La conductivité hydraulique foliaire (KF) est une composante majeure du transport d’eau dans toute la plante. Dans les feuilles de noyer, la KF est stimulée à la lumière et est étroitement liée à l’accroissement du taux des transcrits d’aquaporines JrPIP2s. Par ailleurs, la corrélation entre la stimulation de la KF et des transcrits d’aquaporines à la lumière, n’est pas générale et dépend de l’espèce. Ici, nous étudions cette corrélation chez cinq espèces forestières (Juglans regia, Fagus sylvitica, Quercus robur, Salix alba et Populus tremula) différant par leur réponse à la lumière. Nous démontrons seulement chez le noyer (Juglans regia), la contribution des deux familles d’aquaporines PIP1s et PIP2s. Afin de mieux comprendre le rôle des JrPIP1s et JrPIP2 dans la réponse à la lumière, nous avons isolé 8 nouvelles isoformes dans les feuilles de noyer et nous avons étudié leurs profils d’expression sur une cinétique lumière. Toutes les isoformes étudiées sont accumulées à la lumière et réprimées à l’obscurité. De plus, la KF est dépendante de la qualité de lumière. Elle est réduite de 65% en absence de lumière bleue. Cette diminution serait liée à l’inhibition des transcrits d’aquaporines. Afin de caractériser les mécanismes moléculaires précoces impliqués dans la modulation de KF par la lumière, l’approche globale cDNA-AFLP a été menée sur des feuilles de noyer sous différentes conditions d’éclairement. Nous obtenons 12000 transcrits différentiels dérivés (TDFs) générés par les 128 couples d’amorces. Parmi les 187 séquences obtenues, 93 d’entre elles ont une fonction putative. Leur classification fonctionnelle montre que les gènes relatifs à la régulation cellulaire représentent environ 58% des TDFs identifiés. Les feuilles exposées à la lumière, montrent des changements dans les voies de : signalisation calcique, protéolyse, trafic vésiculaire et l’expression de divers facteurs de transcription et protéines de régulation. Pour mieux comprendre le rôle potentiel de la signalisation calcique dans la modulation de la KF par la lumière, nous avons étudié l’effet d’un inhibiteur des canaux calciques [LaCl3] et d’un antagoniste de calmoduline [W7] sur la KF et les transcrits des 10 JrPIPs. Comparées aux feuilles témoins, les inhibiteurs calciques provoquent une réduction de la KF et de la majorité des JrPIPs étudiées à la lumière. Nos résultats confirment l’implication du complexe Ca2+ /calmoduline dans la transduction du signal lumineux responsable de la stimulation de la KF et des transcrits d’aquaporines chez le noyer. / Leaf hydraulic conductance (Kleaf) takes a significant part in plant water relations. In walnut leaves, Kleaf was stimulated by light and tightly related to accumulation of JrPIP2s aquaporin transcripts. However, the light effect on Kleaf value is not systematically related to aquaporin regulation. Here we investigated the relationship between light, Kleaf and transcript levels of aquaporin in five species (Juglans regia, Fagus sylvitica, Quercus robur, Salix alba and Populus tremula) differing by the response of their Kleaf to light. Only for walnut leaves, we showed that light-increased Kleaf value is closely related to higher stimulation of both PIP1s and PIP2s aquaporins. To further investigate the involvement of aquporins (JrPIP1s and JrPIP2) in the light Kleaf modulation, 8 new full length aquaporins have been identified in walnut leaves and their expression pattern was monitored. All the aquaporin tested was turned up to be upregulated under light condition and downregulated under darkness. Moreover, we showed that the Kleaf response to light is quality-dependant, since it was reduced of 65% in the absence of blue light. Interstingly, this Kleaf reduction was correlated with a high downregulation of almost all aquaporins tested. To give an insight into the early molecular events involved in the light-induced Kleaf regulation, a large-scale transcriptomic analysis consisting of the cDNA-AFLP procedure was carried out on walnut leaves, kept at different light conditions. We obtained a total of 12,000 transcript-derived fragments (TDFs) by cDNA-AFLP with 128 primer pairs. Reliable sequences were obtained for 187 of these TDFs, and functions were attributed to 93 TDFs through BLAST searches in GenBank databases. Most of the 93 TDFs corresponded to genes encoding proteins involved in cellular regulation (58%). Leaves exposed to light showed changes in the Ca2+-signaling pathway, the ubiquitin-proteasome pathway, vesicle trafficking process and the expression of multiple transcription factors and protein regulators. To progress in understanding of a potential role for calcium signalling in light-modulated Kleaf, Kleaf values and transcript accumulation of 10 JrPIPs were monitored in leaves perfused with either a blocker of calcium channels [LaCl3] or a CaM antagonist [W7]. Compared to control, these Ca2+ -effectors led to a significant reduction in Kleaf and transcripts accumulation of almost all JrPIPs under light conditions. Our results indicate that Ca2+ /calmodulin complex may transduce the light signal required for stimulation of Kleaf and its correlated aquaporin expression.

Les maladies des plantes causées par les champignons sont un problème majeur en agriculture. Pour les contrôler, les gènes de résistance (R) qui permettent de développer des variétés de plantes résistantes sont des éléments clés. La majorité des gènes R codent pour des protéines NLRs caractérisées par la présence d'un domaine de liaison aux nucléotides (NB-ARC) et un domaine de répétitions riches en leucines (LRR). Ces protéines agissent comme des récepteurs immunitaires intracellulaires et reconnaissent des facteurs de virulence des agents pathogènes appelés effecteurs. Les champignons phytopathogènes possèdent de vastes répertoires d'effecteurs qui contiennent centaines de protéines sécrétés, de petites tailles et sans similarités de séquence entre elles.La première question abordée dans ma thèse concerne l’origine de l'immense diversité des effecteurs fongiques. Une analyse structurale a identifié une famille d’effecteurs de séquences différentes mais qui possèdent une structure conservée. Cette famille a été appelée MAX-effectors (Magnaporthe Avrs and ToxB like) et elle est particulièrement importante chez Magnaporthe oryzae, l'agent causal de la pyriculariose du riz. Par des analyses d'expression, j'ai confirmé que la majorité des effecteurs MAX de M. oryzae sont spécifiquement exprimés durant la phase précoce de l'infection, suggérant une fonction importante durant la colonisation de la plante. Les effecteurs MAX constituent la première famille d'effecteurs fongiques définis par leur structure. Cette étude apporte donc de nouvelles pistes pour l'identification d'effecteurs chez les champignons et contribue à une meilleure compréhension de l'évolution des effecteurs. En effet, le scénario observé chez les effecteurs MAX suggère que beaucoup d’effecteurs fongiques appartiennent à un nombre restreint de familles d'effecteurs définies par leur structure. La seconde question que j’ai abordée durant ma thèse est le mécanisme moléculaire de la reconnaissance des effecteurs par les NLRs. J'ai abordé cette question en étudiant la reconnaissance de l'effecteur AVR-Pia par le couple de NLRs RGA4/RGA5. Des travaux précédents ont montré que RGA5 agit comme récepteur et se lie directement à AVR-Pia tandis que RGA4 agit comme élément de signalisation constitutivement actif, qui, en absence de l’agent pathogène, est réprimé par RGA5. Un domaine de RGA5, normalement absent chez les protéines NLR et similaire à la chaperonne du cuivre ATX1 (domaine RATX1), interagit physiquement avec AVR-Pia. Il a été suggéré que ce domaine RATX1 puisse agir comme un leurre de la cible de virulence d’AVR-Pia. Ce leurre, intégré dans la structure de RGA5, permettrait de « piéger » l’effecteur par interaction directe et jouerait donc un rôle crucial dans sa reconnaissance spécifique. Grâce à une analyse structurale détaillée d’AVR-Pia j’ai pu confirmer le rôle central de l'interaction AVR-Pia-RATX1 dans la reconnaissance de cet effecteur ce qui conforte le modèle du « leurre intégré ». De plus, j’ai caractérisé la surface d'interaction avec laquelle AVR-Pia lie le domaine RATX1. De plus, j'ai détecté des interactions entre AVR-Pia et d'autres parties de RGA5, indépendantes du domaine RATX1, notamment les domaines NB-ARC et LRR. Ceci a permis de développer un modèle qui explique comment la liaison d’un effecteur à un récepteur NLR comportant un leurre intégré par différentes interactions indépendantes conduit à une reconnaissance très sensible et spécifique qui est peu affectée par des mutations ponctuelles de l’effecteur. En résumé, cette étude a produit des connaissances nouvelles sur la fonction des récepteurs des plantes de type NLRs et sur leur capacité à reconnaitre des effecteurs. Ceci contribue à une meilleure compréhension du système immunitaire des plantes, ce qui est un élément important pour l’obtention de cultures durablement résistantes aux maladies / Plant diseases caused by fungi constitute a worldwide threat to food security and disease resistance (R) genes that allow to breed resistant crops are key elements for efficient disease control. The vast majority of R genes code for NLR multi domain proteins characterized by nucleotide-binding and leucine-rich repeat domains and acting as intracellular immune receptors for pathogen-secreted virulence factors termed effectors. Phytopathogenic fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The first question I addressed in my PhD thesis is: how is the tremendous diversity of fungal effectors generated? A structural analysis had identified the family of sequence-unrelated but structurally conserved MAX-effectors (Magnaporthe Avrs and ToxB like) that has expanded specifically in Magnaporthe oryzae the causal agent of rice blast disease. By expression analysis, I confirmed that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during host colonization. MAX effectors are the first structurally defined family of effectors in fungi and this study gives therefore news clues for the identification of candidate effectors in fungi and constitutes a crucial step towards a better understanding of effector evolution. In fact, the scenario observed for MAX-effectors leads to the hypothesis that the enormous number of sequence-unrelated fungal effectors belong in fact to a restricted set of structurally conserved effector families.The second question I investigated in my PhD thesis is: what are the molecular mechanisms of effector recognition by NLR immune receptors? I addressed this question by studying recognition of the M. oryzae effector AVR-Pia by the rice NLR pair RGA4/RGA5. Previous work has shown that RGA5 acts as a receptor that binds directly to AVR-Pia while RGA4 acts as a constitutively active signaling protein that is, in the absence of pathogen, repressed by RGA5. This functional interaction involves formation of an RGA4/RGA5 receptor complex. By protein-protein interaction studies, I showed that complex formation involves interactions between the RA4 and RGA5 NB-ARC and LRR domains, in addition to previously identified interactions between the coiled-coil domains. AVR-Pia recognition seems not to induce dissociation of the RGA4/RGA5 complex but a ternary RGA4/RGA5/AVR-Pia complex could also not be detected consistently. How effector recognition is translated into receptor complex activation remains therefore to be elucidated in more detail in the future. Previous work has shown that a domain of RGA5 normally not present in NLRs and related to the copper chaperone ATX1 (RATX1 domain) interacts physically with AVR-Pia and may be crucial for effector recognition. The RATX1 domain was hypothesized to mimic the true host targets of AVR-Pia leading to the development of the ‘integrated decoy’ model that states that unconventional domains in NLRs act as decoys in the recognition of effector proteins. By detailed structure-informed analysis of AVR-Pia, I could confirm the pivotal role of the AVR-Pia-RATX1 interaction for effector recognition lending important support to the integrated decoy model. In addition, I could precisely characterize the interaction surface with which AVR-Pia binds to the RGA5 RATX1 domain. Finally, I detected interactions of AVR-Pia with other parts of RGA5, in particular the NB-ARC and the LRR domains. Based on these results, I developed a model that explains how such binding to several independent sites in NLRs leads to high overall affinity and robust effector recognition that is resilient to effector mutations. Taken together, this study provides important novel insight into NLR function and effector recognition and contributes by this to a better understanding of plant immunity which is crucial for generating durable disease resistance in crops.

En esta tesis doctoral se estudia el modo de vida tritrófico del hongo nematófago Pochonia chlamydosporia utilizando técnicas "multiómicas". Pochonia chlamydosporia (= Metacordyceps chlamydosporia) (Goddard) Zare y Gams es un hongo nematófago usado para el control de nematodos agalladores de la raíz (Meloidogyne spp.) (Forghani and Hajihassani, 2020), entre otros. P. chlamydosporia se distribuye por todo el mundo y tiene un modo de vida tritrófico, pudiendo también adoptar estilos de vida endófito y saprófito. El mecanismo que utiliza P. clamydosporia para infectar huevos de nematodo comprende la desacetilación de la quitina de su pared celular a quitosano para facilitar su degradación por quitosanasas (Aranda-Martinez et al., 2016). El quitosano es un biopolímero derivado de la quitina que también se encuentra en el exoesqueleto de artrópodos y crustáceos. El genoma de P. chlamydosporia codifica un elevado número de quitosanasas, gracias a las cuales es resistente a quitosano y puede utilizarlo como fuente de nutrientes (Palma-Guerrero et al., 2010). Ambos pueden combinarse para el control de plagas. En este trabajo de tesis doctoral se pretende estudiar mediante metabolómica, transcriptómica y genómica el modo de vida tritrófico de P. chlamydosporia añadiendo quitosano, para determinar los mecanismos de interacción del hongo en ese entorno. En último término, se pretende sentar las bases para desarrollar un sistema para reducir plagas y enfermedades de forma sostenible.

Plants are fundamental to life on earth, crucially providing the basis of our food supply. As world population continues to grow, so too does the pressure on our agricultural systems, with one of the biggest challenges being the control of plant pathogens to ensure a healthy crop. The interaction between plant and pathogen is complex, with subtleties at the molecular level dictating the boundary between health and disease. This is exemplified by pathogen effectors; secreted proteins which enter the plant cell and interact with host targets to facilitate infection. AVR2 is one such effector, secreted by the oomycete pathogen Phytophthora infestans; the pathogen responsible for potato late blight. AVR2 interacts with a family of kelch-repeat containing phosphatases in potato, the BSLs, implicated in brassinosteroid pathway signalling – a major hormone signalling pathway in plants associated with growth and development. This work investigates the role of AVR2 and its host targets in pathogen virulence, with focus on the link between the brassinosteroid pathway and immunity in S. tuberosum. StBSL1 is shown to be a ‘susceptibility factor’ in P. infestans infection – a host protein with a positive effect on pathogen virulence. AVR2 stabilises BSL1 in planta, and both AVR2 and BSL1 are shown to suppress primary defence responses in the plant. Transcriptional analysis of brassinosteroid-treated S. tuberosum is used to identify a set of marker genes for active BR signalling. Strikingly, AVR2 is shown to upregulatethis pathway, and specifically upregulates the transcription factor StHBI1-like, identified as a suppressor of immunity. These findings reveal a novel mechanism in oomycete effector biology; the exploitation of crosstalk between the brassinosteroid pathway and immune signalling in plants.

Ces travaux concerne le développement de capteurs/effecteurs piézoélectrique et pyroélectrique de faible diamètre intégrable dans des structures textiles par exemple les tissus, ce qui permet soit d’avoir différentes informations sur le comportement mécanique et de température de ces structures (piézoélectricité directe) ou bien de changer les propriétés mécaniques de ces structures (piézoélectricité inverse), ce qui permettrait d’avoir des tissus à contention variable.A ce jour, sur le marché il n’existe pas de capteurs/effecteurs filamentaires piézoélectriques et/ou pyroélectriques flexibles de faibles diamètres intégrables dans les structures textiles. Les instrumentations des structures textiles qui existent se font par le biais de filaments résistifs agissant comme résistances entrecroisées qui permettent seulement d’avoir une information sur la localisation d’une pression sur ces structures textiles. Le but ces travaux est de remédier aux inconvénients des procédés connus ci-dessus. Ce but est atteint grâce à la fabrication de câbles filamentaires à l’aide d’un procédé de filage par voie fondue.Ces composites filamentaires sont caractérisé puis polariser avant de les introduire dans différents textiles par exemple les tissus, ce qui permet soit d’avoir différentes informations sur le comportement mécanique et de température de ces structures (capteurs de déformations et de température) ou bien de changer leurs propriétés mécaniques (effecteurs).Ces travaux ont été brevetés par la cellule Conectus Alsace et ils ont été sélectionnés pour le second prix International Théophile Legrand de l’innovation textile 2012. / The objective of this work is the development of composite filaments with piezoelectric and pyroelectric properties. These composite filaments will be used as sensors or effectors into textile structures (e.g weaving structures). The instrumentation of textile structures with piezoelectric composite filaments (sensors) will give information about the deformations of these structures when they are submitted to stresses. If we focus on the pyroelectric properties, this integration will allow detection of temperature variation of the operating environment. By using inverse piezoelectricity (effectors), we could change mechanical properties of textiles structures. The decision to develop this type of composites filaments was take due to the literature review which shows that composite filamentous sensors or effectors does not exist commercially. The objective of this work was achieved through the development of ferroelectric composite filament by using a melt spinning process. These composite was constituted by a portion of ferroelectric copolymer P(VDF-TrFE), an inner conductive core acting as an inner electrode and an outer conductive layer serving as an outer electrode These composite filaments were characterized with mechanical and rheological tests before being polarized by an alternating field. Then we integrate them into weaving structures using an industrial weaving machine. The result of the stressing of this structure was a variation of the field across the composite electrodes This work has been patented with the help of Conectus Alsace and it was selected for the second International Award Théophile Legrand for Textile Innovation 2012.

Study of cell signalling pathways affected by pathogen entry comprises a fundamental aspect of understanding host-pathogen interactions. In this respect, the current study attempted to ascribe novel roles to Bone Morphogenesis Protein (BMP) signaling during infection. BMP pathway has been majorly studied in context of development where it plays an imperative role and its contribution to immunity has been poorly documented. Subsequent narrative talks about the perturbation of BMP signaling in context of specific signaling networks and its collaboration with other molecular players of host innate armamentarium. There is a pressing need to develop effective chemotherapy against Mycobacterium tuberculosis, the causative agent of tuberculosis, which has garnered the world’s attention as a leading cause of public health emergency. The tyrosine kinase, c-Abl was previously reported to be activated in murine bone marrow derived macrophages infected with mycobacteria. Yet, the identities of host signaling players and mechanisms exploited by mycobacteria in association with c-Abl lacked identification. Here, we deciphered an intricate signaling mechanism linking tyrosine kinase c-Abl, chromatin modifier, lysine acetyl transferase KAT5 and transcription factor, TWIST1 acting at Bmp2 and Bmp4 promoters. This molecular circuitry was observed to affect mycobacterial survival. Emerging studies suggest repurposing of c-Abl inhibitor, Imatinib, as an adjunct to existing anti-tuberculosis therapy. Through the use of Imatinib in an established model of tuberculosis, we demonstrated the ability of c-Abl inhibitors in potentiating innate immune responses. Distinctive instances report the cross regulation among Pattern Recognition Receptors (PRRs). Interestingly, TLR3 signaling cascade induced in response to its cognate ligand was dampened through c-Abl-BMP induced miR27a. TLR3 is known to activate immune surveillance upon viral infections; however, recent studies also suggest its role in tumour regression and induction of apoptosis. Our observation of mycobacteria elicited down regulation of TLR3 pathway corroborated with increased incidences of lung cancer among TB patients and mycobacterial evasion of a well characterized form of cell-death i.e. apoptosis. Further, we utilized a panel of such Mtb mutants associated with virulence and questioned their relevance in the activation of c-Abl-dependent BMP signaling. We found that nitric oxide, hypoxia and carbon monoxide-responsive mycobacterial WhiB3 and DosR, but not the sec-dependent protein secretion pathway, orchestrate mycobacteria driven c-Abl-BMP signaling. Continuing with the theme of exploring roles for BMP signaling during infection, we identified an important role for the C-type Lectin Receptor (CLR), Dectin-2, in activating Candida albicans-driven BMP signaling. Mounting evidences suggest BMP antagonists promote repair and regeneration in cells of varied lineages. We observed a role for BMP signaling in aggravating MMP2 and MMP9, factors that result in chronic non-healing wounds. Wounds are now increasingly recognized as being colonized with fungi along with bacteria. We propose a role for C. albicans orchestrated BMP signaling in contributing to enriched repressive methylation at Egf, Pdgf and Tissue Inhibitors of Matrix Metalloproteases (Timp2/3/4) promoters. Repressive H3K27me3 at these loci impedes the reparative tissue homeostasis, resulting in C. albicans endorsed impaired wound healing. Altogether, we uncovered hitherto unknown roles of BMP signaling during mycobacterial and fungal infections, enabling a better understanding of lesser studied pathways in mediating pathogenesis.

Orientador: Celso Eduardo Benedetti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T05:36:49Z (GMT). No. of bitstreams: 1 Soprano_AdrianaSantos_D.pdf: 24018757 bytes, checksum: 29724fbb81a588e3bb656bf4c2df3390 (MD5) Previous issue date: 2012 / Resumo: O cancro cítrico, causado pela bactéria Xanthomonas citri (X. citri), afeta a maioria das espécies de Citrus, ocorre praticamente em todos continentes e se destaca como uma séria ameaça à citricultura brasileira. O mecanismo molecular pelo qual X. citri causa cancro não é inteiramente conhecido, entretanto, sabe-se que a bactéria utiliza o sistema secretório tipo III para injetar proteínas de patogenicidade, entre elas, PthAs da família AvrBs3/PthA, também conhecidas como efetores TAL (transcriptional activator-like). Os efetores TAL atuam como fatores de transcrição transativando genes específicos da planta que vão beneficiar a bactéria ou desencadear respostas de defesa. Com o objetivo de entender os mecanismos moleculares pelos quais os efetores TAL atuam, a técnica de duplo híbrido foi usada para identificar proteínas de laranja doce (Citrus sinensis) que interagem com PthA4, um dos efetores TAL de X. citri necessário para o desenvolvimento do cancro cítrico. A maioria das proteínas de laranja identificadas como alvos de PthA4 apresenta domínios de ligação à DNA ou RNA e está envolvida no controle da transcrição, estabilização de mRNAs e tradução. Várias dessas proteínas interagem entre si, sugerindo a presença de um complexo multiproteico como alvo de efetores TAL. Entre as proteínas envolvidas no controle da transcrição, destacamos a CsMAF1, uma proteína homóloga à MAF1 humana que atua como regulador negativo da RNA Polimerase III. Os resultados obtidos nesse trabalho revelam que CsMAF1 complementa o fenótipo do mutante maf1 de levedura, reprimindo a expressão de tRNAHis e que a expressão de PthA4 na cepa complementada restaura a síntese desse tRNA. Portanto, os dados mostram que CsMAF1 atua como um repressor da RNA Pol III em levedura e que PthA4 altera o estado repressor de CsMAF1 sobre a RNA Pol III. De forma surpreendente, verificamos que plantas de citros com níveis reduzidos de CsMAF1 apresentaram aumento significativo no número e intensidade de lesões hiperplásticas ou eruptivas quando infiltradas com X. citri, indicando que CsMAF1 desempenha um papel crítico no desenvolvimento dos sintomas do cancro cítrico. O aumento das lesões do cancro nas plantas silenciadas para CsMAF1 se correlaciona com um aumento expressivo de tRNAs, incluindo o tRNAHis, confirmando assim o papel repressor de CsMAF1 sobre a RNA Pol III em citros. Além disso, mostramos nesse trabalho que CsMAF1 é uma fosfoproteína que se encontra na forma dimérica em solução, uma característica singular ainda não descrita para membros dessa família de proteínas. Verificamos que CsMAF1 é fosforilada in vitro pelas quinases PKA e PKC e que apresenta sítios adicionais de fosforilação conservados para a quinase TOR, incluindo o resíduo Thr62. Curiosamente, tais sítios se localizam na interface de dimerização de CsMAF1, sugerindo que a fosforilação desses sítios deve regular a função da proteína e/ou seu estado multimérico. De fato, verificamos que a substituição do resíduo de treonina Thr 62 para ácido aspártico (Asp 62) diminui a proporção dímero:monômero de CsMAF1, indicando que a fosforilação de resíduos na interface do dímero desestabiliza o dímero, e que esse pode ser um mecanismo regulatório novo para essa classe de proteína. Desse modo, esses achados abrem novas perspectivas para o entendimento não só dos mecanismos moleculares envolvidos na regulação da RNA Pol III pela CsMAF1, como também do papel de PthA4 na interação com CsMAF1 e sua modulação da transcrição / Abstract: Citrus canker, caused by Xanthomonas citri (X. citri), is a disease that affects most of the Citrus species, occurs in almost all continents and stands as a threat to the Brazilian citrus industry. The molecular mechanism by which X. citri causes canker is poorly understood, however the bacterium injects pathogenicity proteins via the type III secretion system (T3S) including proteins of AvrBs3/PthA family, also known as transcriptional activator-like (TAL) effectors. TAL effectors have been extensively studied and are known to act as transcription factors that transactivate specific plant genes which either benefit the bacteria or trigger defense responses. To gain insights into the molecular mode of action of TAL effectors, a twohybrid screening was performed to identify sweet orange (Citrus sinensis) proteins that interact with PthA4, one of the X. citri TAL effectors required for citrus canker development. Among the proteins identified as PthA4 interactors, most are DNA and/or RNA-binding factors involved in chromatin remodeling and repair, transcriptional control and mRNA stabilization/modification. Several of these proteins interact with each other, suggesting the presence of a multiprotein complex as a target of TAL effectors. Among the proteins involved in transcription control, we selected for further studies the CsMAF1, a homolog of the human MAF1 that acts as a negative regulator of RNA polymerase III. The results presented here reveal that CsMAF1 complements the yeast maf1 mutant phenotype by repressing the tRNAHis transcription, and that PthA4 expression in the complemented strain restores the tRNAHis synthesis. Thus, the data show that CsMAF1 acts as a RNA Pol III repressor in yeast and that PthA4 somehow suppresses the repressor activity of CsMAF1 upon on the RNA Pol III. Surprisingly, we found that citrus plants with reduced levels of CsMAF1 showed a significant increase in the number, morphology and size of eruptive or hyperplastic lesions when infiltrated with X. citri, indicating the CsMAF1 plays a critical role in canker development. Increased canker lesions in CsMAF1 silenced plants correlated with a significant increase of tRNAs expression, including tRNAHis, thus confirming the repressor role of CsMAF1 upon the citrus RNA Pol III. Furthermore, we showed in this work that CsMAF1 is a phosphorylated and a dimer in solution, a feature that so far has not been reported for any member of this protein family. We found that CsMAF1 is phosphorylated in vitro by PKA and PKC, and has additional phosphorylation sites for the TOR kinase, including the Thr 62 residue. Interestingly, these phosphorylation sites are located at the dimerization interface of CsMAF1, suggesting that phosphorylation of such sites might regulate the function of the protein and / or its multimeric state. Indeed, mutation of threonine residue Thr62 to aspartic acid (Asp62) decreases the dimer:monomer CsMAF1 ratio, indicating that phosphorylation of the residues at the interface of the dimer destabilizes the dimer, and this may be a novel regulatory mechanism for this class of protein. Thus, these findings open new perspectives for the understanding of the molecular mechanisms involved in RNA Pol III regulation by CsMAF1, as well as for the role of PthA4 in the modulation of RNA Pol III transcription mediated by CsMAF1 / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Dans cette thèse, en collaboration avec l'entreprise AéroDRONES, le Laboratoire Bordelais de Recherche en Informatique et l'INRIA, nous abordons le problème de la perception de l'environnement à partir d'une caméra embarquée sur un drone léger. Nous avons conçu, développé et validé de nouvelles méthodes de traitement qui optimisent l'exploitation des données produites par des systèmes de prise de vue aéroportés bas coût. D'une part, nous présentons une méthode d'autocalibrage de la caméra et de la tourelle d'orientation, sans condition spécifique sur l'environnement observé. Ensuite nous proposons un nouvel algorithme pour extraire la rotation de la caméra calibrée entre deux images (gyroscope visuel) et l'appliquons à la stabilisation vidéo en temps réel. D'autre part, nous proposons une méthode de géoréférencement des images par fusion avec un fond cartographique existant. Cette méthode permet d'enrichir des bases de données de photos aériennes, en gérant les cas de non-planéité du terrain. / The last decade has seen the emergence of many Unmanned Aerial Vehicles (UAV) which are becoming increasingly cheap and miniaturized. A mounted video-camera is standard equipment and can be found on any such UAVs. In this context, we present robust techniques to enhance autonomy levels of airborne vision systems based on mini-UAV technologies. First, we present a camera autocalibration method based on central projection based image \dimension{2}-invariants analysis and we compare it to classical Dual Image of the Absolute Conic (DIAC) technique. We present also a method to detect and calibrate turret's effectors hierarchy. Then, we propose a new algorithm to extract a calibrated camera self-rotation (visual gyroscope) and we apply it to propose a real-time video stabilizer with full perspective correction.

L’objectif de ma thèse visait à étudier les processus évolutifs ayant conduit à l’évolution du mode de vie galligène et à rechercher des effecteurs impliqués dans l’induction des galles dans la famille des Gracillariidae (Lepidoptera) avec un accent particulier sur deux espèces, Borboryctis euryae et Caloptilia cecidophora. Nous avons ainsi démontré que ces deux espèces présentent la particularité de posséder un mode de vie intermédiaire entre mineur de feuille et inducteur de galle. Le tissu prolifératif présent dans la mine de B. euryae s’apparente en effet à une galle et les larves de C. cecidophora connaissent une transition du mode de vie mineur de feuille à galligène au cours de leur développement. Des campagnes de terrain ont permis de découvrir de nouvelles espèces de Caloptilia inductrices de galles, et leur étude phylogénétique a montré qu’elles forment un groupe monophylétique. Enfin, exploitant la transition de mode de vie de C. cecidophora ainsi que son contexte phylogénétique, nous avons appliqué une approche de transcriptomique comparative intra- et inter-espèce afin de rechercher des effecteurs candidats impliqués dans la formation de galle. / The aim of my thesis was to study the evolutionary processes that led to the evolution of the gall-inducing lifestyle and to look for effectors involved in the induction of galls in the Gracillariidae family (Lepidoptera) with a particular focus on two species, Borboryctis euryae and Caloptilia cecidophora. We have demonstrated that these two species have a particular intermediate life-style between leaf-miner and gall-inducer. The proliferative tissue in the B. euryae mine is similar to a gall and the larvae of C. cecidophora undergo a transition from leaf-miner to gall-inducer during their development. Field work has uncovered new gall-inducing Caloptilia species, and their phylogenetic study has shown that they form a monophyletic group. Finally, exploiting the transition of feeding habit of C. cecidophora as well as its phylogenetic context, we applied a comparative intra- and inter-species transcriptomic approach to search for candidate effectors involved in gall induction.

La bactérie phytopathogène E. amylovora, est l'agent responsable du Feu bactérien des Spiraeoideae (pommier, poirier, pyracantha), une maladie caractérisée par l'apparition de symptômes nécrotiques des tissus infectés. Le pouvoir pathogène d’E. amylovora repose entre autre sur un système de sécrétion de type III (SSTT) qui permet la sécrétion et l'injection d'effecteurs dans la cellule hôte végétale. Parmi les protéines injectées par le T3SS d'E. amylovora, DspA est essentielle au pouvoir pathogène de la bactérie puisqu’un mutant dspA est non pathogène sur plante (Gaudriault et al., 1997). Le rôle de DspA est dual, d’une part, l’expression de dspA est suffisante pour provoquer des symptômes nécrotiques sur plante et une toxicité chez la levure, d’autre part, DspA est impliquée dans la suppression des réactions de défense telles que la déposition de callose (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA appartient à la famille des effecteurs AvrE qui sont répandus chez les bactéries phytopathogènes et semblent posséder une fonction similaire. Cependant, peu de connaissance existe sur la structure ainsi que la fonction de DspA. L'objectif de ce travail de thèse était de déterminer les domaines ou motifs importants pour la fonction de DspA. Pour cela nous avons choisi d'effectuer une analyse in silico et fonctionnelle de la protéine DspA. L'analyse in silico révèle la présence d'un domaine bêta-propeller au sein de la protéine DspA ainsi que de tous les homologues analysés. De plus, l'analyse fonctionnelle indique que ce domaine est important pour la structure et la fonction de DspA. Dans un second temps, j'ai étudié le mécanisme d'action de DspA dans la levure Saccharomyces cerevisiae. J'ai pu mettre en évidence que l'expression de dspA chez la levure induit un arrêt de croissance et une forte altération du trafic cellulaire. L'étude de mutants de levure suppresseurs de la toxicité de DspA, effectuée avant mon arrivée au laboratoire, montre que les suppresseurs les plus forts sont affectés dans la voie de biosynthèse des sphingolipides, je me suis donc plus particulièrement intéressée au rôle des sphingolipides dans la toxicité générée par DspA. Nos résultats montrent que DspA inhibe la biosynthèse des sphingolipides indirectement via les régulateurs négatifs de la voie, les protéines Orms. / Erwinia amylovora is the causative agent of fire blight of Spiraeoideae (apple, pear, pyracantha), a disease characterized by the apparition of necrotic symptoms on infected tissues. The pathogenicity of E. amylovora relies on a functional type III secretion system (T3SS) that allows secretion and injection of effector proteins into the host plant cell. Among these effector proteins injected by the T3SS of E. amylovora, DspA is essential to the bacteria disease process since a dspA mutant is nonpathogenic on plants (Gaudriault et al., 1997). DspA has a dual role; on the one hand dspA expression is sufficient to induce cell death on plants and toxicity on yeast, on the other hand, DspA is involved on suppression of defense reactions like callose deposition (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA belongs to the AvrE familly of type III effectors which are widespread on phytopathogenic bacteria and likely possess a similar function. However, the structure and function of DspA remain unknown. In the first part of my thesis, I attempted to characterize domains or motifs important for the function of DspA. We performed an in silico and a functional analysis of the DspA protein. In silico analysis predicted a bêta-propeller domain in DspA and all the analysed effectors. In the second part of my thesis, I analysed the mechanism of function of DspA in the yeast Saccharomyces cerevisiae. Results showed that expression of dspA in yeast inhibits cell growth and alters the actin cytoskeleton and endocytosis. Screening of the Euroscarf library for mutants resistant to DspA induced toxicity revealed that mutants impaired in the sphingolipid biosynthetic pathway are the best suppressors. Based on this results, I attempted to determine the role of sphingolipids in the toxicity induced by DspA. Results showed that DspA inhibits indirectly the sphingolipid biosynthetic pathway via the negative regulators, Orm proteins.