Mutagenèse dirigée par la système CRISPR-Cas9

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Analyse intégrative de l interaction Arabidopsis thaliana Plasmodiaophora brassicae (vers l élucidation des mécanismes associés à la résistance partielle)

La hernie des crucifères, provoquée par le biotrophe Plasmodiophora brassicae, est une maladie majeure des Brassica cultivées. L association des méthodes culturales aux résistances génétiques est le seul moyen efficace de contrôler la maladie. Cependant, le succès des stratégies d introduction et la gestion raisonnée des résistances dans un objectif de durabilité nécessitent la compréhension de la fonction et des mécanismes associés aux différents facteurs de résistance. L espèce modèle Arabidopsis thaliana, également hôte de P. brassicae, présente une variabilité naturelle de réponse à la hernie des Crucifères. L objectif de ma thèse est de déterminer quels sont les facteurs génétiques de résistance et les voies métaboliques associées à la résistance partielle en se basant sur le modèle A. thaliana P. brassicae. La résistance partielle portée par l accession Bur-0 a été caractérisée à partir de deux populations en ségrégation (F2/3 et lignées recombinantes) issues de croisements entre Bur-0 et l accession sensible Col-0. Cette résistance partielle, de nature quantitative, est sous le contrôle d au moins quatre QTL additifs et quatre QTL épistatiques. Afin d identifier les voies métaboliques associées à la résistance partielle portée par l accession Bur-0, des analyses différentielles du transcriptome ont été réalisées, à l aide de la puce CATMA. La réponse de résistance partielle se caractérise principalement par l induction des voies de défense, la mise en place de mécanismes prévenant l expansino cellulaire et limitant le détournement du métabolisme carboné de l hôte par l agent pathogène.Clubroot, caused by the obligate biotroph Plasmodiophora brassicae, is one of the economically most important diseases of Brassica crops in the world. The development of resistant cultivars is currently the most efficient way to control clubroot in all Brassica crops. However, successful strategies for breeding and management of durable host-plant resistances require knowledge of clubfoot resistance gene fonctions and associated mechanisms. The model plant resistances require knowledge of clubroot resistance gene functions and associated mechanisms. The model plant Arabidopsis thaliana is also a host for clubroot and shows natural variation in the responses to clubroot. The present work aims to determine the genetic factors and metabolic pathwasys associated with partial resistance, using the A. thaliana P. brassicae pathosystem. A quantitative trait locus approach was carried out using two segregating potpulations (F2/3 and recombinant inbred lines) from crosses between the partially resistant accession Bur-0 and the susceptible one Col-0. Four additive QTLs and four epistatic regions controlling partial resistance to clubroot were identified. A functional genomic approach, using the CATMA whole genoe microarray, was then applied to measure changes in gene expression associated with partial quantitative resistance. We showed that partial clubroot resistance response was characterizd by an induction of classical plant defense responses, an active inhibition of cell enlargement and proliferation and a reduced metabolic diversion by the pathogen. In particular, this work highlighted the involvement of arginine metabolism in partial clubroot resistance.RENNES-Agrocampus-CRD (352382323) / SudocSudocFranceF

The Induction of Mycotoxins by Trichothecene Producing Fusarium Species

In recent years, many Fusarium species have emerged which now threaten the productivity and safety of small grain cereal crops worldwide. During floral infection and post-harvest on stored grains the Fusarium hyphae produce various types of harmful mycotoxins which subsequently contaminate food and feed products. This article focuses specifically on the induction and production of the type B sesquiterpenoid trichothecene mycotoxins. Methods are described which permit in liquid culture the small or large scale production and detection of deoxynivalenol (DON) and its various acetylated derivatives. A wheat (Triticum aestivum L.) ear inoculation assay is also explained which allows the direct comparison of mycotoxin production by species, chemotypes and strains with different growth rates and/or disease-causing abilities. Each of these methods is robust and can be used for either detailed time-course studies or end-point analyses. Various analytical methods are available to quantify the levels of DON, 3A-DON and 15A-DON. Some criteria to be considered when making selections between the different analytical methods available are briefly discussed

Identification of quantitative trait loci controlling partial clubroot resistance in new mapping populations of Arabidopsis thaliana

To date, mechanisms of partial quantitative resistance, under polygenic control, remain poorly understood, studies of the molecular basis of disease resistance have mainly focused on qualitative variation under oligogenic control. However, oligogenic conferred resistance is rapidly overcome by the pathogen and knowledge of the relationship between qualitative and quantitative resistance is necessary to develop durably resistant cultivars. In this study, we exploited the Arabidopsis thaliana-Plasmodiophora brassicae pathosystem to decipher the genetic architecture determining partial resistance. This soil-borne pathogen causes clubroot, one of the economically most important diseases of Brassica crops in the world. A quantitative trait locus (QTL) approach was carried out using two segregating populations (F(2) and recombinant inbred lines) from crosses between the partially resistant accession Burren and the susceptible accession Columbia. Four additive QTLs (one moderate and three minor) controlling partial resistance to clubroot were identified, all the resistance alleles being derived from the partially resistant parent. In addition, four epistatic regions, which have no additive effect on resistance, were also found to be involved in partial resistance. An examination of candidate genes suggested that a potentially diverse array of mechanisms is related to the different QTLs. By fine-mapping and cloning these regions, the mechanisms involved in partial resistance will be identified

Développer des espèces et des variétés permettant la reconception des systèmes de culture

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Développer des espèces et des variétés permettant la reconception des systèmes de culture

Chapitre 5 Disponible gratuitement en ebookInternational audienc

Integration and query of biological datasets with Semantic Web technologies: AskOmics

National audienceOver the past few years, research programs involving genetic, genomic and post-genomic sequencing of various living organisms have become fast growing areas of biology. Once the computational challenges of processing datasets have been dealt with; large and complex biological data still remain in the hands of biologists for interpretation. Projects such as Biomart and Intermine have been developed for the international community to facilitate exchange and comparison of complex biological data. However for non-model organisms, large heterogeneous biological datasets can be difficult to associate in order to obtain a comprehensive view. Overall, access and interrogation remain time consuming for biologists and integrating publicly available data is still an open challenge. Linked data and Semantic Web technologies benefit to biologists. Using RDF (Reference Description Framework); biological data can be described using triples that associate an entity (called subject), a relation (called property) and a value for the relation (called object). Data from different datasets can be integrated and the SPARQL query language support their analysis. Nevertheless; understanding and acquiring the query language can be a daunting task for biologists. Here we present AskOmics, a tool supporting both intuitive data integration and querying while shielding the user from most of the technical difficulties underlying RDF and SPARQL. The virtualization-based deployment of AskOmics makes the tool easy to manage, reliable and simple to install. For data integration, the user loads his data as tabulation-separated files structured according to simple principles. This structure allows AskOmics to generate automatically the corresponding RDF triples, and to store them into a triplestore such as Fuseki or Virtuoso. At this point, the user’s data are available just like in any SPARQL endpoint. AskOmics automatically generates an abstract representation of the dataset based on the types of the subject and object of its triples. For data querying, AskOmics provides a visually intuitive interface compatible with any SPARQL endpoint (that is one generated by AskOmics data generation function, or any regular triplestore). The user can then select a sequence of nodes in this simplified view, and AskOmics generates the corresponding SPARQL query that can be executed on the original dataset. For example; it could be difficult for biologists to identify features such as genes underlying localised genomic regions limited by genetic markers as it requires the users to combine different files. Tabulation-separated files containing genes and genetic markers could be uploaded in AskOmics with the following criteria: genetic markers and genes identified as entity, each entity is related to a chromosome and a position start and end with numerical values. AskOmics interface allows the user, without knowledge in SPARQL language, to either select genomic regions with distinct markers or simply provide numerical values as the lower and upper position. The intersection with additional features could be computed for producing lists of features such as genes underlying specific genomic regions. The result can then be downloaded as a tabulation-separated file. Currently under development, AskOmics will also support the integration of external databases to compare or complete new findings.AskOmics’ principle is generic. It has been applied successfully to the analysis of large scale datasets including genetic, epigenomic, transcriptomic profiles and orthologous relationships to identify genomic regions that are involved in the variability of Brassicaceae (Arabidopsis, cabbage, turnip and oilseed rape) in response to clubroot disease. About 2.6 millions of triples were stored from 370 000 uploaded entities corresponding to genomic positions of genes amongst the four species of the Brassicaeae family as well as relationship data (orthology and transcriptomics). The fast queries allowed to identify lists of genes with specific expression profiles and their corresponding orthologs in the three others species

Attempts to induce homoeologous pairing between wheat and Agropyron cristatum genomes

International audienceAgropyron cristatum (2n = 4x = 28, PPPP) possesses potentially valuable traits that could be used in wheat (Triticum aestivum) improvement through interspecific hybridization. Homoeologous pairing between wheat chromosomes and P chromosomes added to wheat in a set of wheat – A. cristatum addition lines was assessed. First, the Phsuppressing effect of P chromosomes (except 7P) was analyzed. It was concluded that this system is polygenic with no major gene, and consequently, has no prospect in the transfer of alien genes from wild relatives. In a second step, the potential of the deletion ph1b of the Ph1 gene for inducing P–ABD pairing was evaluated. Allosyndetic associations between P and ABD genomes are very rare. This very low level of pairing is likely due to divergence in the repeated sequences between Agropyron and wheat genomes. Development of translocation lines using ionizing radiation seems to be a more suitable technique than homoeologous recombination to exploit the A. cristatum genome in wheat improvementAgropyron cristatum (2n = 4x = 28, PPPP) possède des caractères agronomiques potentiellement intéressants qui pourraient être utilisés dans les programmes d’amélioration du blé tendre (Triticum aestivum) par hybridation interspécifique. L’appariement homéologue entre les chromosomes de blé et les chromosomes P ajoutés dans un ensemble de lignées d’addition blé – A. cristatum a été évalué. L’effet suppresseur du système Ph des chromosomes P (à l’exception du chromosome 7P) a tout d’abord été analysé. De nature polygénique, il ne contient aucun gène majeur et n’offre par conséquent aucune perspective pour le transfert de gènes provenant d’espèces sauvages apparentées. Dans un second temps, le potentiel de la délétion ph1b du gène Ph1 pour l’induction d’appariement P-ABD a été évalué. Les associations allosyndétiques entre les génomes P et ABD sont très rares. Ce très faible niveau d’appariement est certainement lié à une divergence des séquences répétées entre les génomes d’Agropyron et de blé. Le développement de lignées de translocation par radiations ionisantes semble être une technique plus adaptée que la recombinaison homéologue pour exploiter le génome d’Agropyron dans l’amélioration du blé tendr

Differential Regulation of Root Arginine Catabolism and Polyamine Metabolism in Clubroot-Susceptible and Partially Resistant Arabidopsis Genotypes

The hypertrophy and hyperplasia of infected roots into clubs are the intrinsic characteristics of clubroot, one of the economically most important diseases in Brassica crops worldwide. Polyamines, arginine (Arg)-derived metabolites, have long been recognized as cell proliferation and differentiation regulators in plants and consequently are suitable candidates for potential gall development factors. Furthermore, Arg catabolism, through arginase, which is strongly connected to polyamine metabolism, would play an important role in response to wound trauma and pathogen infection. In this study, we exploited the Arabidopsis (Arabidopsis thaliana)-Plasmodiophora brassicae pathosystem to investigate the involvement of polyamine metabolism and Arg catabolism in host responses to the pathogen infection and in partial clubroot resistance mechanisms. We demonstrated at the transcriptional, enzymatic, and metabolic levels that polyamine metabolism and Arg catabolism are induced during the later stages of disease in compatible Arabidopsis-P. brassicae interactions. However, susceptible and partially resistant plants showed strikingly different Arg metabolism signatures. Susceptible plants were characterized by a transient agmatine production, a massive induction of arginase, and a strong accumulation of proline. The potential functions of this marked activation of the arginase pathway in the P. brassicae pathogenicity strategy are discussed. Partially resistant plants showed a continuous agmatine production and a weaker arginase pathway activity than the susceptible genotype. Results suggest that the symptom severity was strongly associated to the differential regulation of root polyamine metabolism and Arg catabolism. Further work using arginase transgenic plants will provide insight into the physiological function of the arginase pathway in partial clubroot resistance

Differential regulation of root arginine catabolism and polyamine metabolism in clubroot-susceptible and partially resistant Arabidopsis genotypes

The hypertrophy and hyperplasia of infected roots into clubs are the intrinsic characteristics of clubroot, one of the economically most important diseases in Brassica crops worldwide. Polyamines, arginine (Arg)-derived metabolites, have long been recognized as cell proliferation and differentiation regulators in plants and consequently are suitable candidates for potential gall development factors. Furthermore, Arg catabolism, through arginase, which is strongly connected to polyamine metabolism, would play an important role in response to wound trauma and pathogen infection. In this study, we exploited the Arabidopsis (Arabidopsis thaliana)-Plasmodiophora brassicae pathosystem to investigate the involvement of polyamine metabolism and Arg catabolism in host responses to the pathogen infection and in partial clubroot resistance mechanisms. We demonstrated at the transcriptional, enzymatic, and metabolic levels that polyamine metabolism and Arg catabolism are induced during the later stages of disease in compatible Arabidopsis-P. brassicae interactions. However, susceptible and partially resistant plants showed strikingly different Arg metabolism signatures. Susceptible plants were characterized by a transient agmatine production, a massive induction of arginase, and a strong accumulation of proline. The potential functions of this marked activation of the arginase pathway in the P. brassicae pathogenicity strategy are discussed. Partially resistant plants showed a continuous agmatine production and a weaker arginase pathway activity than the susceptible genotype. Results suggest that the symptom severity was strongly associated to the differential regulation of root polyamine metabolism and Arg catabolism. Further work using arginase transgenic plants will provide insight into the physiological function of the arginase pathway in partial clubroot resistance