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International audience; Leptosphaeria maculans is a phytopathogenic fungus being responsible for a damaging disease of oilseed rape (Brassica napus): stem canker. The disease is mainly controlled by plant genetic resistance: single-gene specific resistance or quantitative, adult-stage resistance. During its particularly complex and long infectious cycle, L. maculans colonizes asymptomatically the stems of oilseed rape, producing late effectors specific to this colonization stage. In the context of a strong need to identify new sources of disease resistance, we exploited the repertoire of ‘late’ effectors to identify genes in the plant that could contribute to quantitative disease resistance. Our hypothesis was that quantitative resistance partly rely on gene-for-gene interactions, with fungal effectors produced during stem infection being recognized by resistance proteins. Using an innovative strategy of early expression of late effector genes, we validated that the interaction between the late effector LmSTEE98 and the resistance RlmSTEE98 obeys a typical gene-for-gene interaction, occurring during the colonization of oilseed rape stems by L. maculans, that contributes partly to quantitative resistance, in controlled conditions. We then used the same strategy to search for new sources of resistance after having established criteria to select the most relevant late effectors, and chosen ten of these for screening. Our screening approach of 130 diversified genotypes representative of the available diversity of B. napus, allowed us to identify new sources of resistance, displaying diversified interaction phenotypes. The next steps of this project now are further validation of the efficacy of the new sources of resistance in the field and of the validity of the quantitative resistance markers. However, as it stands, our results demonstrate the existence of unsuspected sources of resistance that are potentially more durable than the classic major genes expressed early after penetration in plant tissues.

Cet ouvrage collectif propose une réflexion de politistes et de sociologues qui s’adosse aux travaux de Daniel Gaxie. C’est l’occasion de reprendre le débat autour des nombreux domaines qu’il a défrichés, qu’il s’agisse de travaux sur la représentation et la professionnalisation politiques, sur la politisation des « profanes », sur le militantisme et ses rétributions ou encore sur les luttes au principe de l’action publique. Le pari était le suivant : montrer comment cette sociologie qui, dans les années 1970, dévoilait les ressorts sociaux d’un enchantement largement partagé de la politique démocratique, montrant la faible démocratisation du recrutement des représentants, définissant les contours d’un « cens caché », critiquant une vision héroïsée de l’action publique, pouvait analyser aujourd’hui un monde politique nouveau, largement désenchanté (« crise » de la représentation, suspicions autour de l’activité politique, jugements désabusés sur son impuissance…). La fameuse lutte contre les prénotions qui habitait le métier de sociologue doit désormais emprunter des chemins beaucoup plus escarpés. Les textes réunis posent frontalement la question de l’actualisation des apports de cette sociologie politique et s’interrogent sur les manières de la renouveler en soulignant des manques, en suggérant des prolongements et en proposant des pistes pour l’avenir.

Située à l’intersection de deux aires de recherche qui tendent à s’ignorer, l’enquête à l’origine de cette contribution vise à combler une double cécité. La première touche la sociologie électorale qui néglige de prendre en considération les groupes d’intérêt et les mouvements sociaux. La seconde atteint les travaux sur les groupes d’intérêt qui n’intègrent pas les agents politiques à leurs analyses et tendent à les concevoir comme passifs dans la relation d’influence ou de « lobbying ». Il s...

La mobilisation électorale des groupes d’intérêt est largement ignorée de la littérature académique européenne. Ce chapitre explore les différents types d’intervention des groupes, en revenant sur l’histoire des mobilisations électorales. Exposant les biais analytiques des travaux sur l’influence, il propose de réintégrer les agents politiques et leur entourage dans l’étude de cette relation et de se pencher, enfin, sur les pratiques concrètes du jeu d’influence, qui relèvent d’une forme de division du travail de production des enjeux électoraux.

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National audience; Des décennies de casse sans relâche : les dernières crises sanitaire et économique en montrent l’ampleur et les dangers. Mais qui veut la peau du service public ? Pourquoi, et au détriment de qui ? Qui sont les commanditaires et les exécuteurs du massacre en cours au nom de la modernisation ? Quels sont leurs certitudes, leur langage, leurs bonheurs et leurs tourments ? Comment s’en tirent les agents du service public quand leurs métiers deviennent missions impossibles ? Comment s’en sortent les usagers quand l’hôpital est managérialisé, quand les transports publics sont dégradés ? Ce livre raconte les services publics : ceux qui ont fait vivre des villages et ceux qui ont enrichi des entreprises, les guichets où on dit « non » et ceux qui donnent accès à des droits. Il combat les fausses évidences qui dévalorisent pour mieux détruire – les fonctionnaires trop nombreux, privilégiés, paresseux. Il mène l’enquête pour dévoiler les motifs des crimes et leurs modes opératoires, des projets de réforme à leurs applications. On entre dans les Ehpad, aux côtés des résidents et du personnel soignant, on pousse la porte des urgences, on se glisse dans les files d’attente de la CAF ; on s’aventure dans les grandes écoles, on s’infiltre dans les clubs des élites, au gré de récits et d’images qui présentent les recherches universitaires les plus récentes.

Early Access; International audience; The control of stem canker disease of Brassica napus (rapeseed), caused by the fungus Leptosphaeria maculans is largely based on plant genetic resistance: single-gene specific resistance (Rlm genes) or quantitative, polygenic, adult-stage resistance. Our working hypothesis was that quantitative resistance partly obeys the gene-for-gene model, with resistance genes "recognizing" fungal effectors expressed during late systemic colonization. Five LmSTEE (stem-expressed effector) genes were selected and placed under the control of the AvrLm4-7 promoter, an effector gene highly expressed at the cotyledon stage of infection, for miniaturized cotyledon inoculation test screening of a gene pool of 204 rapeseed genotypes. We identified a rapeseed genotype, 'Yudal', expressing hypersensitive response to LmSTEE98. The LmSTEE98-RlmSTEE98 interaction was further validated by inactivation of the LmSTEE98 gene with a CRISPR-Cas9 approach. Isolates with mutated versions of LmSTEE98 induced more severe stem symptoms than the wild-type isolate in 'Yudal'. This single-gene resistance was mapped in a 0.6 cM interval of the 'Darmor_bzh' x 'Yudal' genetic map. One typical gene-for-gene interaction contributes partly to quantitative resistance when L. maculans colonizes the stems of rapeseed. With numerous other effectors specific to stem colonization, our study provides a new route for resistance gene discovery, elucidation of quantitative resistance mechanisms, and selection for durable resistance.This article is protected by copyright. All rights reserved.

Osteoarthritis, the leading cause of chronic joint pain, is studied through different animal models, but none of them is ideal in terms of reliability and translational value. In this pilot study of female rats, 3 surgical models of osteoarthritic pain,L’arthrose, la principale cause de douleur chronique articulaire, est étudiée à travers différents modèles animaux, mais aucun d’eux n’est idéal en termes de fiabilité et de valeur translationnelle. Trois modèles chirurgicaux de douleur arthrosique, c’est-à-dire, la déstabilisation du ménisque médial, la transsection du ligament croisé crânial et la combinaison des deux, ainsi qu’un modèle chimique (injection intraarticulaire de mono-iodoacétate de sodium) ont été comparés dans cette étude pilote chez des rattes quant à leurs impacts sur les évaluations fonctionnelles de la douleur (distribution pondérale statique, évaluation ponctuelle de l’allodynie tactile) et les neuropeptides spinaux (substance P, peptide relié au gène de la calcitonine, bradykinine et somatostatine). Six rats ont été assignés à chacun des modèles et un groupe Sham. Autant le modèle du mono-iodoacétate de sodium que celui de la combinaison chirurgicale ont tous les deux induits des altérations fonctionnelles de la distribution pondérale statique et du seuil de retrait de la patte suite à une stimulation ponctuelle tactile, mais avec des changements plus persistants dans le groupe de la combinaison chirurgicale. Ces deux modèles ont également engendré une augmentation des niveaux en neuropeptides pro-nociceptifs et anti-nociceptifs à différents moments. Un intérêt du modèle chirurgical a été démontré suite à la comparaison de la douleur avec le modèle du mono-iodoacétate de sodium, en particulier la déstabilisation du ménisque médial combinée à la transsection du ligament croisé crânial, tandis que les inductions chirurgicales unique entraînaient des altérations fonctionnelles temporaires avec aucun changement neuropeptidomique.(Traduit par les auteurs).

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Leptosphaeria maculans is a pathogenic fungus, responsible for one of the main diseases of oilseed rape (Brassica napus), the stem canker disease. The infectious life cycle of L. maculans is especially complex. After the primary infection of leaves and cotyledons, the fungus develops a long endophytic stage in the stem. This infection stage, which is entirely asymptomatic, lasts several months before necrosis develops at the stem base, responsible of yield loss. At this stage, the oilseed rape may exhibit "adult resistance" limiting the onset and severity of symptoms. While the fungal genes expressed during the primary infection are extensively studied, very little knowledge was available concerning the systemic colonization. To explain the ability of the fungus to colonize the stem without inducing symptom, we have therefore hypothesized that L. maculans expressed effectors, i.e. small secreted proteins, interfering with the plant defense system.The objective of my thesis was to identify such effectors and to characterize them to better understand the systemic colonization of oilseed rape by L. maculans. One of the underlying challenges of this thesis was also to identify new resistances allowing the specific recognition of these effectors expressed during stem colonization, and which may explain, at least in part, the adult resistance observed in some varieties.Using a transcriptomic approach, I was able to identify 307 "late" effector candidates specifically expressed during stem colonization and 107 "early" effector candidates specifically expressed during cotyledon colonization. I confirmed that the genes encoding early effectors of L. maculans are specifically localized in gene-poor regions and rich in repeated elements of the fungal genome. Conversely, late candidate effectors are absent from these regions and are located in regions rich in genes of the genome. L. maculans effectors have thus a distinct genomic localization based on their expression profile.A detailed analysis of five of these late effectors showed their conservation in the natural populations of L. maculans and their involvement in the suppression of plant cell death. These results, associated with the analysis of their expression profile in stem samples from the fields during a growing season, allowed us to propose the following model: L. maculans would colonize systemically the oilseed rape stem by secreting effectors suppressing cell death and thus interfering with plant defenses. At the end of the growing season, the decreased expression of these effectors would allow the fungus to switch from a biotrophic to a necrotrophic lifestyle and to induce stem canker. This transition between the two ways of life would therefore be based on a balance between effectors suppressing and effectors inducing cell death.In order to identify new specific sources of resistance and / or to facilitate the identification of quantitative resistances in plant material, I created fungal strains over-expressing late effectors during cotyledon colonization. With these transformed strains I evaluated by cotyledonary test a large collection of oilseed rape genotypes to identify potential gene-for-gene. A variety with a hypersensitive response to a late effector was thus identified, the monogenic control of this response was validated and its genetic mapping carried out in two progenies. This approach therefore effectively enables the identification of new sources of resistance for effective control of L. maculans.; Leptosphaeria maculans est un champignon pathogène, responsable de l’une des principales maladies du colza (Brassica napus), la nécrose du collet. Le cycle de vie infectieux de L. maculans est particulièrement complexe. Après l’infection primaire des feuilles et des cotylédons, le champignon développe une longue phase de vie endophytique dans la tige. Cette phase de vie, qui est entièrement asymptomatique, dure plusieurs mois, avant que la nécrose ne se développe à la base de la tige, préjudiciable à l'élaboration du rendement. Durant cette phase, le colza peut présenter une « résistance adulte » limitant l'apparition et la gravité des symptômes. Alors que les gènes fongiques exprimés au cours de l’infection primaire du colza sont largement étudiés, très peu de connaissances étaient disponibles concernant la phase de colonisation systémique. Pour expliquer la capacité du champignon à coloniser la tige sans induire de symptômes, nous avons donc émis l’hypothèse que L. maculans exprimait à ce stade des effecteurs, c'est-à-dire des petites protéines sécrétées, interférant avec le système de défense de la plante. L’objectif de ma thèse était d'identifier de tels effecteurs et de les caractériser afin de mieux comprendre la colonisation systémique du colza par le champignon. Un des enjeux sous-jacents de cette thèse était aussi d'identifier de nouvelles résistances permettant la reconnaissance spécifique de ces effecteurs, qui pourraient expliquer, au moins en partie, la résistance adulte observée dans certaines variétés.Par une approche transcriptomique, j'ai pu identifier 307 effecteurs candidats "tardifs", spécifiquement exprimés lors de la colonisation de la tige et 107 effecteurs "précoces", spécifiquement exprimés lors de la colonisation des cotylédons. J’ai confirmé que les gènes codant des effecteurs précoces de L. maculans sont spécifiquement localisés dans les régions pauvres en gènes et riches en éléments répétés du génome fongique. A l'inverse les gènes codant des effecteurs candidats tardifs sont absents de ces régions et sont localisés dans les régions riches en gènes du génome. Les effecteurs de L. maculans ont donc une localisation génomique distincte en fonction de leur profil d'expression.Une analyse approfondie de cinq de ces effecteurs tardifs a permis de montrer leur conservation dans les populations naturelles de L. maculans et leur implication dans la suppression de la mort cellulaire végétale. Ces résultats associés à l'analyse de leur profil d'expression dans des échantillons de tige issus du champ au cours d'une saison culturale ont permis de proposer le modèle suivant: L. maculans coloniserait la tige de colza en sécrétant des effecteurs supprimant la mort cellulaire et donc interférant avec les défenses de la plante. A la fin de la saison culturale, la diminution de l'expression de ces effecteurs permettrait au champignon de passer d'un stade de vie biotrophe à un stade de vie nécrotrophe et d’induire la nécrose au collet. Cette transition entre les deux modes de vie serait donc basée sur un équilibre entre effecteurs supprimant la mort cellulaire et effecteurs induisant la mort cellulaire.Dans le but d'identifier de nouvelles sources de résistance spécifiques et/ou faciliter l’identification de résistances quantitatives dans le matériel végétal, j'ai créé des souches fongiques sur-exprimant précocement des effecteurs tardifs. Avec ces souches transformées j'ai évalué par test cotylédonnaire une grande collection de génotypes de colza pour identifier de potentielles relations de type gène-pour-gène. Une variété présentant une réponse hypersensible à un effecteur tardif a ainsi pu être identifiée, le contrôle monogénique de cette réponse a été validé et sa cartographie génétique effectuée dans deux descendances. Cette approche permet donc effectivement d'identifier de nouvelles sources de résistances pour lutter efficacement contre la nécrose du collet.

L’étiologie exacte de la douleur arthrosique n’est que partiellement comprise et le recours à des modèles animaux est fondamental dans l’investigation physiopathologique du modèle nociceptif et le développement de modalités thérapeutiques, en particulier lors de la création d’un modèle de douleur chronique pathologique. Dans l’investigation de la douleur arthrosique du genou chez le rat, l’utilisation d’un modèle par injection intra-articulaire de mono-iodo-acétate de sodium (MIA) est surreprésentée. Cependant, ce modèle ne représente pas la maladie naturelle chez l’humain puisque l’atteinte est aiguë, induite chimiquement, et temporaire. Nous émettons l’hypothèse que les modèles chirurgicaux possèdent l’avantage d’utiliser des mécanismes mimant la condition naturelle dans l’arthrose post-traumatique. Le modèle de transsection du ligament croisé crânial, de déstabilisation du ménisque médial (par méniscotomie médiale partielle) et leur combinaison ont été évalués lors d’une étude pilote avec l’objectif de comparer les données fonctionnelles et les neuromarqueurs spinaux entre les modèles chirurgicaux et MIA. L’induction chirurgicale a été accompagnée de changements neurophysiologiques quantifiables liés à la douleur pathologique et persistant plus longtemps que le modèle MIA, représentant ainsi plusieurs avantages pouvant être appliqués lors de l’évaluation de modalités thérapeutiques. Les modèles chirurgicaux simples se sont révélés sans intérêt car ils présentent des atteintes fonctionnelles temporaires et sans modification neuroprotéomique., The exact aetiology of osteoarthritic pain is only partially understood and the use of animal models is fundamental in the pathophysiological investigation and the development of therapeutic modalities, particularly the creation of a pathological chronic pain model. In the investigation of OA knee pain in rats, the intra-articular injection of sodium mono-iodo-acetate (MIA) is an overrepresented model. However, this does not represent the natural disease in humans since the disease is acute, chemically induced, and temporary. We hypothesized that surgical models have the advantage of using mechanisms with better resemblance to the natural post-traumatic osteoarthritis. The model of transection of the cranial cruciate ligament, destabilization of the medial meniscus (partial medial meniscotomy) and their combination were evaluated in a pilot study with the objective to compare the pain behaviour and spinal neuromarkers between the surgical and MIA models. Surgical induction was accompanied by measurable neurophysiological changes associated with pathological pain and lasting longer than the MIA model, an advantage that can be applied in the evaluation of therapeutic modalities. Simple surgical models have proved to be without interest as they present temporary functional impairment without neuroproteomics modification.

The stem canker disease, caused by Leptosphaeria maculans, is one of the most devastating diseases of oilseed rape (canola). It colonizes the plant in two stages: a short and early colonisation stage corresponding to cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically and symptomlessly the plant during several months before stem canker appears. To date, determinants of the late colonisation stage remain poorly understood. By a transcriptomic approach, we previously identified two waves of effector candidate expression during the early and late colonisation stages (Gervais et al, 2016). The late effector candidates are located in gene-rich genomic regions, whereas the early effector genes are located in gene-poor regions of the genome. Among the late effector candidates identified, we selected 6 genes for further characterization. We created mutants silenced for these effector candidates. For one of these genes, its expression level correlated negatively with the size of the necrosis observed in the stem. The identification of new effector genes would contribute to the identification of new resistance genes specific to these effectors. To easily identify matching resistance genes in oilseed rape, we created transgenic isolates expressing these 6 late effectors at the early steps of infection to provide medium-throughput strategies to screen more efficiently different cultivars. Preliminary results indicate that some cultivars with adult resistance were more resistant to these transgenic isolates in cotyledon assays. With this approach, we also identified a cultivar carrying a specific resistance to one these 6 effector candidates. Reference Gervais, J., Plissonneau, C., Linglin, J., Meyer, M., Labadie, K., Cruaud, C., Fudal, I., Rouxel, T. and Balesdent, M.H. (2016) Different waves of effector genes with contrasted genomic location are expressed by Leptosphaeria maculans during cotyledon and stem colonization of oilseed rape. Mol. Plant Pathol.

National audience; Plant pathogens secrete effector proteins into host tissues to promote infection through the manipulation of host processes. Sequencing and analyses of the genomes of fungal phytopathogens have shown that they contains tens to hundreds of genes predicted to encode putative effectors. Moreover, global analyses of gene expression revealed that several waves of concerted expression of effector genes take place during host invasion. In sharp contrast with the situation described inplanta, the expression of the effectors is difficult to detect and quantify in axenic cultures because their genes are expressed at a very low level. In the present study, we investigate biotic and abiotic factors that may relieve suppression of expression of effectors during axenic growth. Biotic factors (such as carbon source, nitrate source, antibiotics) as well as abiotic factors (pH, temperature) can influence their expression. Of major interest, incubation of the fungal mycelium with 1ug/ml of an antibiotic of the aminoglycoside family allowed an increase of effector gene expression 20-fold to 60- fold compared to regular axenic growth. An RNAseq analysis aiming at identifying the set of effectors up- and down-regulated in a culture medium supplemented or not with the antibiotic has been performed and results obtained will be presented. This simple system could be a good starting point to characterize the plant signals that trigger fungal effector gene expression.

Leptosphaeria maculans, the fungus responsible for stem canker of oilseed rape (Brassica napus), presents a particular genome structure divided into gene-rich and transposable element (TE)-rich regions. The latter are proposed to be heterochromatic domains, as they are gene-poor, TE-rich and present lower recombination rates than gene-rich regions. While overall gene-poor, TE-rich regions are enriched for putative effector genes specifically expressed during the early stages of plant infection (‘early’ effector genes). In contrast, effector genes expressed during the late stages of infection (endophytic growth and stem necrosis, ‘late’ effector genes) are located in gene-rich regions. We investigated the concerted expression of ‘early’ effector genes by assessing whether TE-rich regions could be targets of reversible chromatin modifications that affect the regulation of effector gene expression. We first examined the involvement of one histone modification, histone H3 lysine 9 tri-methylation (H3K9me3) in chromatin-based regulation of effector gene expression. For this purpose, we silenced expression of two key players in heterochromatin assembly and maintenance, HP1 and KMT1, by RNAi. Silencing of HP1 and KMT1 led to an over-expression of ‘early’ effector genes in TE-rich regions during in vitro growth (but no induction of ‘late’ effector gene expression). The increase of expression was associated with a reduction of H3K9 tri-methylation at two effector-encoding gene loci. Our data strongly suggest that a chromatin-based control, mediated by HP1 and KMT1, represses the expression of at least part of the ‘early’ effector genes located in TE-rich regions during growth in axenic culture. We are currently investigating the role of another key player in heterochromatin assembly, KMT6, involved in the heterochromatic-associated histone modification H3K27me3, on the control of effector gene expression

International audience; Fungal effector genes are very diverse and typically encode small proteins, predicted to be secreted, with no or low homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. On these bases, the StructuraLEP project aims at elucidating the involvement of L. maculans effectors in pathogenicity through the structural and functional characterization of a few major effector proteins and the determination of their interactants. We are investigating six L. maculans effectors 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 have to be recognized by a specific R gene). We present here the strategies developed within the project to answer two questions: (i) “Where do L. maculans effectors act during plant infection?” and (ii) “Which proteins interact with L. maculans effectors?”. In order to localise L. maculans effectors into plant cells and to identify their plant targets, we will transiently express effectors with a fluorescent tag into tobacco leaf epidermal cells and observe effector localisation by confocal microscopy. Tobacco leaves expressing effectors will be used to perform pull-down assays and tobacco proteins interacting with effectors will be identified through mass spectrometry. A more exploratory but biologically relevant strategy will also be tested. L. maculans transformants stably expressing effectors with HA-tag will be used to infect oilseed rape leaves. Localisation of effectors in oilseed rape leaves will be performed by immunocytolocalisation using antibodies against the HA-tag. The infected oilseed rape leaves will also be used to perform pull-down assays in order to identify plant proteins targeted by L. maculans effectors.

Leptosphaeria maculans, the causal agent of stem canker disease, colonizes oilseed rape (Brassica napus) in two stages: a short and early colonization stage corresponding to cotyledon or leaf colonization, and a late colonization stage during which the fungus colonizes systemically and symptomlessly the plant during several months before stem canker appears. To date, the determinants of the late colonization stage are poorly understood; L. maculans may either successfully escape plant defences, leading to stem canker development, or the plant may develop an ‘adult‐stage’ resistance reducing canker incidence. To obtain an insight into these determinants, we performed an RNA‐sequencing (RNA‐seq) pilot project comparing fungal gene expression in infected cotyledons and in symptomless or necrotic stems. Despite the low fraction of fungal material in infected stems, sufficient fungal transcripts were detected and a large number of fungal genes were expressed, thus validating the feasibility of the approach. Our analysis showed that all avirulence genes previously identified are under‐expressed during stem colonization compared with cotyledon colonization. A validation RNA‐seq experiment was then performed to investigate the expression of candidate effector genes during systemic colonization. Three hundred and seven ‘late’ effector candidates, under‐expressed in the early colonization stage and over‐expressed in the infected stems, were identified. Finally, our analysis revealed a link between the regulation of expression of effectors and their genomic location: the ‘late’ effector candidates, putatively involved in systemic colonization, are located in gene‐rich genomic regions, whereas the ‘early’ effector genes, over‐expressed in the early colonization stage, are located in gene‐poor regions of the genome.

International audience; Leptosphaeria maculans, a hemibiotrophic fungus responsible of stem canker, colonises oilseed rape in two stages: an early stage of cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically without visible symptom the plant before stem canker appears. L. maculans presents a bipartite genome structure alternating gene-rich and transposable element (TE)-rich regions. TE-rich regions, which encompass one third of the genome, are enriched in putative effector-encoding genes that present the same expression pattern (no or a low expression level during in vitro growth and a strong over-expression during early infection in cotyledons and leaves; ‘early’ effectors). In contrast, gene-rich regions were recently reported to contain putative effector-encoding genes specifically expressed during the late stages of stem infection (‘late’ effectors). We have previously investigated the involvement of the chromatin structure of repeat-rich regions on the expression of ‘early’ effector genes: RNAi silencing of two genes encoding key players in heterochromatin assembly through histone modification H3K9me3, HP1 and KMT1, induced an over-expression of genes located in AT-isochores, particularly ‘early’ effector genes but no modification of ‘late’ effector genes expression. Here, we performed analysis of nucleosome positioning, chromatin structure and gene expression at the genome scale combining MAINE-seq, ChIP-seq and RNA-seq data during in vitro growth of L. maculans. We analysed in vitro ChIP-seq data targeting heterochromatin modifications, H3K9me3 and H3K27me3, and a euchromatin mark, H3K4me2 and found that gene-rich regions are associated with H3K4me2 and H3K27me3 while TE-rich regions are associated with H3K9me3. Effector genes are also associated with distinct heterochromatin marks according to their genomic location and expression kinetics: while ‘early’ effector genes located in TE-rich regions are associated with H3K9me3, ‘late’ effector genes located in gene-rich region are associated with H3K27me3. Genome-wide nuclesome positioning was analysed using MAINE-seq data, showing distinct nucleosome organization for genes located in TE-rich or gene-rich regions, and according to gene expression level. Finally, we recently investigated the role of another key player in heterochromatin assembly, KMT6, involved in the heterochromatic-associated histone modification H3K27me3, on the control of effector gene expression. RNAi silencing of KMT6 leads to a deregulation of genes not only associated with H3K27me3 in the wild type strain, suggesting a relocation of different histone modifications

National audience

National audience; Fungal effector genes are very diverse and typically encode small proteins, predicted to be secreted, with no or low homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. On these bases, the StructuraLEP project aims at elucidating the involvement of L. maculans effectors in pathogenicity through the structural and functional characterization of a few major effector proteins and the determination of their interactants. We are investigating six L. maculans effectors 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 have to be recognized by a specific R gene). We present here the strategies developed within the project to answer two questions: (i) “Where do L. maculans effectors act during plant infection?” and (ii) “Which proteins interact with L. maculans effectors?”. In order to localise L. maculans effectors into plant cells and to identify their plant targets, we will transiently express effectors with a fluorescent tag into tobacco leaf epidermal cells and observe effector localisation by confocal microscopy. Tobacco leaves expressing effectors will be used to perform pull-down assays and tobacco proteins interacting with effectors will be identified through mass spectrometry. A more exploratory but biologically relevant strategy will also be tested. L. maculans transformants stably expressing effectors with HA-tag will be used to infect oilseed rape leaves. Localisation of effectors in oilseed rape leaves will be performed by immunocytolocalisation using antibodies against the HA-tag. The infected oilseed rape leaves will also be used to perform pull-down assays in order to identify plant proteins targeted by L. maculans effectors.

Ecotoxicological impact of an antibiotic mixture on soil nitrogen cycling microbial guilds. First International Conference on Risk Assessment of Pharmaceuticals in the Environment (ICRAPHE)

Background and objectivesThe fate and effects of human and veterinary antibiotics in the environment have been the subject of intense investigation for nearly two decades. While the occurrence and persistence of antibiotics and antimicrobial resistance in environment have become major human health and research issues, their impacts on microbial ecosystem processes (i.e. nutrient recycling) are not well-understood. Many gaps still persist regarding the ecotoxicological assessment of antibiotics as related to environmental risk assessment (ERA). ERA researches have been mainly focused on normalized single species bioassay, and current ecotoxicological studies should aim at bridging the gap existing in the understanding and assessment of antibiotic impact on microbial ecosystem processes. The ERA methodologies for antibiotics have been developed based on the current standard ecotoxicology tests existing guidelines for other chemicals, but such regulatory assessment has been questioned (Boxall et al., 2012). Actually, the modes of action of antibiotics are mainly specific for bacteria, but for bacterial toxicity tests play only a minor role during the ecotoxicological assessment of antibiotics (Kümmerer et al., 2004; Brandt et al., 2015). Consequently, this may overlook adverse effects of antibiotics on microbial ecosystem functions. This issue additionally requires the characterization of the community – function relationships, to identify sensitive, resistant and resilient populations, and the main edaphic factors controlling antibiotic availability and thus microbial exposure to antibiotic. The growing interest for organic wastes recycling as soil amendments, can lead to various diffuse antibiotic contaminations, requiring an ecotoxicological assessment of such agricultural practice. The scope of this work aims at identifying microbial community-based tests and endpoints to improve targeted protection of key microbial soil processes and also unravel the diversity – function relationships involving in the resilience of soil microbial functioning, following application of contaminated amendments. Methods and resultsHere, we investigated the effects of sulfamethoxazole (SMX) on microbial nitrogen transformations. A dose-effect approach was performed with different doses of SMX added to different organic wastes (compost of sewage sludge - green waste (SGW), farmyard manure (FYM)), before their mixing with soil, in microcosms. The final concentrations of SMX in amended soils ranged from 0.022 to 2.22 mg kg-1dw, with control soil microcosms amended with SGW or FYM without SMX. Nitrogen forms, potential nitrification and denitrification activities were determined in soils, after 8, 28 and 84 days following amendments. Abundances and composition of specific microbial guilds (ammonia oxidizing bacteria and archae and some denitrifiers) were determined with molecular tools. The total and available concentrations of SMX were extracted with organic and aqueous solutions, respectively, and then quantified by UHPLC-MS/MS.Discussion and conclusionsOnly the nitrification activity was adversely impaired by SMX, following a dose-response pattern, in SGW amended soils, but not in FYM amended soils. These effects had short-term outcomes for nitrogen nutrient dynamic, decreasing soil nitrate content, SGW amended soils, from 0.22 mg SMX kg-1dw. Organic matter is a key environmental factor influencing the bioavailability and the effects of antibiotics on soil microorganisms. In this experiment, the chemical assessment of SMX availability did not show strong differences between the SGW or FYM amended soils. Actually, the acute toxicity of SMX, following a bacteriostatic action, can be only detected on growing microbial populations, such as nitrifiers, in SGW amended soils with high NH4+ levels. Denitrification activity was only slightly affected by high SMX doses, in SGW amended soils, despite an obvious non limitation of nutrients (nitrate and organic carbon) improving population growth. Different level of taxonomic diversity can explain this different sensitivity between nitrification and denitrification. In addition, the magnitude of effects of low doses of SMX (from 0.22 to 0.66 mg kg-1dw) slightly decreased after 84 days of exposure, compared to days 8 and 28, in accordance with the decrease of SMX availability, measured in SGW amended soils. Analytical chemistry (See presentation Goulas et al.) improves our understanding of community responses, with the determination of the exposure to antibiotics. Furthermore, modification of community composition could contribute to the functional resilience against antibiotic stress, through the substitution of sensitive taxa by more tolerant taxa which can colonize vacant niche (Ollivier et al., 2013).This work reveals that, in high nutrient context, soil nitrification appeared as a sensitive indicator of SMX effects on soil microbial functioning, while denitrification was more resistant. Also, close to realistic environmental concentration, the results highlighted resilience of soil nitrification, following a short-term impairment. The dissipation of SMX, leading to a decrease of soil microorganism exposure, promoted this resilience, in addition to changes in community composition. The availability of antibiotic and ecological characteristics of microbial communities (resistance and resilience) must be integrated in ERA methodologies.Boxall A, Rudd M.A, Brooks B.W., …Topp E, Van Der Kraak G. 2012. Pharmaceuticals and personal care products in the environment: what are the big questions? Environ Health Perspect. 120, 1221–1229.Brandt KK, Topp E, et al., 2015. Ecotoxicological assessment of antibiotics: A call for improved consideration of microorganisms. Environ Inter. 85, 189–205.Kümmerer K, Alexy R, Hüttig J, Schöll A. 2004. Standardized tests fail to assess the effects of antibiotics on environmental bacteria. Water Res. 38, 2111–2116.Ollivier J, Schacht D, Kindler R, Groeneweg J, Engel M, Wilke B-M, Kleineidam K and Schloter M. 2013. Effects of repeated application of sulfadiazine-contaminated pig manure on the abundance and diversity of ammonia and nitrite oxidizers in the root-rhizosphere complex of pasture plants under field conditions. Front Microbio. 4, 22.