Le PTGS (post-transcriptional gene silencing) est un mécanisme de défense qui cible les acides nucléiques invasifs d’origines endogènes (transposons) ou exogènes (pathogènes, transgènes). Des mutations dans les gènes JMJ14 et NAC52 ont été isolées lors d’un crible génétique visant à identifier des mutants déficients en PTGS. JMJ14 code une histone déméthylase ciblant la lysine 4 bi- ou tri-méthylée de l’histone H3, tandis que NAC52 code un facteur de transcription. Ces deux protéines forment un complexe qui régule la transcription de centaines de gènes endogènes. Toutefois, le rôle de ce complexe chromatinien dans l’expression des transgènes et surtout dans le PTGS reste incompris. JMJ14 interagit avec NAC52 mais aussi avec une protéine de type guanine exchange factor de la famille RCC1. Des mutations dans l’un ou l’autre des membres du complexe RCC1-JMJ14-NAC52 réduisent la transcription des transgènes. JMJ14 se fixe au promoteur de façon indépendante de NAC52, tandis que NAC52 a besoin de JMJ14 pour se fixer à la région transcrite. Toutefois, JMJ14 et NAC52 ne semblent pas requis pour la transcription proprement dite. En effet, un niveau normal de transcription est restauré chez le double mutant jmj14 drm2, indiquant que le rôle du complexe RCC1-JMJ14-NAC52 semble être d’empêcher la méthylation de novo du promoteur par DRM2.L’effet des mutations jmj14 et nac52 sur la transcription des transgènes ne peut expliquer leur effet sur certaines formes de PTGS. En effet, les mutations jmj14 et nac52 n’affectent pas le PTGS induit constitutivement. Par contre, elles empêchent la systémie du PTGS induit localement. Des mutations dans le gène SPCL45 codant une Serine Carboxy Peptidase-Like qui interagit avec NAC52, mais pas JMJ14, ont le même effet. En revanche, la mutation rcc1 n’affecte pas la systémie du PTGS, suggérant que c’est au sein d’un complexe JMJ14-NAC2-SPCL45 que JMJ14 et NAC52 contrôlent le PTGS systémique. Ce complexe pourrait agir directement sur la chromatine du transgène pour permettre d’enclencher le PTGS en réponse à la perception du signal systémique, ou indirectement en contrôlant l’expression d’un gène endogène codant une protéine régulant la systémie du PTGS. Afin de mieux comprendre le rôle de JMJ14 dans la systémie du PTGS, un crible génétique visant à isoler des suppresseurs de la mutation jmj14 a été réalisé. Seize mutants correspondants à sept gènes codant des protéines ayant un rapport avec la chromatine et une action antagoniste à JMJ14 ont été caractérisés. Les mutations dans ces sept gènes pourraient supprimer l’effet de jmj14 en augmentant la transcription du transgène cible et donc la quantité du signal systémique de PTGS. Un 17ème mutant pourrait quant à lui affecter qualitativement le signal systémique de PTGS ou la perception du signal dans les cellules qui le reçoivent. Le gène correspondant reste à identifier. / Post-transcriptional gene silencing (PTGS) is a defense mechanism that targets invading nucleic acids from endogenous (transposons) or exogenous (pathogens, transgenes) origins. Mutations in JMJ14 and NAC52 have been retrieved from a genetic screen aiming to identify PTGS deficient mutants. JMJ14 encodes an histone demethylase targeting the bi- or tri-methylated lysine 4 of histone H3, while NAC52 encodes a transcription factor. Both act in a complex that regulates the transcription of hundreds endogenous genes. However, the function of this chromatin complex in transgene expression and in PTGS is not known. JMJ14 interacts with NAC52 but also with a guanine exchange factor of the RCC1 family. Mutations in any member of the RCC1-JMJ14-NAC52 complex reduce transgene transcription. JMJ14 binds to the transgene promoter independently of NAC52, whereas NAC52 requires JMJ14 to bind on the transcribed region. However, JMJ14 and NAC52 do not seem to be required for transcription itself. Indeed, a wild-type level of transcription is restored in the jmj14 drm2 double mutant, suggesting that the complex RCC1-JMJ14-NAC52 prevents de novo DNA methylation of the promoter by DRM2. The effects of jmj14 and nac52 mutations on transgene transcription cannot explain their specific effect on some forms of PTGS. Indeed, jmj14 and nac52 do not affect constitutively-induced PTGS, but prevent the systemic spreading of locally-induced PTGS. Mutations in SCPL45, encoding a Serine-Carboxy Peptidase-Like that interacts with NAC52, but not JMJ14, have the same effect. In contrast, rcc1 does not affect the systemic PTGS, suggesting that a JMJ14-NAC52-SCPL45 complex is involved in the control of systemic PTGS. This complex could act directly on transgene chromatin to trigger PTGS in response to the PTGS signal, or indirectly by controlling the expression of an endogenous gene encoding a protein regulating systemic PTGS. To better understand the function of JMJ14 in systemic PTGS, a genetic screen aiming to identify suppressors of jmj14 have been performed. Sixteen mutants corresponding to seven genes encoding proteins related to chromatin and having an antagonist function to JMJ14, have been characterized. Mutations in theses seven genes could suppress jmj14 by increasing transgene transcription and consequently the quantity of the PTGS systemic signal. A seventeenth mutant could have a qualitative effect on the PTGS systemic signal or could affect the perception of this signal in recipient cells. The corresponding gene remains to identify.

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Throughout evolution, plants have developed a sophisticated network of signaling pathways allowing the activation and regulation of immune responses. The identification of metabolic pathways which are involved in modulating the intensity of that immune responses is an important challenge in the field of plant-pathogen interaction. With this aim, we performed two genetic approaches in Arabidopsis thaliana against the disease caused by the hemibiotroph bacterial pathogen Pseudomonas syringae DC3000. We demonstrate that the regulation of two pathways, related between them, is crucial to activate an effective immune response. By means of a genetic screening of regulators components of plant immunity, we identified the mutant scs9 (suppressor of csb3) which shows an affected resistance that triggers a enhanced susceptibility to P.s. DC3000 through an independent pathway of salicylic acid (SA)-mediated immune response. The cloning and characterization of SCS9 reveals that it codes for 2'-O-ribose tRNA methyltransferase. Our results indicate that the SCS9-mediated methylation of nucleosides N32 and N34, located in the tRNAs anticodon loop, is crucial for the plant immunity effectiveness. On the other hand, with a chemical genetic screening of agonist molecules of the immune response, we identified the sulfonamides as priming inducer molecules that exhibit a faster and/or stronger activation of SA-related defense responses and enhanced resistance to P.s. DC3000. Analysis of the mechanism of action of these molecules reveals that synthesis and accumulation of folates exert a SA-independent negative control on the immune response to P.s. DC3000. Through comparative proteomic analysis we identified the 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase 1 (methione synthase, here named as METS1), enzyme responsible of the methionine synthesis in the folate-dependent 1C metabolism and overaccumulated in scs9 mutants, as modulator component in the immune response to P.s. DC3000. We observed that the overexpression of METS1 in transgenic plants of Arabidopsis suppresses plant immune responses and promotes enhanced susceptibility to P.s. DC3000. This repressor effect is due to a genome-wide increase in DNA methylation level, which is mediated by the overaccumulation of METS1 and the consequent increase of folate-dependent methionine synthesis. Therefore, the findings of this work provide a deeper knowledge about the mechanisms by which the DNA methylation and epigenetic regulation exert an influence on plant immunity through folate metabolism, particularly by METS1, whose synthesis is regulated through specific tRNA modifications mediated by SCS9. / Las plantas, a lo largo de la evolución, han desarrollado un sofisticado entramado de rutas de señalización que permiten la activación y el control de la respuesta inmune. Identificar qué procesos metabólicos participan en modular la amplitud de dicha respuesta inmune es un reto en el campo de la interacción planta-patógeno. Con este propósito, se han utilizado dos aproximaciones genéticas llevadas a cabo en Arabidopsis thaliana contra la infección por la bacteria hemibiotrofa Pseudomonas syringae DC3000. Los resultados ponen de manifiesto la importancia de la regulación de dos mecanismos, a su vez relacionados, para la activación de una respuesta inmune efectiva. Mediante un rastreo genético en busca de componentes reguladores de la inmunidad, identificamos el mutante que denominamos scs9 (supresor de csb3). scs9 muestra una resistencia afectada que conlleva un incremento en la susceptibilidad a P.s. DC3000 a través de un mecanismo independiente a la respuesta inmune mediada por ácido salicílico (SA). La clonación y caracterización de SCS9 revela que codifica una 2'-O-ribosa metiltransferasa de tRNA. Nuestros resultados indican que la modificación por metilación mediada por SCS9 de los nucleósidos N32 y N34 de la región anticodón de los tRNAs, es clave para la inmunidad de la planta. Por otro lado, mediante un rastreo de genética química en busca de moléculas agonistas de la respuesta inmune, identificamos un grupo de sulfonamidas como moléculas activadoras de un mecanismo de priming. Este conlleva una más rápida y/o más intensa activación de la respuesta defensiva dependiente de SA y de un incremento de la resistencia frente a P.s. DC3000. El análisis del mecanismo de acción de dichas moléculas revela que la síntesis y acumulación de folatos ejerce un control negativo sobre la respuesta inmune frente a P.s. DC3000; y ese control es ejercido de manera independiente a la ruta de señalización mediada por SA. A través de un análisis proteómico comparativo identificamos la proteína 5-metiltetrahidropteroiltriglutamato homocisteína metiltransferasa 1 (metionina sintasa, denominada aquí METS1), responsable de la síntesis de metionina en el metabolismo C1 dependiente de folatos y sobreacumulada en los mutantes scs9. Esta proteína participa entonces como componente modulador de la respuesta inmune a P.s. DC3000. La sobreexpresión de METS1 en plantas transgénicas observamos que suprime la respuesta inmune y conlleva a un incremento en la susceptibilidad frente a P.s. DC3000. Dicho efecto represor de la resistencia acontece a raíz de un incremento del nivel de metilación de DNA en todo el genoma mediado por la sobreacumulación de METS1 y del consiguiente posible aumento en la síntesis de metionina dependiente de folatos. Por tanto, estos resultados ahondan en el conocimiento de cómo la metilación de DNA y el control epigenético ejercen una influencia sobre la respuesta inmune. Esta influencia puede ser controlada a través del metabolismo de folatos, y en particular a través de METS1, enzima cuya síntesis está a su vez controlada por determinadas modificaciones de tRNA mediadas por SCS9. / Les plantes, al llarg de l'evolució, han desenvolupat un sofisticat entramat de rutes de senyalització que permeten l'activació i el control de la resposta immune. Identificar quins procesos metabòlics participen en la modulació de l'amplitud d'aquesta resposta immune és un repte en el camp de la interacció planta-patogen. Amb aquest propòsit, s'han utilitzat dues aproximacions genètiques en Arabidopsis thaliana en resposta a la infecció pel bacteri hemibiotrofo Pseudomonas syringae DC3000. Els resultats posen de manifest la importància de la regulació de dos mecanismes, al seu torn relacionats, per a l'activació d'una resposta immune efectiva. Mitjançant un rastreig genètic per a la recerca de components reguladors de la immunitat, es va identificar el mutant que denominem scs9 (supresor de csb3). scs9 mostra una resistència afectada que comporta un increment en la susceptibilitat a P.s. DC3000 fent ús d'un mecanisme independent a la resposta immune mediada per l'àcid salicílic (SA). La clonació i caracterització de SCS9 revela que codifica una 2'-O-ribosa metiltransferasa de tRNA. Els nostres resultats indiquen que la modificació per metilació mediada per SCS9 dels nucleòsids N32 i N34 de la regió anticodó dels tRNAs, és clau per a la immunitat de la planta. D'altra banda, per mitjà d'un rastreig de genètica química per a la recerca de molècules agonistes de la resposta immune, es va identificar un grup de sulfonamidas com a molècules activadores d'un mecanisme de priming. Aquest, comporta una més rápida i/o més intensa activació de la resposta defensiva dependent de SA i d'un increment de la resistència enfront de P.s. DC3000. L'anàlisi del mecanisme d'acció d'aquestes molècules revela que la síntesis i acumulació de folats exerceix un control negatiu sobre la resposta immune davant el bacteri P.s. DC3000; i eixe control és exercit de manera independent a la ruta de senyalització mediada per SA. Amb un anàlisi proteòmic comparatiu es va identificar la proteïna 5-metiltetrahidropteroiltriglutamato homocisteína metiltransferasa 1 (metionina sintasa, denominada ací METS1), responsable de la síntesi de metionina al metabolisme C1 dependent de folats i sobreacumulada en els mutants scs9. Aquesta, així doncs, es troba participant com a component modulador de la resposta immune a P.s. DC3000. La sobreexpressió de METS1 en plantes transgèniques suprimeix la resposta immune i comporta a un increment en la susceptibilitat per P.s. DC3000. L'efecte repressor de la resistència succeïx arran d'un increment del nivell de metilació de DNA en tot el genoma, mediat per la sobreacumulació de METS1 i del consegüent posible augment en la síntesi de metionina dependent de folats. Per tant, aquests resultats aprofundixen en el coneixement de com la metilació de DNA i el control epigenètic exerceixen una influència sobre la resposta immune. Aquesta influència pot ser controlada mitjançant el metabolisme de folats, i en particular a través de l'enzim METS1, la síntesi de la qual està al seu torn controlada per determinades modificacions de tRNA mediades per SCS9. / González García, B. (2017). Implicación de las modificaciones de tRNA y del metabolismo de los folatos en la respuesta inmune de Arabidopsis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/86162

RNA interference (RNAi) is one of the key mechanisms that are involved in many biological processes such as control of plant gene expression, influence on chromatin arrangement or providing protection against invasive DNA or RNA transposons, viruses and transgenes. In plants, RNAi is triggered by double stranded RNA (dsRNA) that is cleaved by DICER LIKE (DCL) proteins to small RNAs (sRNAs). The size of these sRNAs is in range of 21 - 24 nucleotides (nt). Small RNA acts in the place of origin and they are also a mobile signal which in plants can move to a short distance through plasmodesmata and to a long distance trough phloem. sRNA and Argonaute (AGO) protein form RNA-induced silencing complex (RISC). Together, they recognize the target RNA molecule and contribute to an efficient RNAi phase which may be exhibited by gene silencing at posttranscriptional level (PTGS) or transcriptional level (TGS). The purpose of this study was to compare the effects of silencing constructs, witch in a controlled way differently trigger RNAi directed against the expression of the GFP reporter gene in the model organism Arabidopsis thaliana. Silencing constructs were placed under an inducible promoter activated by the presence of 17-β-estradiol (XVE system). They differed in the way of the dsRNA formation and in the...

Les éléments transposables (ETs) sont des composants ubiquitaires des génomes eucaryotes, parfois prépondérants chez les plantes. Ce sont des séquences mobiles, potentiellement mutagènes, reconnues comme des acteurs de l’évolution des génomes. Cependant, la plupart des ETs sont aujourd’hui inactifs car réprimés par des mécanismes épigénétiques très efficaces. Néanmoins, ces derniers peuvent être relâchés par des stress, conduisant à la réactivation d’ETs. De tels stress sont-ils suffisants pour activer la transposition dans les populations naturelles? L’application répétée d’un stress peut-elle expliquer les pics d’activité transpositionnelle qui ont eu lieu en conditions naturelles? De récents travaux chez un mutant d’Arabidopsis thaliana, affecté dans une voie de répression d’ETs, le RdDM (RNA-directed DNA Methylation), ont démontré qu’un stress thermique conduisait à la réactivation transpositionnelle d’un ET. Mes travaux de thèse portent sur l’étude de riz sauvage et d’un mutant non décrit, affecté dans le RdDM, cultivés en conditions normales ou de stress thermique sur plusieurs générations. Les objectifs de mes travaux ont été de déterminer (1) l’impact de la mutation sur les différentes étapes d’activation rétrotranspositionnelle et (2) l’activation rétrotranspositionnelle en réponse à un stress thermique. Une part importante de ce travail a été consacrée au développement et à la comparaison de méthodes d’identification des mouvements d’ETs et différentes approches « omiques » ont été utilisées. La réactivation de 5 ETs dans les plantes mutantes, dont la mobilité n’avait pas encore été observée, suggère que la voie RdDM est impliquée dans le contrôle de leur répression. De plus, nos résultats confirment que les ETs ne sont pas tous réprimés par les mêmes voies de régulation. / Transposable elements (TEs) are ubiquitous among eukaryotic genomes sometimes overriding in plants. Due to their ability to replicate and transpose, they are potentially mutagenic and recognized as actors of genome evolution. However, the analysis of the transpositional activity of TEs in different plant species have shown that most of them are maintained in a transcriptionally inactive state through powerful and specific epigenetic mechanisms. These silencing processes can nevertheless be allievated under stress conditions, leading to TE reactivation. Are these stress sufficient to activate transposition in natural populations? Are repeated heat stress able to trigger transposition and therefore lead to bursts of transposition? In recent reports, reactivation of retrotransposons has been shown in Arabidopsis thaliana mutants impaired in the RdDM pathway (RNA-directed DNA Methylation) and submitted to heat stress. My PhD works reports the study of of a wild rice and a new rice mutant, affected in the RdDM, cultivated under optimal or heat stress conditions over generations. Here, we propose to determine (1) the impact of the mutation at the different levels leading to the retrotranspositional activation and (2) the retrotranspositional activity in response to heat stress. An important part of this work has been devoted to the development and the comparison of different methods to identify TE movements, and different -omics approaches have been used. The reactivation of 5 new TEs in mutants, suggests that the RdDM pathway is involved in the control of the repression of these TEs. Furthermore, our result confirm that all TEs are not regulated through the same pathways but are under the control of different lock.

Genetic information must be protected, maintained and copied from cell to daughter cells, from generation to generation. In plants, most of the cells contain complete genetic information, and many of these cells can regenerate to a whole new plant. Such a feature leads to the need for precise control of which genes will be active and which not because in growth and differentiation, only the activity of specific genes for the individual cells, tissues, organs are required. One of the mechanisms controlling the gene activity is RNA interference (RNAi), which down- regulates or blocks the expression of specific genes at the transcriptional or post-transcriptional level. The crucial part of the RNAi is guiding the RNAi machinery to the target. It is mediated via sequence complementarity of the target with a small RNA (sRNA), which is diced from a double- stranded RNA (dsRNA) precursor. The molecular mechanism of dsRNA and sRNA formation and also the target origin predestinates the subsequent silencing pathway. In transcriptional gene silencing (TGS), the gene expression is regulated through chromatin epigenetic modifications. One of the epigenetic marks is cytosine methylation, which is established mainly by RNA-directed DNA-methylation (RdDM) pathway. Although the protein machinery was relatively...

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The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...

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