major function for this family of factors in the regulation of 3D chromatin domains. It will now be important to test how the other remodelers affect CTCF binding, if CTCF itself plays a role on the recruitment of remodelers and how changes in CTCF occupancy may cause changes in the 3D organization of the ES cell genome.