Fungi are the most devastating pathogens of plants, including crops of major economic importance and have a significant impact in economics, human health and environment. In addition, fungi show an incredible plasticity and ability to disseminate and adapt rapidly to drastic changes in their environment. These processes of adaptation are exemplified by Leptosphaeria maculans which is an Ascomycete responsible for an economically important disease of oilseed rape, behaves as an invasive species and shows an extremely high evolutionary potential. Control of fungal crop diseases necessitates a global understanding of fungal pathogenicity determinants. During infection, pathogens, including fungi, secrete an arsenal of molecules, collectively called effectors, key elements of pathogenesis which modulate innate immunity of the plant and facilitate infection. Effectors have a dual role in microbe-plant interactions, both targeting plant components and being targeted by resistance (R) proteins and then termed avirulence (AVR) proteins. Fungal effector genes are very diverse and typically encode small proteins, predicted to be secreted (SSPs, Small Secreted Proteins), with no or low homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown and structure information provides an elegant way to resolve functional traits. From a limited set of data, the patterns that begin to emerge for possible fungal effector functions include interference with plant immunity, inhibition of programmed cell death, neutralization of host defence molecules and debilitation of plant tissues. On these bases, we propose a basic research project, StructuraLEP, to elucidate the involvement of L. maculans effectors in pathogenicity. We want to reach this objective through the structural and functional characterization of a few major effector proteins and the determination of their interactants. StructuraLEP is an interdisciplinary project involving two teams with internationally acknowledged expertise in the complementary fields of fungal genomics / fungal effector biology (Partner 1) and structural biology (Partner 2). We will investigate six L. maculans effectors corresponding to promising candidates previously identified by Partner 1 and chosen for their biological significance (involvement in fungal fitness, cognate R gene identified) or because they may represent novel modes of interaction with their plant target (two AVR genes necessary to be recognized by a specific R gene). The StructuraLEP project will combine structural biology, functional genomics, plant transformation and cytology and is organised in 3 main tasks (plus one coordination task) ranging from an initial task aiming to structurally and functionally characterise L. maculans effectors (Task 2), an exploratory task that relies on the screening of plant proteins and molecules potentially interacting with L. maculans effectors (Task 3) to a more focused task aiming to functionally and physically understand the identified interactions (Task 4). The effectors and their interactants will be analysed according to these complementary approaches to address the following questions: (i) What is the 3-D structure and the associated biochemical function of L. maculans effectors? (ii) Where do L. maculans effectors act during plant infection? (iii) Which molecules interact with L. maculans effectors? and (iv) Which plant processes are manipulated and which host proteins are targeted by L. maculans effectors? Integration of knowledge on effector 3-D structure, localization, targeting of host proteins and host processes by L. maculans effectors studied in the StructuraLEP project will give insight into the fundamental processes governing the close association of a pathogenic fungus with its host plant. Progresses in these fields are necessary for sustainable crop production through the development of novel strategies to control fungal diseases.

In addition to their role in protein synthesis by the ribosomes, aminoacyl-tRNAs participate in various metabolic pathways as a source of ester-activated amino acids. Among the tRNA-dependent aminoacyl transferases, enzymes of the Fem family catalyze an essential step of peptidoglycan synthesis in pathogenic bacteria and are considered as attractive targets for the development of novel antibiotics. FemX, the model enzyme of the family, transfers L-Ala from Ala-tRNA to the epsilon-amino group of L-Lys in the peptidoglycan precursor UDP-MurNAc-pentapeptide (UM5K). The crystal structures of the apo-enzyme and of a UM5K-FemX complex have been determined but co-crystallization with Ala-tRNA has not been obtained. We propose to develop the semi-synthesis of highly modified aminoacyl-tRNAs and bi-substrates to explore the catalytic mechanism of FemX. We will synthesize chemical probes that will specifically interact with FemX and its substrates. Azides and alkynes will be introduced into the tRNA and in UM5K, respectively. The Huisgen-Sharpless Cu(I)-catalyzed cycloaddition reaction will afford bi-substrates containing the tRNA covalently linked to the peptidoglycan precursor. In parallel, the active center of FemX will be used to catalyze the same reaction. By this approach, we will obtain molecules suitable for co-crystallization with FemX. Because the in situ generated reaction products are likely to trap a single conformational state of FemX corresponding to the catalytically active form of the enzyme, this approach is likely to be more powerful than the conventional crystallogenesis screens made with the substrates or products of the reaction. Phospho-derivatives of the tRNA will be synthesized to mimic the putative tetrahedral intermediate resulting from the intramolecular nucleophilic attack of the carbonyl of Ala-tRNA by the vicinal ribose hydroxyl. These phospho-derivatives will also be used to trap a relevant conformation of the enzyme that allows the trans-acylation reaction of the amino acid between the 2’ and 3’ positions of Ala-tRNA to occur within the active site. The enzyme-catalyzed cycloaddition reaction will be further investigated both to identify inhibitors of FemX and to decipher the mechanism of the enzyme-assisted catalyzed cycloaddition reaction, which is poorly understood. We will assess and compare the contributions of substrate binding and substrate activation (i) in the CuI- and FemX-catalyzed cycloaddition reactions using the functionalized substrates, and (ii) in the amino acid transfer reaction catalyzed by FemX with the “natural” substrates. The information gathered on the catalytic mechanism of FemX and on the structure of its active site should provide the critical information for the rationale design of drugs active on Fem transferases from pathogenic bacteria such as methicillin-resistant staphylococci. The approach will be of broad application in RNA biology.

The main goal of this french-taïwanese project is to identify genes and biochemical pathways involved in the development of lymphoproliferation leading to auto-immune responses in mice. The french coordinator of this project has produced, in collaboration with his french partner, a novel strain of C57Bl/6 (B6) mice bearing the lpr mutation (deficiency of death receptor Fas) and knock-out for the purinergic receptor P2X7 (P2X7R). On B6 genetic background, the lpr mutation induces a mild lymphoproliferation with limited autoimmune responses. In contrast, MRL/lpr mice develop a systemic autoimmune disease resembling systemic lupus erythematosus. The lack of P2X7R, in C57Bl/6, does not lead to any apparent disease. Interestingly, in B6/lpr-P2X7R KO mice, a strong lymphoproliferation appears with auto-immune responses currently under study. To determine which cell sub-populations are contributing to the disease found in B6/lpr, P2X7R KO, the french partners will produce mice which do not express P2X7R in T lymphocytes or dendritic cells (DC). Thus, CD4-Cre recombinase and CD11c-Cre recombinase mice will be mated with B6/lpr-P2X7R KO animals. In these new strains of mice, the disease severity will be evaluated by quantifying lymphoproliferation, measuring antibody and cytokine concentrations and assessing tissue damages. Thus, the respective roles of T lymphocytes and DC in the disease will be tested in these mouse strains. Recently, deep-sequencing technologies of cDNAs in order to determine a sample's RNA content, named RNA-Seq, have been developed as an approach for analyses of gene expression. By obtaining millions of reads of transcribed sequences, an RNA-Seq experiment can provide a comprehensive survey of the population of transcribed genes and their isoforms, revealing the molecular components of normal or pathological tissues. Statistical methods and bioinformatics for RNA sequence analyses have helped in the interpretation of this large amount data. The 2 taiwanese partners have a strong expertise in RNA seq and bioinformatics to analyze the levels of transcripts which are amplified or decreased in lymphoid organs or DC or sub-populations of T lymphocytes. These methods will be used to compare the transcriptomes of B6, B6/lpr, B6 P2X7R KO and B6 /lpr-P2X7R KO allowing us to identify the transcribed genes which are up- or down-modulated in the "autoimmune" B6/lpr-P2X7R KO mice. The same methods will be used to determine which genes, among those already identified, are preferentially up- or down-regulated in T lymphocytes or DC. Furthermore, the analyses performed in B6/lpr lacking P2X7R in T lymphocytes or DC only should help in defining which biological pathways are deregulated in cells lacking P2X7R and may unravel cellular synergies. Demonstrating that the identified genes ("driver genes") are involved in the autoimmune responses found in B6/lpr-P2X7R KO mice will require in vivo validation. To this end, several strategies may be used to determine whether these genes are involved in lymphoproliferation and/or autoimmunity :1) if a KO mouse for one of the identified genes exists, it will be mated with B6/lpr-P2X7R KO mice and the autoimmune responses will be evaluated in the B6 lpr/lpr, P2X7R KO carrying a novel nul allele; 2) injection into B6 lpr/lpr, P2X7R KO mice of lentiviruses encoding specific shRNA able to silence the expression of identified "driver gene"; 3) injection into B6 lpr/lpr, P2X7R KO mice of neutralizing monoclonal antibodies specific for the driver gene product(s). This ambitious collaborative project between taiwanese and french partners with complementary and multidisciplinary expertises should bring important information on the biological pathways involved in auto-immune responses and pathologies.