Orientador: Renate Krause Sakate / Resumo: A espécie Arachis pintoi, conhecida como amendoim forrageiro, é uma leguminosa nativa do Brasil. Muitas são as utilidades atribuídas ao amendoim forrageiro, sendo seu uso mais comum como espécie forrageira, fornecendo alimento em grande quantidade e qualidade aos animais, em plantios puros ou consórcio com gramíneas. A ocorrência de sintomas de viroses em genótipos e cultivos de amendoim forrageiro tem sido observada por pesquisadores em diferentes estados brasileiros. No Brasil, apenas duas espécies virais já foram relatadas: o Peanut mottle virus- PeMoV e o Cowpea mild mottle virus - CpMMV. O objetivo deste trabalho foi estudar os vírus de plantas de amendoim forrageiro do Banco de Germoplasma da Embrapa Acre e detectar viroses em plantas de pimenta Cumari-do-Pará. Para detectar possíveis vírus nesses genótipos, uma análise de sequenciamento de nova geração foi realizada. A extração total de RNA dos 22 acessos foi realizada com o kit RNA Viral PureLink (Invitrogen) seguida de preparação da biblioteca e sequenciamento do transcriptoma utilizando a plataforma Illumina HiSeq2500. A montagem de novo das leituras de 24.659.442 foi realizada usando o software CLC Genomics Workbench v7.0.3. Os 9.709 contigs obtidos foram submetidos a uma pesquisa BLASTn usando o software Geneious v.9.1.5. A análise metagenômica permitiu a identificação de sete espécies de vírus: Peanut mottle virus - PeMoV (Potyvirus), Cucumber mosaic virus sub-grupo IB (Cucumovirus), Cowpea chlorotic mottle virus... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The species Arachis pintoi, known as forage peanuts is a native legume from Brazil. There are many uses attributed to forage peanut, being its most common use as a forage species, providing food in large quantity and quality to the animals, in single crops or consortium with grasses. The occurrence of virus symptoms in forage peanut genotypes and crops has been observed by researchers in different Brazilian states. In Brazil, only two viral species have been reported: Peanut mottle virus-PeMoV and Cowpea mild mottle virus-CpMMV. The objective of this work was to study the forage peanut plant viruses of the Embrapa Acre Germplasm Bank and to detect viruses in Cumari-do-Pará pepper plants. To detect possible viruses in these genotypes, a new generation sequencing analysis was performed. Total RNA extraction from the twenty-two accessions was performed with the PureLink Viral RNA Kit (Invitrogen) followed by library preparation and transcriptome sequencing using the Illumina HiSeq2500 platform. Re-assembly of the 24,659,442 readings was performed using the CLC Genomics Workbench v7.0.3 software. The 9,709 contigs obtained were subjected to a BLASTn search using the Geneious v.9.1.5 software. Metagenomic analysis allowed the identification of seven virus species: Peanut mottle virus - PeMoV (Potyvirus), Cucumber mosaic virus subgroup IB (Cucumovirus), Cowpea chlorotic mottle virus - CCMV (Bromovirus) and a probable new member of the Potyviridae family, and also new species of the... (Complete abstract click electronic access below) / Doutor

Les variétés modernes de canne à sucre sont d'origine bispécifique et possèdent une structure génétique complexe, aneuploïde et hautement polyploïde rendant difficile les études de résistance génétique. La feuille jaune de la canne a sucre est une maladie dont l'agent causal est le sugarcane yellow leaf virus (scylv). Ce virus a une large diversité. Seuls trois génotypes viraux, différenciables par rtpcr, ont été trouves en Guadeloupe. Les objectifs de l'étude sont d'évaluer: l/la possibilité de marquer la résistance de la plante au scylv grâce a une étude d'association pan-génomique 2/l'impact de la diversité de l'agent pathogène sur la résistance de la canne a sucre au scylv. Les études d'association ont été menées avec plus de 4000 marqueurs aflp et d'art sur quatre types de données phénotypiques (intensité et densité virale dans les feuilles et les tiges). Les phénotypes ont été mesures sur 189 variétés de cannes à sucre dans deux essais successifs dans un dispositif en trois blocs randomises. De ces variétés, 40 ont été sélectionnées et ont permis d'obtenir 10 croisements biparentaux. Les descendances obtenues ont été suivies sur deux essais. L'incidence et la diversité du scylv ont été évaluées pour les 40 variétés et les descendances. L'héritabilité au sens strict de la résistance aux scylv a été déterminée. Six marqueurs de résistance au scylv ont été identifies ainsi que deux gènes ayant potentiellement un rôle dans la résistance au virus. L'étude montre également que la résistance de la plante est variable en fonction du génotype du scylv et que cette résistance est en partie transmise aux descendances. Créer des variétés résistantes au scylv est donc possible. / Modern varieties of sugarcane have a bispecific origin and a complex genetic structure, aneuploid and highly polyploid, maklng genetic resistance study uneasy to perform. Yellow leaf of sugarcane is a viral disease whose causal agent is the sugarcane yellow leaf virus (scylv). This virus has a wide range of diversity. Only three viral genotypes, distinguishable by rt-pcr, were found in guadeloupe. The objectives of this srudy are to assess: l/the possibility to find markers associated with plant resistance to scylv through a genome wide association study 2 1 the impact of the pathogen diversity on the resistance of sugarcane to scylv. Association studies have been conducted with more than 4000 aflp and dart markers on four types of phenotypic data (virus intensity and density in leaves and canes). Phenotypes were measured on 189 varieties of sugarcane in two successive trials in a three randomized complete block design. From these varieties, 40 were selected and allowed to obtain 10 biparental crosses. The offspring were followed during two trials. The incidence and the diversity of scylv were evaluated in the 40 varieties and the offspring. The narrow sense heritability of the resistance to the scylvs was determined. Six markers of the resistance to the scylv and two genes, with potential contribution in virus resistance, have been identified. The study also shows that the resistance of the plant is variable depending on the scylv genotype and that this resistance is partly transmitted to the offspring. Breeding for scylv resistance is practicable.

Le projet de thèse consistait à étudier le mouvement du Virus de la jaunisse du navet (TuYV) dans le système vasculaire. Le premier objectif était d’identifier la nature du complexe viral cheminant dans les tubes criblés : virions et/ou complexes ribonucléoprotéiques. L’analyse du mouvement de mutants viraux dans différentes espèces végétales, en absence ou en présence de protéines de capside de type sauvage apportées en trans, a permis de démontrer une étroite relation entre la formation de virions et le transport à longue distance. Le second objectif de cette étude portait sur l’identification de partenaires cellulaires de la protéine P4 du TuYV. Deux protéines ont été identifiées par un criblage de banques d’ADNc d’A. thaliana par le système du double hybride dans la levure, et l’analyse de leur implication dans le cycle viral a été amorcée par des expériences de localisation subcellulaire et de validation fonctionnelle in planta. / In the project, Turnip yellows virus (TuYV) transport in the phloem was analysed. The first objective was to identify the nature of the viral complex involved in vascular movement: virions and/or ribonucleoprotein complexes. Mutant viruses were modified in the capsid protein gene to inhibit formation of virions. By analyzing their movement in different host plants, in the absence or in the presence of the wild-type capsid proteins brought in trans, we demonstrated a strong relation between virion formation and virus long-distance movement. The second objective was to identify cellular partners of the TuYV-P4 protein, a putative movement protein which is host-specific. Two proteins were identified by screening a cDNA library of A. thaliana using the yeast two hybrid technique, and their function in the virus cycle was assessed by performing sub-cellular localizations and infection of A. thaliana KO mutants.

Les polérovirus infectent une large gamme de plantes d’intérêt économique. Ils sont transmis par un insecte vecteur, le puceron, selon le mode circulant non-multipliant. Le virus, acquis par le puceron lors de l’ingestion de sève sur une plante infectée, traverse l’épithélium des cellules intestinales puis celui des glandes salivaires par un mécanisme de transcytose impliquant des récepteurs encore inconnus. Le récepteur de l’éphrine (Eph) est une protéine membranaire dont un domaine est capable de se lier dans la levure aux protéines structurales des polérovirus. En développant des techniques basées sur l’ARN interférence, nous avons montré que l’acquisition orale d’ARN double brin ciblant Eph chez le puceron Myzus persicae permet de réduire de manière reproductible l’internalisation des polérovirus dans le corps du puceron. Les pucerons ainsi traités transmettent le virus avec une efficacité réduite. Eph pourrait donc assurer la fonction de récepteur des polérovirus chez M. persicae. / Poleroviruses infect a wide range of economically important plants. They are transmitted in a circulative and non-propagative mode by an insect vector, the aphid. The virus particles are acquired by aphids when ingesting the sap from an infected plant and cross successively the epithelia of the midgut and the salivary gland cells by a transcytosis mechanism that relies on the presence of unknown receptors.The ephrin receptor (Eph) is a membrane protein which contains a domain able to bind in yeast to the structural proteins of poleroviruses. By developing methods based on RNA interference, we have shown that oral acquisition of double-stranded RNA targeting Eph in the aphid Myzus persicae can reproducibly reduce polerovirus internalization into the aphid's body. Such treated aphids transmit the virus to plants with a lower efficiency. Eph could therefore function as a receptor for poleroviruses in M. persicae.