The Arabidopsis zygotic embryo, like that of almost all angiosperms, develops buried deep within the seed, nourished and protected by the maternally derived seed-coat and a second zygotic tissue, the endosperm. Although the endosperm plays important roles in ferrying nutrients from the maternal tissues to the developing embryo, and in determining the final size of the seed, recent work from our laboratory suggests that its presence may also generate developmental problems, which have had to be surmounted during angiosperm evolution. The angiosperm endosperm is thought to be a sexualized homologue of the gymnosperm female gametophyte, which also serves a nutritive role, but which develops before fertilization. In angiosperms seeds the simultaneous and closely juxtaposed development of the embryo and endosperm following fertilization may make it difficult for the embryo to establish a well-defined boundary, in the form of a properly cuticularized epidermal cell wall. This hypothesis is supported by recent published and unpublished data from our laboratory showing that a signalling pathway involving an endosperm specific protease (ALE1), and two embryo specific receptor kinases (GSO1 and GSO2) is necessary for the formation of a functional embryonic cuticle, and indeed for the stabilization of embryonic epidermal cell fate. This signalling pathway is not required for cuticle formation during post-germination growth, indicating that it is necessary only in the context of zygotic embryo development. Our recent transcriptional analyses of mutants defective in this signalling pathway, have provided tantalizing and novel clues that at least part of this cuticle reinforcing signalling pathway is closely allied to signalling pathways involved in innate immune responses, and particularly to systemic acquired resistance mechanisms in plants. The hypothesis that a form of “autoimmune” response is necessary for the formation of the boundary between the embryo and the endosperm in plants is entirely novel, and is basis for the proposed project. In this work we will develop novel tools allowing biochemical characterization of the seedling cuticle in Arabidopsis, a structure which remains almost entirely unstudied despite its important role in seedling survival. These tools will subsequently be applied to the analysis of mutant lines studied in this project. Using transcriptomic analysis, combined with spatial analysis of gene expression, we will determine where, both spatially and genetically, the ALE1-dependent innate immune response fits into the cuticle reinforcing signalling pathway which we have established. A candidate gene approach, combined with the analysis of a novel protein which we have recently shown is essential for the ALE1/GSO1/GSO2 signalling pathway, will permit the identification and characterization of further pathway components. Finally, we will establish the identities and biochemical functions of the downstream targets of the ALE1/GSO1/GSO2 signalling pathway, in order to understand more about the mechanisms underlying the biogenesis of the embryonic cuticle. In summary, our multidisciplinary approach will allow us not only to dissect in detail an entirely new and very striking example of how immune responses may have been harnessed by plants in order to fulfil developmental roles during the evolution of increasingly complex organs, but also provide novel and much needed understanding both of the composition of the embryonic cuticle, and of the effectors necessary for its biogenesis.

The development and use of cultivars genetically resistant to plant viruses, notoriously difficult to treat and combat, is a critical factor for sustainable agriculture. Potyvirus is one of the largest genera of plant viruses responsible for serious diseases in important vegetable and fruit crops. To invade plants, those obligatory parasites have developed tactics to reroute host cellular functions for their own benefits. The completion of the viral cycle results from a complex interplay between virus- and host-encoded factors, also called susceptibility factors. In this scheme, absence or non-adequacy of a single susceptibility factor leads to full or partial resistance to viruses. PotyMove partners were among the first to demonstrate this concept of loss-of-susceptibility resistance genes through the identification of eukaryotic initiation factors as key players in plant-potyvirus interactions, and demonstrated the “transferability” of a potential recessive resistance from the model plant Arabidopsis thaliana to crop plants. Considering the high adaptative potential of plant viruses and resulting resistance breakdown, a more extensive screen of host-virus protein-protein interactors will lead to the identification of new host factors conferring recessive resistance to potyviruses. The ability to combine in the same plant, mutations affecting eukaryotic initiation factors and newly identified genes involved in other steps of the viral cycle, will lead to higher durability of the resistance. In this context, the aim of PotyMove is to identify new plant factors involved in the virus cell-to-cell movement, a key-step of the viral infection. It is considered to be a major putative obstacle to viral exponential expansion in the plant by generating population bottlenecks and thus, an excellent target for resistance: indeed a defection of plant factors required by the virus for its cell-to-cell movement would cause the virus to be confined or restricted to its primary infection focus, thus delaying or preventing its systemic spreading. Also, by increasing the genetic drift, reduced cell-to-cell virus movement could increase resistance durability. Plasmodesmata (PD) are symplasmic tunnels between cells that are the gateway for plant virus movement. Pant virus genome encodes a class of proteins called Movement Protein that interact with host proteins to modify the PD for cell-to-cell movement. For the potyvirus genus, this key step is still very little documented. In particular, no dedicated movement protein has been identified, but three viral proteins with other known functions have been reported to participate in potyvirus movement. Based on a unique consortium with high complementary and transdisciplinary skills (virology, biochemistry, PD proteomics, and genetics), the PotyMove project aims at identifying membrane-associated and/or PD proteins that interact with these three key viral proteins. In parallel, a high throughput genetic approach complementary to PD proteomics will be performed: using forward genetic based on natural diversity or induced diversity, and on quantitative phenotyping, we will identify candidate genes involved in cell-to-cell movement. Furthermore, the PotyMove project aims at providing experimental evidence that the identified candidate genes can afford a broad-range durable resistance to potyviruses and can be transferred to a crop species, tomato, using genome editing strategy. This will be applied to the two initial available candidates, Remorin and PrepP, at the beginning of the PotyMove project. The potential higher durability of those new resistance genes will rely on the fact that they target viral movement proteins known to be under evolutionary constraint and can be further used in crop species for gene pyramiding with previously identified translation initiation factors, in order to implement multifaceted, durable and consumer-acceptable resistances to virus infection.

Trypanosoma brucei is a unicellular and extracellular parasite transmitted by the tsetse fly (Glossina genus) and causing Human African Trypanosomiasis (HAT) in Sub-Saharan Africa. If untreated, this neglected tropical disease has a case fatality rate close to 100%. There is no vaccine and the currently available drugs present significant side-effects, variable efficacies and/or do not cross the blood brain barrier. Other related parasite species are responsible for animal African trypanosomiases that remain a major constraint to productive livestock rearing in countries throughout Sub-Saharan Africa, causing major economic losses. It has been considered for decades that trypanosomes propagate exclusively in the body fluids of their mammalian hosts and mostly in the blood. In a break with this dogma, three recent publications showed that most parasites actually reside in the extravascular compartment of mouse models, especially in the adipose tissues and the skin, from which transmission can occur. Within the skin, some parasites seem to tightly interact with adipocytes, the major constituent of fat, suggesting that the adipocytes-trypanosomes interactions might confer a selective advantage to the parasites. In resonance with this major discovery, we recently broke another strong dogma considering that T. brucei relied exclusively on the glucose provided by the mammalian host blood to feed its central carbon metabolism. Indeed, we established long-term conditions for growth of the parasite in glucose-free medium containing glycerol. Since adipocytes excrete large amounts of glycerol from lipolysis and glycolysis, we hypothesised that extravascular trypanosomes could take advantage of this glycerol production to (i) feed their central carbon metabolism and (ii) colonize the glycerol-rich tissues by chemotactism. Our preliminary data presented in details in the application support these two hypotheses. The objectives of this project are (i) to evaluate the importance of the glycerol metabolism for parasites in vivo, including a potential role of the glycerol as a metabolic sensor to attract trypanosomes in specific tissues, (ii) to develop an in vitro assay to study the metabolic interactions between adipocytes and trypanosomes, (iii) to determine the probable alternative(s) to FBPase, the key gluconeogenic enzyme, as well as the role of the parasite gluconeogenesis in vivo in the mammalian host, (iv) to confirm the existence and determine the role of ß-oxidation of fatty acids previously proposed to be activated in tissue-dwelling parasites, and (v) to identify the most relevant metabolic step(s) targeted by suramin. All these questions will be tackled by leading experts in complementary fields forming a long-standing network. This project will have major impacts in understanding the biology of tissue trypanosomes. It will also pave the way for the development of therapeutic approaches targeting extravascular parasites.

Plasmodesmata (PD) are nano-scaled membrane-lined channels PD that span the thick cell wall of virtually all plant cells, establishing both cytoplasmic and membrane continuity throughout the entire plant body (Fig.1). In recent years, PD have emerged as key elements of the cell-to-cell communication machinery and as such have been implicated in processes guaranteeing the collaborative functioning of the cells, and controlled developmental events. Plant viruses but also fungus can exploit PD transport machinery to establish infection and the emerging view is that PD may well represent a consensus target for pathogens and constitute key element in defence signalling and plant development. Understanding of how PD dictate cellular connectivity is dependent on comprehensive knowledge of the composition of PD and functional characterization of their constituents. Although some progress has been made in the identification of PD proteins, the role played by major membrane constituents, e.g. the lipids, remains totally uncovered. Yet PD are primarily membranous structures, defined by specialised membrane domains of the endoplasmic reticulum (desmotubule) and the plasma membrane (PD-PM) and the majority of identified PD proteins are membrane-associated. Research in biological membrane over the last decade has unequivocally demonstrated that lipids are functional units that can modulates membrane organisation and cellular function. Just as proteins, lipids are likely to be key elements of PD specialised membrane domains and as such contribute to proper functionality at PD. In line with that recent data suggests that the PM domain lining PD may share similar properties to membrane rafts, high ordered sterol- and sphingolipid-enriched nanodomains of the PM involved in signalling, protein targeting and lateral segregation of membrane constituents. Hence, several raft markers have been shown not only to localise to microdomains at the PM per se but also accumulates at PD. Moreover recent data suggest that rafts could play a central role in the control of viral cell-to-cell spray through the PD channels. Altogether, this raises questions about the lipidic nature of the PD-PM. Do rafts contribute to the PD-PM? What is the role of lipids in defining PD membrane structurally and functionally? The CONNECT project concentrates on the functional relationship that may exist between PD and rafts, as well as the implication of lipids in PD function and structure. Using suspension-cultured cells, to isolate pure PD, we propose for the first time to analyse the lipid composition of these unique membrane structures. Coupling lipidomic, biochemical approaches, spectrometric analyses as well as high-resolution microscopy we would like to establish whether or not raft-like domains are enriched at PD. Is the specialised PD-PM domain sharing properties similar to rafts; i.e. highly organised and enriched in sterols and sphingolipids? Is lipid segregation along PD-PM fundamental for proper PD function? In particular the role of lipids in PD targeting will be investigated by modifying their sterol composition. Last we propose to examine the involvement of rafts in the control of Potato virus X movement via PD. In particular, we make the hypothesis that rafts may be involved in the control of PD permeability and that raft aggregation occurs around PD during viral infection.

Plant life depends on cell-to-cell connectivity through plasmodesmata (PD) channels. PD are indispensable for plant development, environmental adaptation and defence signalling. A striking feature of PD organisation, setting them apart from animal cell junctions, is a strand of endoplasmic reticulum (ER) running through the pore, tethered extremely tight (~10nm) to the plasma membrane (PM) by unidentified “spokes”. This classifies PD as a specialised type of membrane contact site (MCS), whose function remains a mystery. Combining interdisciplinary approaches in membrane biophysics, molecular cell biology and electron tomography, we propose a pioneer research program, which will for the first time address the mechanisms and function of ER-PM tethering at PD. We aim to 1)Identify/characterise PD membrane-tethering elements at atomic level 2)defines the role of anionic lipids in PD MCS regulation 3)Uncover the function of ER-PM apposition for plant intercellular communication