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Identification and Study of Promoters Induced by Asian Soybean Rust : Application in an Artificial Cell Death System Lisa Cabre

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Identification and Study of Promoters Induced by Asian Soybean Rust : Application in an Artificial Cell Death System Lisa Cabre Identification and study of promoters induced by Asian soybean rust : application in an artificial cell death system Lisa Cabre To cite this version: Lisa Cabre. Identification and study of promoters induced by Asian soybean rust : application inan artificial cell death system. Microbiology and Parasitology. Université de Lyon, 2019. English. NNT: 2019LYSE1054. tel-02281993 HAL Id: tel-02281993 https://tel.archives-ouvertes.fr/tel-02281993 Submitted on 9 Sep 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. N°d’ordre THESE de DOCTORAT DE L’UNIVERSITE DE LYON opérée au sein de l’Université Claude Bernard Lyon 1 Ecole Doctorale 341 (Evolution Ecosystème Microbiologie Modélisation) Spécialité de doctorat : BIOLOGIE Disciplines : Microorganismes, interactions, infections Soutenue publiquement le 25/04/2019 par : Lisa Cabre Identification and study of promoters induced by Asian soybean rust. Application in an artificial cell death system Devant le jury composé de : MARCELINO-GUIMARAES HDR (Embrapa, Brazil) Rapporteure Francismar Correa VOEGELE Ralf PR, HDR (Hohenhiem University, Germany) Rapporteur DUMAS Bernard DR, HDR (Université Toulouse III) Examinateur PRAT Daniel PR, HDR (Université Lyon1) Examinateur POUSSEREAU Nathalie MDC, HDR (Université Lyon 1) Directrice de thèse PELISSIER Bernard PhD (Bayer Cropscience Lyon) Co-encadrant 1 2 3 4 THANKS Thank you to Daniel Prat for agreeing to be the jury’s president. I’d like to extend my thanks to Mrs Marcelino-Guimaraes Francismar Correa and Professor Voegele Ralf for agreeing to be my thesis reporter and reviewing my scientific work. Thank you to Dumas Bernard for agreeing to be a jury examinator. Thank you to Sarra Mazzotta for agreeing to be a gest member of the jury. I’d like to extend a special thanks to Ulrich Schaffrath, Marc-henri Lebrun & Frank Meulewaeter for agreeing to be part of my thesis comity and for providing me with valuable insight and stimulating scientific discussions. Thanks to Ulrich for welcoming me in his laboratory at Aachen for a three weeks internship on transient soybean transformation. Thank you for making this experience enriching on the scientific level as well as on the human level. Thanks to Marco for guiding me for these three weeks through the soybean rust and through the German culture so I’d feel at home. Thank you to Steven Engelen to followed the progression of the project and to have make the link with BASF when it was needed. Merci à Philippe Peret de m’avoir accueillie au sein de son équipe et de d’avoir tout mis en œuvre pour que ma thèse se déroule dans des conditions optimales au sein de Bayer Crop Science. Merci à Bernard Pelissier, Nathalie Poussereau et Sophie Ducerf pour leur encadrement, leur implication et leur soutien tout au long de ces trois ans de thèse. Merci à vous encore, pour vos relectures impliquées durant la rédaction de ce manuscrit. Merci à toi Bernard pour t’être toujours montré disponible, même durant ces derniers mois alors que tu commençais ta nouvelle vie à Toulouse. Merci pour ton implication entière dans ce projet et pour m’avoir aidée dans mes tranfos soja quand j’en ai eu besoin. Merci à Sophie pour son implication sans limites dans ce projet.et pour m’avoir permis de découvrir le monde de la recherche en entreprise. Merci Nathalie d’avoir accepté d’être ma directrice de thèse et d’avoir participé à ce projet scientifique. Merci à toi pour avoir toujours fait le lien avec la perspective universitaire de ce travail. Merci à toute l’équipe du labo mixte pour leurs conseils et discussions avisés. J’aimerais aussi remercier Matthias et Vincent pour avoir pris de leur temps pour partager leurs expertises scientifiques. Merci à l’ensemble de l’équipe Biochimie & Biotechnologies pour leur accueil chaleureux, leur bienveillance et leur disponibilité. 5 Merci à Catherine pour m’avoir incluse dans le projet de l’équipe ASR, pour son enthousiasme sur le projet de recherche et pour avoir sacrifié de son temps libre pour m’aider au cours de mes longues soirées de travail. Merci à Lucie pour m’avoir formée en transformation de soja et pour sa bonne humeur communicative au quotidien. Merci à Cécile pour m’avoir partagé son expertise en Biologie Moléculaire et pour tous ses conseils avisés et toutes ses discussions éclairantes Merci à Charlotte de m’avoir initiée à l’inoculation Phakpa au sein de Bayer. Merci à Christelle pour m’avoir fait découvrir les mille et une facettes du Macrofluo ainsi que toutes les possibilités qu’il a à offrir. Merci à Stéphane et Pierrick pour avoir partagé avec moi leur savoir de bioinformatique et m’avoir initié à cette sphère privée. Merci d’avoir toujours su m’éclairer et me guider dans mes analyses. Merci à Aurélia pour avoir fait preuve de pédagogie en me formant au confocal et pour sa passion débordante et stimulante face à la recherche scientifique. Un merci tout particulier à toi Daphné pour m’avoir tenu compagnie au cours des ces journées au labo biomol, merci pour toutes tes contributions à mon travail, mais par-dessus tout merci pour ton soutien et ton partage. Merci à toi Florent, pour ce que tu m’as apporté sur le plan humain, merci de t’être toujours montré disponible pour me donner de pertinents conseils sur mes interrogations scientifiques. Un immence merci pour ton aide de valeur lors de la rédaction et la relecture du papier. Merci à Didier pour son travail exceptionnel en serre et pour avoir pris soin de mes sojas, de les avoir choyés sans réserve et d’avoir tendrement pris soin d’eux jusqu’à leur maturité. Merci aussi pour tous les éclas de rire partagés. Merci à tous ceux qui de près ou de loin ont participé à ce projet de recherche et sans qui rien n’aurait été possible. Merci également à ma famille et mes amis qui sont toujours restés à mes côtés dans les bons jours comme dans les mauvais. Merci à mon fiancé pour m’avoir soutenu moralement au cours de ces trois ans et pour son soutien sans faille tout au long de cette aventure. Merci d’avoir toujours été là quand j’en ai eu le plus besoin. Merci pour ton aide précieuse pour la redaction de ce document. Merci d’avoir toujours cru en moi et dans ce projet dans lequel je me suis lancée. À mon grand père qui comme moi partageait l’amour des sciences, ... 6 7 Résumé Titre : Identification et etude de promoteurs induits par la rouille asiatique du soja. Application à un système de mort cellulaire artificielle. Mot clés : Soja, Phakopsora pachyrhizi, promoteurs inductibles, GFP, mort cellulaire. RESUME : Phakopsora pachyrhizi Syd.& P.Syd. est le plus promoteur induit par le pathogène, permettant important fléau du soja (Glycine max (L.) une régulation spatiotemporelle. Merrill). Introduit au Brésil dans les années 2000, ce champignon s’est rapidement répandu La recherche de tels promoteurs a été effectuée sur les deux continents Américains. Seule en utilisant des données transcriptomiques et l’utilisation de fongicides associée à des bibliographiques. Des sojas stables exprimant pratiques culturales strictes permet de maintenir les différentes fusions promoteur:GFP ont été le niveau de production. L’utilisation répétitive créées afin d’étudier l’ expression de ces fongicides ainsi que la plasticité spatiotemporelle de ces promoteurs en présence génétique de ce champignon ont rapidement du champignon. Les promoteurs pGmCHIT1 entraîné une diminution d’efficacité de (de G. max) et pgst1 (de Solanum tuberosum) certaines molécules. Par ailleurs, la plupart des contrôlant respectivement l’expression d’une résistances verticales identifiées dans les chitinase et d’une glutathione-S-transférase ont ressources naturelles du soja restent inefficaces été identifiés comme induits par le pathogène. contre certains isolats du champignon. La L’impact de différents stress sur ces deux compréhension des mécanismes de l’immunité promoteurs a été évalué. des plantes permet de proposer des solutions biotechnologiques pour le contrôle des Les constructions génétiques maladies. « barnase/barstar » comprenant les différentes combinaisons des promoteurs ont été générées. L’utilisation antérieure du système Nicotiana benthamiana a été utilisé pour barnase/barstar induisant une mort cellulaire exprimer transitoirement les construits et artificielle, a permis de générer des pommes de évaluer leur phytotoxicité en absence du terre résistantes à Phytophtora infestans. Cette pathogène. Un seul construit contenant le technologie est basée sur l’expression de la promoteur gst1 s’est avéré non phytotoxique. Il barnase une ribonucléase toxique pour les a été transféré avec succès dans le soja. Ces cellules, et la barstar un inhibiteur de la barnase. sojas n’ont pas montré de gain de tolérance à la Il a été proposé d’évaluer ce système pour le rouille. Une proposition d’amélioration du contrôle de P. pachyrhizi. Le point critique de système barnase/barstar est discutée afin de cette approche est de trouver le bon rapport de mieux cerner les possibilités et les limites de ce l’expression des gènes barnase/barstar. Pour ce système pour le contrôle de la rouille du soja. faire la barnase sera placée sous le contrôle d’un . 8 Summary Title: Identification and study of promoters induced by Asian soybean rust. Application in an artificial cell death system. Keywords: Soybean, Phakopsora pachyrhizi, inducible promoters, GFP, cell death.
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