As sessile organisms, plants cannot escape from adverse environmental conditions. In particular, drought and salinity are the major abiotic constraints that affect crop yield and productivity. Their impact on agriculture is expected to increase in the near future due to global climate changes. Thus, deciphering the molecular mechanisms of plant stress perception and adaptation is essential to improve crop tolerance and meet the increasing human demands. Plants have developed robust and efficient signaling networks to quickly sense stress stimuli and induce the adaptive cellular responses required for their survival, such as metabolic changes and transcriptional reprogramming. Protein kinases, which modify the activity, stability or localization of their targets by reversible phosphorylation, are key players of plant stress signaling. The model plant Arabidopsis thaliana contains more than a thousand of protein kinases that display specific as well as partially overlapping biological functions, to fine tune plant stress responses and ensure the fundamental cellular processes in any environmental condition. Besides the well-known and most studied mitogen-activated protein kinases (MAPKs), the calcium-dependent protein kinases (CDPKs) are emerging as central regulators of both biotic and abiotic stress responses. They exhibit the unique feature of combining calcium sensing and protein kinase activity in a single protein to efficiently transduce calcium signals that are the most widespread mediators of plant signaling. The Arabidopsis genome encodes 34 CDPKs that exhibit some functional redundancy, which has limited their discovery by classical genetic screens. Only few isoforms have been assigned a specific biological role and most of the substrates remain unknown. New strategies are thus required to elucidate CDPK functions in vivo. In the context of basic research, the goals of the present project are to understand and evaluate the roles of CDPKs in abiotic stress responses. We will focus on two key closely related Arabidopsis isoforms, CPK5 and CPK6, which function at the crossroad of biotic and abiotic stress signaling. In a first part, I propose to decipher the molecular bases of the hypersensitive stress phenotype of the cpk5cpk6 double mutant through a multi-level omic approach, combining transcriptomics, metabolomics and phosphoproteomics. In a second part, I propose to use an integrative approach combining phosphoproteomics, biochemistry, cellular and molecular biology to identify in vivo substrates of CPK5 and CPK6. Additional physiological assays combined with genetic approaches will be developed to validate the candidate genes and characterize their roles in abiotic stress responses mediated by CPK5 and CPK6. Thus, the present project will contribute to elucidate the molecular mechanisms of plant stress signaling.

Due to climate change, heat stress is going to become a major source of yield loss in Europe in the coming years. There is thus urgent need for the elucidation of cellular mechanisms involved in heat stress response to be able to produce new varieties with improved tolerance. Although the mechanisms involved in heat tolerance have been previously explored, little attention has been given to the contribution of chromatin dynamics to this process, particularly in crops. Major advances have been made regarding the epigenetics regulation of biological processes in model species, such as Arabidopsis thaliana. However, despite this, the extrapolation of the funding to polyploid species, such as wheat, is still a challenge. Also, epigenetic phenomena cannot be understood simply by determining the DNA sequences of genes or focusing on specific epigenetics mark. The 3DWheat project aims to bring new insight on the epigenetic regulation of heat stress resistance. An integrative approach of different layers of regulation will be investigated instead focusing on a specific epigenetics marks. We hope through this integration approach to answer the question how the epigenome of a polyploid species such as wheat, contribute to the adaptation of crop plants in a fluctuant environment. In this context, the 3Dwheat project has for objectives (i) to decipher the chromatin modification and changes in nuclear architecture that preside to heat stress response in wheat and (ii) to use knowledge acquired in model plants to engineer the wheat genome and create mutant lines for epiregulators with altered heat tolerance.

The production of membrane vesicles (MVs) is a universal but poorly understood mechanism for cell-cell communication that has been only recently appreciated. Outer membrane vesicles in Bacteria, as well as exosomes/ectosomes in Eukarya can transfer toxins, quorum sensing agents, pathogenicity factors, RNA and possibly DNA. Recently, the group of Patrick Forterre in Orsay (Biologie Moléculaire du Gene chez les Extrêmophiles, BMGE, partner 1) discovered that hyperthermophilic archaea of the order Thermococcales produce high amount of MVs. The aim of the ThermoVesicle project is to study MVs produced by several groups of hyperthermophilic archaea (Thermococcales, Methanococcales) and bacteria (Thermotogales). Special emphasis will be on the interaction of MVs with plasmids and viruses and their possible role in genes transfer between different groups of microbes living in the same environment. The project groups two partners who have been working for a long time on hyperthermophiles, Patrick Forterre, professor at the “Université Paris-Sud” (coordinator of the project) and Claire Geslin, partner 2, assistant professor at the Université de Bretagne-Occidentale, member of the team, “Laboratoire de Microbiologie des Environments Extrême » (LMEE). Preliminary results from partner 1 have shown that MVs from Thermococcales can recruit endogeneous or exogeneous plasmids and transfer them between cells. A newly strain isolated by scientists from BMGE, Thermococcus nautilus, whose genome has been recently sequenced, produces MVs harbouring a viral genome. Claire Geslin has isolated the only two viruses presently known from Thermococcales. Importantly, TPV1 (Thermococcus prieurii Virus 1) can infect Thermococcus kodakaraensis, a model organism for which genetic tools are available in BMGE. Claire Geslin has also observed that Methanococcales and Thermotogales are strong MVs producers and isolated the first two viruses infecting species of Thermotogales. The two partners will combine their expertises, biological materials and equipment to start a new collaborative effort to make a breakthrough in MVs study and to open new research lines in studying interactions between MVs and viruses. Production of MVs by Thermococcales and infection by TPV1 will be further characterized, using the genetically tractable strain Thermococcus kodakaraensis as model. The objectives will be to study in parallel the molecular mechanisms of MVs and virion production and fusion. A comparative analysis of MVs in Thermococcales, Methanococcales and Thermotogales will be performed with the aim to determine what are the group specific and universal properties of MVs in hyperthermophiles. Claire Geslin has recently observed that MVs from T. nautilus, prepared by partner 1, inhibit specifically the growth of some Thermococcales, including T. kodakaraensis. In this project, we will try to determine the nature of MV-associated toxicity factors and their spectrum of antimicrobial activity. We will study the mechanisms of DNA recruitment in MVs and DNA transfer, and determine if MVs could also be used to transfer genomic DNA. In silico analyses have documented horizontal gene transfers (HGT) between hyperthermophilic archaea and bacteria, but their mechanism remain mysterious. We will explore the possibility to use MVs to transfer DNA between Archaea and Bacteria. The study of functional and evolutionary relationships between MVs and viruses will be an important and original aspect of the project. We will use TPV1, previously identified MVs carrying a viral genome, and two recently discovered viruses infecting Thermotogales as model systems to determine if MVs can interfere with viral infection. For a long time, the importance of MVs in biology has been underestimated. Several actions will be undertaken to remedy this situation. In particular, we would like to organise at the end of this proposal the first international meetings on the MVs in the three domains of life.

Large sets of data have been accumulated over the course of the last 5 years describing the important role of the lipopolysaccharides [(LPS)/CD14-TLR4] dyad in the control of the onset of metabolic diseases. A change to high-fat diet is associated with a change in intestinal human and animal microflora, leading to an increased ratio of Gram negative to Gram positive bacteria, thereby increasing the probability of LPS diffusion into the blood. This concept is responsible at least in part for the initiation of the low grade metabolic inflammation characterizing obesity and type 2 diabetes and linking the major role played by intestinal microbiota to metabolic diseases. Importantly, although LPSs share a common architecture, they vary with each species of a genus and each structural detail exert a strong influence on the activities. LPS are made of a lipid moiety called lipid A carrying most of the biological activities of the molecule. This moiety is substituted by a core oligosaccharide, itself carrying the O-specific antigens made of repetitive oligosaccharide subunits. The latter is the most variable LPS moiety also called O-chain, and the number and structure of its units is highly variable. Another important source of heterogeneity is the number of fatty acids in the lipid A moiety, this, added to the length of their carbon chains are crucial points for the recognition of the molecule at the level of LPS receptors leading to inflammatogenic activities. Therefore, we are planning to determine i. whether type 2 diabetes is associated with structural changes in plasma LPS and what are the corresponding mechanisms, ii. whether there is a differential distribution of LPS within their main transporters: the lipoproteins. To answer these questions two Teams are gathering their skills and specialties: - Team 1 is specialized in LPS structural analysis in connection to biological activities, having a unique expertise in France for detection and analyses of LPS structures. Team 2, which discovered the increase of LPS diffusion into the blood of diabetes patients, has unique mouse models, as well as, in vivo, and in vitro lipidomics and phenotyping procedures to determine the causal link between the onset of metabolic diseases and LPSs. The preliminary data demonstrate that changes in LPS moieties are related to different inflammatory actions and that Rasltonia is a genus present in large amount in the adipose tissues from obese patients at risk to become type 2 diabetic. These joint efforts should allow a better understanding of the relationship between the structure of the LPSs and their function in diabetes. This project should lead to the identification of targets and biomarkers for the treatment and early diagnosis of metabolic diseases.

Nitrogen-fixing symbioses between legumes and rhizobia are extremely valuable in sustainable agriculture. They require the formation of a specific organ, the root nodule, where nitrogen fixation and nutritional exchanges between the plant and the bacteria take place. While spectacular progress has been made on early symbiotic stages (plant-rhizobium recognition, infection and initiation of nodule formation), the understanding of how later stages of nodule development are controlled is still very limited. A key process in nodule development is a developmental transition, the coordinated differentiation of plant and bacterial cells, in order to generate the appropriate microenvironment for symbiotic nitrogen fixation. This transition results from a massive reprogramming of gene expression, with thousands of genes affected in successive waves. We have discovered that epigenetic regulations, involving plant DNA (de)methylation and small interfering RNA (siRNA) populations, are essential to produce nitrogen-fixing nodules. Our working hypothesis in the EPISYM project is that these epigenetic regulations play an important role in gene expression reprogramming associated with nodule differentiation. The dynamics of the methylome and siRNA populations during nodulation will be analyzed at genome-wide level. The role of 3 genes coding for key enzymes in the control of DNA methylation and heterochromatic-siRNA production will be characterized, both in the model legume Medicago truncatula and pea, a closely related crop species, to better understand their regulation, their impact on nodule development and their target genes. Their response to environmental conditions known to impact nodule development will also be examined. In parallel, the genome-wide dynamics of histone marks will be analyzed, in connection with symbiotic gene expression, DNA methylation as well as small and long non-coding RNAs (lncRNAs). On one hand, the integration of these data will be highly valuable for the symbiosis community via a web-accessible genome browser linking expression data (mRNAs, siRNAs and lncRNAs) with epigenetic marks (DNA and histone methylation). On the other hand, we will analyse the chromatin topology of specific symbiotic genes to dissect the relationship between 3D chromatin structure and epigenetic regulation on the reprogramming of specific expression patterns in nodule differentiation. This project will thus take advantage of the strengths and complementarity of the two Partners to conduct innovative research on this emergent theme of broad interest in biology as well as on novel types of symbiotic regulators in order to provide new strategies to improve the efficiency of nitrogen-fixing symbioses.