Une analyse du transcriptome d’Arabidopsis thaliana soumis à différents stress biotiques a révélé l’activation de certains membres de la famille CYP76, particulièrement celle de CYP76C2 (≈ 50 fois). La caractérisation fonctionnelle de la famille CYP76, et plus particulièrement celle de CYP76C2 a donc fait l’objet de cette thèse. Après confirmation de l’activation sélective de CYP76C2 en réponse aux pathogènes par qRT-PCR, le phénotype de ses mutants d’insertion et de surexpression a été caractérisé sous différentes conditions d’infection par: Pseudomonas syringae pv. tomato DC3000, P. syringae pv. tomato DC3000 avrRpm1 et par Botrytis cinerea. Afin d’identifier la voie métabolique faisant intervenir CYP76C2, un profilage métabolique ciblé et non ciblé a été entrepris, centré sur le(s) métabolite(s) différentiellement accumulés dans les différents mutants en condition d’infection. Alors que des différences subtiles de sensibilité des mutants de CYP76C2 aux pathogènes semblent confirmer son rôle dans la réponse aux pathogènes, les lignées affectées dans son expression ne présentent pas de phénotypes clairement différents de ceux des plantes sauvages. Une analyse non–ciblée en UPLC-MS (Orbitrap) a permis d’identifier un composé absent dans le mutant cyp76c2 qui pourrait correspondre à un dérivé conjugué en C11, sans que sa structure ne puisse pour l’instant être identifiée (formule brute C17H28O9). CYP76C2 ne semble pas impliqué directement dans la synthèse d’une molécule cruciale pour la mise en place du processus de défense, mais exerce plus probablement une fonction spécialisée ou partiellement redondante de défense ou de détoxication. / A transcriptome analysis of Arabidopsis thaliana subjected to biotic stresses has revealed the activation of members of the CYP76 family, especially of CYP76C2 (≈ 50 times). The functional characterization of CYP76C2, has been the objective of this thesis. After confirmation of the selective activation of CYP76C2 by pathogens, the phenotype of its insertion and overexpressor mutants was characterized under infection by Pseudomonas syringae pv. tomato DC3000, P. syringae pv. tomato DC3000 avrRpm1 and Botrytis cinerea. In order to identify the metabolic pathway involving CYP76C2, targeted and non-targeted metabolic profiling was focused on differentially accumulated compounds in the different mutants after infection. Whereas subtle differences of response of the CYP76C2 mutant lines in response to pathogens seemed to confirm its involvement in response to biotic stress, phenotypes strikingly different from those of wild-type plants were not observed. A non-targeted analysis by UPLC-MS (Orbitrap) identified a compound absent in the cyp76c2 line that may correspond to an oxygenated C11 conjugate (raw formula C17H28O9), but its structure was not identified. CYP76C2 thus does not seem directly involved in the synthesis of a molecule crucial for defense responses, but more likely has a role in the synthesis of a potentially redundant specialized defense compound or in a detoxification process.

L'induction du métabolisme secondaire fait partie du système de défense des plantes lors d’une attaque par un microorganisme pathogène. Les propriétés antimicrobiennes ou de signalisation des métabolites secondaires (MS) peuvent être régulées par un processus majeur et efficace, la glycosylation. Cette réaction est catalysée par les glycosyltransférases (UGTs) qui assurent le transfert d’une molécule de sucre sur un MS. Mon travail de thèse a porté sur l’étude de trois UGTs d’Arabidopsis thaliana (Arabidopsis), UGT76E12 d’une part, et UGT73B3 et UGT73B5 d’autre part, et a montré que 1) UGT76E12 est impliquée dans la mise en place de la résistance basale à la souche virulente de la bactérie Pseudomonas syringae pv. tomato, Pst DC3000, et participe au dialogue entre les voies de signalisation hormonale de l’acide jasmonique (JA) et de l’acide salicylique (SA). Des analyses par clustering hiérarchique ont révélé une co-expression forte entre le gène UGT76E12, et deux gènes codant une terpène synthase (TPS4) et un cytochrome P450 (CYP82G1), impliqués dans la biosynthèse d’un diterpène, le TMTT ((E,E)-4,8,12-triméthyletridéca-1,3,7,11-tetraène), suggérant un rôle pour UGT76E12 dans la régulation de la voie de biosynthèse des terpènes ; 2) UGT73B3 et UGT73B5 sont impliquées dans la mise en place de la résistance spécifique d’Arabidopsis à la souche avirulente Pst DC3000-AvrRpm1, notamment par la régulation de MS intervenant dans le maintien de l’état redox cellulaire au cours de la réaction d’hypersensibilité (HR). Ces deux gènes appartiennent à la classe des « SA-early induced-genes » et présentent un profil co-expression avec le gène TOLB-related et deux gènes codant des glutathion S-transférases (GSTU7 et GSTU24) impliqués dans la réponse au stress oxydant et la détoxication des MS. Nos résultats suggèrent un rôle pour UGT73B3 et UGT73B5 dans la glycosylation de métabolites oxydants associés à la mise en place de la HR. / Plant secondary metabolism induction is part of an integrated defense system after pathogen infection. Signalling and antimicrobial properties of secondary metabolites (SM) can be regulated by an efficient process named, glycosylation. This reaction is ensured by glycosyltransferases (UGTs) which catalyze the transfer of a sugar moiety on a SM. My PhD work was divided in two parts, the first on UGT76E12 and the second part on UGT73B3 and UGT73B5, three UGTs of Arabidopsis thaliana (Arabidopsis) and it demonstrated that 1) UGT76E12 is involved in basal resistance establishment during plant challenge with the virulent strain of Pseudomonas syringae pv. tomato (Pst DC3000), and participates to the hormonal crosstalk between jasmonic acid (JA) and salicylic acid (SA). Clustering analyses revealed a strong co-expression between UGT76E12 and two genes encoding a terpene synthase (TPS4) and a cytochrome P450 (CYP82G1), both involved in the biosynthesis of a diterpene named TMTT ((E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene), which suggests a role for UGT76E12 in terpene biosynthesis pathway regulation. 2) UGT73B5 and UGT73B3 are involved in the establishment of Arabidopsis specific resistance against the avirulent strain of Pst DC3000-AvrRpm1, especially in the regulation of MS playing a role in redox homeostasis status during the hypersensitive response (HR). These two genes are part of SA-early induced-genes and showed co-expression patterns with the TOLB-related gene and two genes encoding glutathione S-transferases (GSTU7 and GSTU24) involved in oxidative stress responses and in MS detoxification. Our results suggest a role for UGT73B3 and UGT73B5 in the glycosylation of oxidant MS associated with HR establishment.

Atualmente, o HLB é considerado a principal doença que acomete as plantas cítricas. Diante desse fator, pesquisas por cultivares resistentes a esta doença são necessárias. A transformação genética via Agrobacterium, juntamente com o uso de plantas modelos, tem sido uma alternativa para verificação do funcionamento dos genes em resposta a patógenos, isto porque as plantas modelos possuem como característica ciclo de vida curto e alto poder de regeneração. Assim sendo, objetivou-se com o presente trabalho, a transformação genética via Agrobacterium tumefaciens, de tomate Micro-Tom (Solanum lycopersicum L.) e de laranja doce, com os genes que codificam as proteínas PR-8 e CDR-1, isolados a partir de Citrus simensis. Os cotilédones provenientes de sementes germinadas in vitro de tomate Micro-Tom foram utilizados como fonte de explante para os experimentos de transformação genética com os genes PR-8 e CDR-1. Esses explantes foram subcultivados até o aparecimento de brotos regenerantes e posteriormente plantas transgênicas, as quais foram aclimatizadas e levadas a casa-de-vegetação. A transgenia foi confirmada por PCR e o número de inserções do gene por Southern blot. As plantas foram cultivadas até a obtenção da geração T1. Simultaneamente, foram realizados experimentos de transformação genética em segmentos de epicótilo, provenientes de sementes de laranja ‘Hamlin’ germinadas in vitro, com o gene CDR-1, a fim de se obter plantas transgênicas e sua caracterização. Paralelamente, foi realizada a construção da curva padrão pela análise de qPCR para identificação de Pseudomonas syringae pv. tomato. Foram obtidas treze plantas transgênicas de tomate Micro-Tom com o gene PR-8 e três com o gene CDR-1. As eficiências de transformação foram em torno de 0,38 a 1,98%. Três plantas de tomate Micro-Tom transgênicas com o gene PR-8 foram caracterizadas por Southern blot e o número de inserções variou de 1 a 3. Dezenove plantas transgênicas de laranja ‘Hamlin’ com o gene CDR-1 foram obtidas através dos experimentos de transformação genética. A eficiência de transformação foi de 2,06 a 5,96%. Dessas, apenas uma foi caracterizada por Southern blot apresentando 1 número de cópia do DNA no genoma da planta. / HLB is currently considered the main disease affecting citrus plants. Given this factor, research for cultivars resistant to this disease is needed. Genetic transformation via Agrobacterium with the use of model plants has been an alternative for checking the gene function in response to pathogens, because these model plants have as characteristic a short life cycle and high power of regeneration. Therefore, the aim of this work was to produce transgenic plants, via Agrobacterium tumefaciens, of Micro-Tom tomato (Solanum lycopersicum L.), and sweet orange, with the genes encoding the PR-8 and CDR-1 proteins isolated from Citrus sinensis. The cotyledons from in vitro germinated Micro-Tom tomato seeds were used as explants source for genetic transformation experiments with PR-8 and CDR-1 genes. These explants were subcultured until the appearance of regenerating shoots and after transgenic plants, which were acclimatized and taken to a greenhouse. The transgenic plants were confirmed by PCR and the number of gene insertions by Southern blot. The plants were grown until T1 generation was obtained. Simultaneously genetic transformation experiments were performed with epicotyl segments from \'Hamlin\' sweet orange seeds germinated in vitro with CDR-1 gene in order to obtain transgenic plants and their characterization. Simultaneously, the standard curve construction was performed by qPCR analysis for identification of Pseudomonas syringae pv. tomato. Thirteen transgenic plants of Micro-Tom tomato with PR-8 gene and three with CDR-1 gene were obtained. The transformation efficiencies were around 0,38 to 1,98%. Three transgenic plants of Micro-Tom tomato with PR-8 gene were characterized by southern blot, and the number of inserts ranged from 1 to 3. Nineteen transgenic \'Hamlin\' sweet orange plants with CDR-1 gene were obtained through genetic transformation experiments, and the transformation efficiency was 2,06 to 5,96%. One plant was characterized, by Southern blot and has one DNA copy number in the plant genome.

Parmi les Prunus, genre botanique d’intérêt économique important, l’abricotier (Prunusarmeniaca L.) est une culture emblématique du Bassin Méditerranéen. Il y est soumis à des contraintes biotiques importantes, parmi lesquelles le dépérissement bactérien, causé par Pseudomonas syringae (Psy), peut mener à des phénomènes de mortalité en verger au niveau des régions à hivers froids et humides. La mise en évidence de différences variétales en verger offre potentiellement des perspectives de contrôle de la maladie à travers le levier génétique. Aussi, ce travail de thèse avait pour principaux objectifs (i) d’identifier chez la plante des régions génomiques liées à la résistance partielle à la bactérie et (ii) d’étudier un plan factoriel d’interaction entre les diversités de la plante et de la bactérie (GxG) afin d’apprécier la généricité de la résistance et sa durabilité. Afin de répondre au premier objectif, deux approches complémentaires ont été mobilisées : une cartographie de QRLs (Quantitative Resistance Loci) sur quatre populations biparentales dont trois sont issues du croisement avec un géniteur commun ainsi qu’une analyse d’association sur une core-collection. Les données phénotypiques mobilisées correspondent à des symptômes issus d’inoculations contrôlées ainsi que des notes de mortalité obtenues suite à infection naturelle en verger. Ces deux approches (analyse de liaison et d’association) ont permis de mettre en évidence 22 QRLs de résistance, parmi lesquels seuls 2 QRLs sur les chromosomes 6 et 7 colocalisent entre les deux approches. Deux régions majeures détectées en étude d’association sur les chromosomes 5 et 6 se sont révélées être en déséquilibre de liaison et contrôlent près de 26 et 43% de la variation des symptômes. Deux mécanismes complémentaires reposant sur le blocage de l’infection de Psy et sur la limitation de la progression locale de la bactérie dans les tissus ont été mis en évidence à travers la détection de QRLs sur les chromosomes 3, 6, 8 d’une part et 1,4et 6 d’autre part. Le second objectif a été abordé grâce à une étude d’un plan factoriel d’interaction entre 20 accessions d’abricotier et 9 souches de Psy, échantillonnées d’après la connaissance de l’épidémiologie de la maladie en verger. L’analyse statistique de ce dispositif mis en œuvre à la fois en verger et en laboratoire a démontré la prédominance de l’effet du facteur souche dans la variabilité des symptômes étudiés et la très faible importance du facteur d’interaction GxG, indiquant une potentielle généricité des facteurs de résistance et des perspectives favorables à leur durabilité en verger.Les résultats issus de cette thèse contribuent à offrir une meilleure compréhension des mécanismes de résistance partielle au dépérissement bactérien de l’abricotier et fournissent des marqueurs et haplotypes, potentiellement mobilisables dans le cadre de programmes d’innovation variétale. / Within the genus Prunus, that contains highly valuable species, apricot (Prunusarmeniaca L.) is an emblematic Mediterranean crop. But apricot cultivation is constrainedby many biotic stresses, among which bacterial canker caused by Pseudomonas syringae(Psy) is particularly severe and can lead to the death of the trees in regions with humidand cold winters. Differences of susceptibilities have been observed between cultivars inorchards and create opportunities for disease management through genetic improvement.This thesis aimed to (i) identify genetic determinants linked to partial resistance to thebacterium and to (ii) study a factorial interaction design between both diversities of theplant and the pathogen (GxG interaction) in order to assess resistance genericity anddurability. With regard to the first objective, two complementary approaches were used :QRL (Quantitative Resistance Loci) mapping over four biparental progenies, amongwhich three were obtained with a cross involving a common genitor, and a genome-wideassociation study on a core-collection. The phenotypic data mobilized in this work rely onsymptoms issued from controlled inoculations and on mortality notations followingnatural infections in the orchard. These approaches led to the detection of 22 QRLs amongwhich only 2 QRLs, located on chromosomes 6 and 7, co-localized between the twomethods. Two main regions detected in the association study, over the chromosomes 5and 6, appeared to be in linkage disequilibrium and controlled 26 and 43% of the variationof the symptoms. A complementarity between two mechanisms, one that involves blockingthe infection of Psy and the other that limits bacterial mobility in the tissues has beenrevealed through the detection of QRLs over chromosomes 3, 6, 8 for one mechanism and1,4, 6 for the other, respectively. The second objective was fulfilled with a study of afactorial interaction design between 20 apricot accessions and 9 Psy strains, which weresampled according to the previous knowledge of the disease epidemiology in the orchard.Statistical analyses of phenotypic data obtained both from the orchard and a laboratorytest showed a clear predominance of the strain effect on symptom variability and a weakimportance of the GxG interaction factor. This last result highlighted a potentialgenericity of the resistance factors and favorable perspectives of durability in the orchard.The results issued from this thesis contribute to a better understanding of the mechanismsunderlying partial resistance of apricot to bacterial canker. Moreover, it provide markersand haplotypes of interest which could be mobilized in breeding programs.

La capacité des plantes à répondre aux stress de l'environnement, qu'ils soient de nature biotique ou abiotique, tient au fait qu'elles sont capables d'intégrer les signaux perçus grâce à des mécanismes de transduction du signal rapides et efficaces. La perception, le décodage et la mise en place de réponses biologiques adaptées font appel à de nombreux acteurs moléculaires tels que le calcium (Ca2+), second messager majeur de la signalisation Eucaryote. Parmi les "senseurs de calcium", la calmoduline (CaM) est la protéine la plus connue. Il existe des protéines apparentées à la CaM, spécifiques aux plantes, les Calmodulin-like (CMLs). Les CMLs sont très peu étudiées et la caractérisation de leurs rôles ouvre de larges perspectives sur l'identification des réseaux de régulation. L'objectif de ce travail de thèse a concerné un partenaire nucléaire d'une de ces CMLs, AtCML9, le Pseudo-Response Regulator 2 (PRR2), une protéine atypique contenant un domaine de liaison à l'ADN de type GARP et de fonction inconnue. Au cours de ce travail, des analyses moléculaires et biochimiques ont permis de caractériser le rôle de PRR2 dans l'immunité végétale, et en particulier en réponse à Pseudomonas syringae. L'étude de lignées perte ou gain de fonction a permis de mettre en évidence que PRR2 agit comme un régulateur positif des défenses lors de l'infection par la bactérie pathogène hémibiotrophe Pst DC3000 à travers la modulation de l'acide salicylique, de composés de défense tels que la protéine PR1 et la camalexine. Les analyses phénotypiques réalisées en réponse à différentes souches de Pseudomonas ont permis de préciser que PRR2 contribue à la mise en place des défenses à travers la signalisation dépendante de l'acide salicylique et de l'injection des effecteurs bactériens. Dans une deuxième partie, l'interaction entre PRR2 et des facteurs de transcription spécifiques aux plantes, les TCPs (Teosinte Branched 1, Cycloidea and PCF), a été étudié. Ces analyses ont montré une spécificité d'interaction entre PRR2 et TCP19 ou TCP20. Ces interactions stabilisent et relocalisent PRR2 dans des domaines nucléaires spécifiques. Ces données suggèrent une forte régulation post-traductionnelle de la protéine PRR2 qui pourrait s'avérer nécessaire à sa fonction biologique. / Plants are able to perceive and respond to diverse biotic or abiotic environmental cues. This ability relies on efficient signalling pathways that are ultimately associated with genetic reprograming. These responses involve various actors of the signalling pathways such as calcium (Ca2+) transients which act as a second messenger in eukaryotic cells. The variations in intracellular Ca2+ concentrations are perceived by calcium sensors. The calmodulin (CaM) is an ubiquitous Ca2+ sensor well studied both in animal and plant cells. Comparatively, plants also possess CaM-related proteins called Calmodulin-like (CMLs) which are less studied and their role in plant physiology are emerging. The objective of this PhD work was to perform the functional analysis of PRR2 (Pseudo-Response Regulator 2), a plant specific transcription factor (with a GARP DNA binding domain) previously identified as an AtCML9-interacting partner. Using diverse genetic tools, we were able to study the role of PRR2 in plant immunity using the model plant Arabidopsis thaliana and a phytopathogenic bacteria, Pseudomonas syringae. Our study has shown that PRR2 acts as a positive regulator of plant defenses upon bacterial infection. We show that PRR2 could act by modulating the biosynthesis of the salicylic acid (SA), and the production of defense-associated compounds such as PR1 and camalexin. Collectively our data indicate that PRR2 acts as a positive regulator of plant defense associated with SA. In the aim to better understand how PRR2 could be involved in different physiological responses, we search for PRR2-interacting partners. We have more precisely worked on the interactions between PRR2 and the TCPs (Teosinte branched 1, Cycloidea and PCF) which are also plant specific transcriptions factors involved in different biological processes. We showed that PRR2 specifically interact with TCP19 or TCP20. As consequences, these interactions stabilize PRR2 and relocalize the complex in specific nuclear subdomains.

Atualmente, o HLB é considerado a principal doença que acomete as plantas cítricas. Diante desse fator, pesquisas por cultivares resistentes a esta doença são necessárias. A transformação genética via Agrobacterium, juntamente com o uso de plantas modelos, tem sido uma alternativa para verificação do funcionamento dos genes em resposta a patógenos, isto porque as plantas modelos possuem como característica ciclo de vida curto e alto poder de regeneração. Assim sendo, objetivou-se com o presente trabalho, a transformação genética via Agrobacterium tumefaciens, de tomate Micro-Tom (Solanum lycopersicum L.) e de laranja doce, com os genes que codificam as proteínas PR-8 e CDR-1, isolados a partir de Citrus simensis. Os cotilédones provenientes de sementes germinadas in vitro de tomate Micro-Tom foram utilizados como fonte de explante para os experimentos de transformação genética com os genes PR-8 e CDR-1. Esses explantes foram subcultivados até o aparecimento de brotos regenerantes e posteriormente plantas transgênicas, as quais foram aclimatizadas e levadas a casa-de-vegetação. A transgenia foi confirmada por PCR e o número de inserções do gene por Southern blot. As plantas foram cultivadas até a obtenção da geração T1. Simultaneamente, foram realizados experimentos de transformação genética em segmentos de epicótilo, provenientes de sementes de laranja ‘Hamlin’ germinadas in vitro, com o gene CDR-1, a fim de se obter plantas transgênicas e sua caracterização. Paralelamente, foi realizada a construção da curva padrão pela análise de qPCR para identificação de Pseudomonas syringae pv. tomato. Foram obtidas treze plantas transgênicas de tomate Micro-Tom com o gene PR-8 e três com o gene CDR-1. As eficiências de transformação foram em torno de 0,38 a 1,98%. Três plantas de tomate Micro-Tom transgênicas com o gene PR-8 foram caracterizadas por Southern blot e o número de inserções variou de 1 a 3. Dezenove plantas transgênicas de laranja ‘Hamlin’ com o gene CDR-1 foram obtidas através dos experimentos de transformação genética. A eficiência de transformação foi de 2,06 a 5,96%. Dessas, apenas uma foi caracterizada por Southern blot apresentando 1 número de cópia do DNA no genoma da planta. / HLB is currently considered the main disease affecting citrus plants. Given this factor, research for cultivars resistant to this disease is needed. Genetic transformation via Agrobacterium with the use of model plants has been an alternative for checking the gene function in response to pathogens, because these model plants have as characteristic a short life cycle and high power of regeneration. Therefore, the aim of this work was to produce transgenic plants, via Agrobacterium tumefaciens, of Micro-Tom tomato (Solanum lycopersicum L.), and sweet orange, with the genes encoding the PR-8 and CDR-1 proteins isolated from Citrus sinensis. The cotyledons from in vitro germinated Micro-Tom tomato seeds were used as explants source for genetic transformation experiments with PR-8 and CDR-1 genes. These explants were subcultured until the appearance of regenerating shoots and after transgenic plants, which were acclimatized and taken to a greenhouse. The transgenic plants were confirmed by PCR and the number of gene insertions by Southern blot. The plants were grown until T1 generation was obtained. Simultaneously genetic transformation experiments were performed with epicotyl segments from \'Hamlin\' sweet orange seeds germinated in vitro with CDR-1 gene in order to obtain transgenic plants and their characterization. Simultaneously, the standard curve construction was performed by qPCR analysis for identification of Pseudomonas syringae pv. tomato. Thirteen transgenic plants of Micro-Tom tomato with PR-8 gene and three with CDR-1 gene were obtained. The transformation efficiencies were around 0,38 to 1,98%. Three transgenic plants of Micro-Tom tomato with PR-8 gene were characterized by southern blot, and the number of inserts ranged from 1 to 3. Nineteen transgenic \'Hamlin\' sweet orange plants with CDR-1 gene were obtained through genetic transformation experiments, and the transformation efficiency was 2,06 to 5,96%. One plant was characterized, by Southern blot and has one DNA copy number in the plant genome.

Orientador: Ione Salgado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T08:31:31Z (GMT). No. of bitstreams: 1 Vitor_SimoneCespedes_M.pdf: 2469178 bytes, checksum: 5b012d0f044b319a6fb8e2dc382c8242 (MD5) Previous issue date: 2013 / Resumo: O NO é uma molécula sinalizadora versátil muito importante em diversos processos em plantas. Uma de suas principais atuações é na sinalização celular durante o processo de defesa contra o ataque de patógenos. Plantas de Arabidopsis thaliana do genótipo mutante nia1 nia2, deficientes para os dois genes estruturais que codificam para a enzima nitrato redutase (NR), são susceptíveis à bactéria Pseudomonas syringae. Foi sugerido que a resposta de defesa prejudicada no mutante NR-deficiente seria resultante de seus reduzidos níveis de NO, quando comparados àqueles do genótipo selvagem. Em um trabalho recente de nosso grupo, empregando microarranjo de DNA, foi observado que a fumigação com gás NO no mutante nia1 nia2 foi capaz de modular diversos genes relacionados à defesa, alguns dos quais não previamente documentados como responsivos a esse radical. Neste trabalho se analisou por PCR em tempo real o efeito do gás NO na expressão de genes relacionados à defesa em plantas selvagem e no mutante nia1 nia2 infectados com uma linhagem avirulenta da bactéria P. syringae. Genes de defesa, como PR1, foram induzidos pela bactéria e a expressão destes foi maior no genótipo selvagem quando comparado ao nia1 nia2, o que é consistente com a susceptibilidade do mutante. A fumigação com NO também modulou genes relacionados à biossíntese de lignina (CAD1) e à sinalização de auxina (TIR1, ILL1, GH3) e etileno (ACCS7). Análises de quantificação de lignina mostraram uma pequena redução desse composto no genótipo mutante em relação ao selvagem, além de uma diferença em sua composição. Ademais, a fumigação com NO atenuou a expressão de PR1 e outros genes relacionados à via do ácido salicílico em plantas inoculadas e preveniu o crescimento bacteriano em folhas nia1 nia2. Já foi demonstrado que a inoculação do patógeno no mutante induz uma baixa produção de NO e no presente trabalho observou-se uma alta produção de H2O2 comparado ao selvagem. O H2O2 potencializou o efeito microbicida do NO fumigado na suspensão de bactéria. Os resultados sugerem que um efeito microbicida direto do NO, em conjunto com H2O2, pode resultar na atenuação da resposta de defesa na planta, reduzindo o gasto de energia associado à transcrição de genes relacionados à defesa / Abstract: NO is an important signaling and versatile molecule which plays important roles in many processes in plants. One of its main actions is in cell signaling during defense response against pathogen attack. Arabidopsis thaliana plants of the nia1 nia2 mutant genotype, deficient in the two structural genes encoding for the enzyme nitrate reductase (NR), are susceptible to the avirulent bacteria Pseudomonas syringae. It has been suggested that the impaired defense response in the NR-deficient mutant would result from their low NO levels when compared to those of the wild type. Indeed, in a recent study from our group, it was observed through a DNA microarray that fumigation of nia1 nia2 mutant with NO gas was able to modulate many genes related to defense, some of which not previously documented as responsive to this radical. In this work we analyzed by real-time PCR the effect of NO gas on the expression of genes related to defense in the wild type and nia1 nia2 mutant infected with an avirulent strain of Pseudomonas syringae. Defense genes such as PR1 were induced by the bacteria and its expression was higher in wild type when compared to nia1 nia2, which is consistent with the susceptibility of the mutant. NO fumigation also modulated genes related to the biosynthesis of lignin (CAD1) and the auxin (Tir1, ILL1, GH3) and ethylene pathways (ACCS7). Analysis of lignin showed a reduction of this compound in the mutant genotype compared to wild type, and a difference in its composition. In addition, fumigation with NO attenuated the expression of PR1 and other genes related to salicylic acid signaling in infected plants and prevented bacterial growth in nia1 nia2 leaves. Furthermore, pathogen infection is known to induce a low production of NO in nia1 nia2 and here we also observed that there is a higher production of H2O2 in the mutant compared to the wild type. H2O2 potentiated the microbicidal effect of NO fumigated in bacterial suspensions. The results suggest that a direct microbicidal effect of NO, together with H2O2, may result in attenuation of the defense response in the plant, reducing energy expenditure associated with the transcription of genes related to defense / Mestrado / Biologia Vegetal / Mestra em Biologia Vegetal

Doctor of Philosophy / Genetics Interdepartmental Program, Plant Pathology / Xiaoyan Tang / Pseudomonas syringae bacteria utilize the type III secretion system (T3SS) to deliver effector proteins into host cells. The T3SS and effector genes (together called the T3 genes hereafter) are repressed in nutrient rich medium but are rapidly induced after the bacteria are transferred into minimal medium (MM) or infiltrated into the plant. The induction of the T3 genes is mediated by HrpL, an alternative sigma factor that recognizes the conserved hrp box motif in the T3 gene promoters. The induction of hrpL is mediated by HrpR and HrpS, two homologous proteins that bind the hrpL promoter. To identify additional genes involved in regulation of the T3 genes, P. s. pv. phaseolicola (Psph) NPS3121 transposon insertion mutants were screened for reduced induction of avrPto-luc and hrpL-luc, reporter genes for promoters of effector gene avrPto and hrpL, respectively. Determination of the transposon-insertion sites led to the identification of genes with putative functions in signal transduction and transcriptional regulation, protein synthesis, and basic metabolism. A transcriptional regulator (AefRNPS3121) identified in the screen is homologous to AefR, a regulator of the quorum sensing signal and epiphytic (plant-associated) traits that was not known previously to regulate the T3 genes in P. s. pv. syringae (Psy) B728a. AefRNPS3121 in Psph NPS3121 and AefR in Psy B728a are similar in regulating the quorum sensing signal in liquid medium but different in regulating epiphytic traits such as swarming motility, entry into leaves, and survival on the leaf surface. The two component system RhpRS was identified in Pseudomonas syringae as a regulator of the T3 genes (Xiao et al. 2007). In the rhpS- mutant, the response regulator RhpR represses the induction of the T3 gene regulatory cascade, but induces its own promoter in a phosphorylation-dependent manner. Deletion and mutagenesis analyses revealed an inverted repeat (IR) element GTATC-N6-GATAC in the rhpR promoter that confers the RhpR-dependent induction. Computational search of the P. syringae genomes for the putative IR elements and Northern blot analysis of the genes with a putative IR element in the promoter region uncovered five genes that were upregulated (PSPTO2036, PSPTO2767, PSPTO3477, PSPTO3574, and PSPTO3660) and two genes that were down-regulated (PSPTO0536 and PSPTO0897) in an RhpR-dependent manner. ChIP assays indicated that RhpR binds the promoters containing a putative IR element but not the hrpR and hrpL promoters that do not have an IR element, suggesting that RhpR indirectly regulates the transcriptional cascade of hrpRS, hrpL, and the T3 genes. To identify additional genes involved in the rhpRS pathway, suppressor mutants were screened that restored the induction of the avrPto-luc reporter gene in the rhpS- mutant. Determination of the transposon-insertion sites led to the identification of rhpR, an ATP-dependent Lon protease, a sigma 70 family protein (PSPPH1909), and other metabolic genes. A lon- rhpS- double mutant exhibited phenotypes typical of a lon- mutant, suggesting that rhpS acts with or through lon. The expression of lon was elevated in rhpS- and other T3-deficient mutants, indicating a negative feedback mechanism. Both the lon- rhpS- and the PSPPH1909- rhpS- double mutant displayed enhanced transcription of hrpL in MM than did the rhpS- mutant.

La réussite de la mise en place d’une résistance efficace chez la plante hôte requiert la présence d’un dialogue entre les différentes voies signalétiques menant à l’induction de la défense. L’implication de l’extinction génique post-transcriptionnelle dans la résistance contre des agents pathogènes, autres que les virus, est une nouvelle avenue qui va permettre de disséquer le lien qui peut exister entre les deux voies immunitaires de la plante : celle qui est déjà importante pour lutter contre les bactéries et les champignons (voie induite à la suite de la reconnaissance des éliciteurs) et celle de l’extinction génique post-transcriptionnelle. Les résultats que nous avons obtenus ont une signification très importante concernant l’implication de l’extinction génique post-transcriptionnelle dans la résistance des plantes contre les agents pathogènes d’une manière générale et Botrytis cinerea, Alternaria brassicicola et Pseudomonas syringae en particulier. Mon travail de recherche a permis d’identifier le miR472 comme étant un acteur qui favorise la mise en place de la maladie causée par des agents pathogènes du type nécrotrophe et hémibiotrophe. De plus j’ai pu, en utilisant le modèle P. syringae pv tomato DC3000-Arabidopsis, disséquer dans le détail, comment la bactérie manipule la machinerie de la plante pour activer la voie des microARNs menant à l’accumulation du miR472, responsable de la suppression de la résistance basale et du PTI. Cette suppression passe par l’inhibition de sa cible, la protéine NB-LRR; RDS1 un nouveau régulateur impliqué dans le PTI induit par la Flg22. Cette étude permettra une avancée remarquable dans les connaissances reliées à l’immunité des plantes, ce qui constituerait une étape clé dans la découverte de nouvelles stratégies de lutte contre les agents phytopathogènes. Sur le plan fondamental, cette étude a permis de consolider le dialogue qui existe entre les voies immunes de la plante et de mettre en évidence un nouvel acteur impliqué dans la résistance basale. En matière d’application, comprendre les mécanismes de défense et de résistance des plantes aux agents pathogènes est primordial pour développer des plantes génétiquement résistantes et des stimulateurs de défense naturelle (Vaccin de plantes). Ceci constitue un enjeu majeur dans l'objectif de concevoir de nouveaux moyens de lutte phytosanitaire, à la fois plus respectueux de l’environnement et ayant moins d’impact sur la santé humaine.

The apoplast is the site of infection for many important bacterial crop pathogens, including the model pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The chemical environment within the plant apoplast can determine the outcome of bacterial infection and the composition of this compartment is known to change in response to the presence of invading organisms. However, this metabolically dynamic environment has received little attention in the literature, and even less is known about how metabolites in the apoplast influence the expression of virulence genes. In this study, several aspects of the metabolic context of virulence were assessed. First, a broad-scale analysis of the tomato apoplast was undertaken, which identified metal ions, sugars, organic acids and amino acids, the most abundant of which was the non-protein amino acid gamma-aminobutyric acid (GABA). The impact these components had on the expression of virulence genes and metabolism in Pst DC3000 were then tested. Components such as fructose and aspartate caused high levels of virulence gene expression which correlated with the accumulation of intracellular glutamate, whereas repressive components, such as GABA and threonine, resulted in lower glutamate levels. Second, metabolic flux analysis showed that Pst DC3000 underwent major changes in central carbon metabolism in response to virulence gene inducing conditions. The identification of altered internal metabolism in Pst DC3000 cells expressing virulence genes led to the conclusion that Pst DC3000 may understand its external environment by sensing intracellular metabolites or metabolic fluxes. Third, the role of GABA assimilation in virulence was explored, and it was found that high internal GABA levels resulted in virulence gene repression. In addition, previously unidentified mechanisms for GABA uptake and transport were detected by the use of a novel ‘unlabelling’ experiment.