Die Unfolded Protein Response (UPR) ist ein in Eukaryoten konservierter Signalweg, der durch Akkumulation von un-/fehlgefalteten Proteinen im endoplasmatischen Retikulum (ER) aktiviert wird um die Proteinhomöostase zu gewährleisten. In <i>Saccharomyces cerevisiae</i> wird die UPR durch den ER-Stresssensor Ire1p und den bZIP Transkriptionsfaktor Hac1p, oder XBP1 in höheren Eukaryoten, reguliert. In dieser Arbeit konnten die Homologe der zentralen UPR-Regulatoren im biotrophen Pilz <i>Ustilago maydis</i> charakterisiert und die UPR als essenzieller Koordinator der pathogenen Entwicklung identifiziert werden. Die Komplementation der <i>∆HAC1</i> Mutante durch <i>cib1</i> (Homolog von <i>HAC1</i>) und umfassende Expressionsanalysen zeigten, dass die Regulationsmechanismen der UPR in <i>U. maydis</i> weitestgehend konserviert sind, das Spektrum der regulierten Zielgene jedoch sekretierte Virulenzfaktoren beinhaltet die für die pathogene Entwicklung notwendig sind. So konnten durch <i>in silico</i> Vorhersage möglicher Cib1 Bindestellen (UPRE) mit pit1/pit2 und tin1-1 drei bereits charakterisierte Effektorgene als direkt regulierte UPR-Zielgene identifiziert werden. Die gezielte Deletion des vorhergesagten UPREs führt zu einer Aufhebung der ER-Stress induzierten und Cib1 abhängigen Expression von pit2 und verringert die Virulenz signifikant. Darüber hinaus konnte gezeigt werden, dass eine funktionelle UPR sowohl notwendig für eine verstärkte Expression wie auch für die korrekte Prozessierung des Pit2-Effektors innerhalb des ERs ist. Im Gegensatz zur Bäckerhefe <i>S. cerevisiae</i> und den filamentösen Ascomyceten <i>Aspergillus niger</i> und <i>Trichoderma reseei</i> kodiert die ungespleißte XBP1 mRNA in höheren Eukaryoten für einen negativen Regulator der UPR. Mit den vorliegenden Untersuchungen in <i>U. maydis</i> konnte erstmals für niedere Eukaryoten gezeigt werden, dass die ungespleißte cib1 mRNA für einen negativen Regulator kodiert, der darüber hinaus eine bislang unbeschriebene und vermutlich konservierte Funktion in der Antwort auf ER-Stress besitzt. Die genaue Kontrolle der UPR-Aktivität ist Voraussetzung für die korrekte Ausführung der verschiedenen Schritte innerhalb der pathogenen Entwicklung von <i>U. maydis</i>. Während eine vorzeitige UPR-Aktivierung zur Inhibition des zur Pflanzeninfektion notwendigen filamentösen Wachstums führt, ist die gezielte Aktivierung der UPR nach erfolgreicher Penetration der Pflanzenoberfläche und ihre andauernde Aktivität während des Wachstums <i>in planta</i> notwendig für die pathogene Entwicklung. Die direkte Interaktion zwischen Cib1 und dem Entwicklungsregulator Clp1 während dieser Entwicklungsphase führt zur Stabilisierung von Clp1 und der Modulation der Cib1 abhängigen Genexpression. Auf diese Weise wird die Proliferation <i>in planta</i> ermöglicht und eine erhöhte ER-Stressresistenz vermittelt. Zusammenfassend zeigen die gewonnenen Ergebnisse, dass die UPR in <i>U. maydis</i> als Kontrollpunkt dient, um die zelluläre Physiologie, den Entwicklungsverlauf und die Sekretion von Effektoren aufeinander abzustimmen.

Orientador: Celso Eduardo Benedetti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T05:56:46Z (GMT). No. of bitstreams: 1 Pereira_AndreLuizAraujo_D.pdf: 43759919 bytes, checksum: 379266575f1db46c7d620232efb9ba13 (MD5) Previous issue date: 2011 / Resumo: O cancro cítrico resulta da interação compatível entre a bactéria Xanthomonas axonopodis pv. citri e Citrus spp. A doença não tem cura, é de fácil disseminação e difícil controle. O cenário é preocupante, pois a doença diminui drasticamente o rendimento e a qualidade dos frutos de plantas infectadas, ocasionando um forte impacto econômico na citricultura mundial. Os principais sintomas do cancro cítrico, resultantes dos processos de hipertrofia (aumento do volume celular) e hiperplasia (aumento da divisão celular), são dependentes da proteína efetora PthA de X. citri. PthA integra a família de fatores de transcrição conhecida como efetores ativadores de transcrição (transcription activator-like ou TAL). O principal homólogo de PthA é o efetor AvrBs3 de X. campestris pv. vesicatoria que atua regulando a transcrição de genes do hospedeiro em benefício do patógeno. A similaridade entre estas proteínas gira em torno de 97%, sugerindo, portanto, função semelhante para PthA. Através de uma série de microarranjos, investigou-se o perfil de expressão gênica de laranja doce (Citrus sinensis) dependente de PthA (X. citri) e de PthCs de X. aurantifolii, uma bactéria que causa cancro cítrico apenas no limão galego e que, em laranja doce, induz uma reação de hipersensibilidade. Desta forma, verificou-se a regulação positiva ou negativa de uma série de genes. Os PthCs regularam negativamente genes associados à sinalização por auxina e induziram a expressão de genes de defesa e silenciamento gênico. Em contrapartida, PthAs induziram uma série de genes intimamente relacionados aos sintomas de cancrose, incluindo: genes associados aos processos de aumento e divisão celular, síntese e remodelamento de parede celular, bem como genes envolvidos na sinalização por auxina e giberelina. Neste sentido, efetuou-se o isolamento de regiões promotoras de cinco genes, os quais são potencialmente regulados por PthA. A análise destas regiões revelou a presença de um possível TATA-box notavelmente semelhante àquele encontrado no gene upa20, denominado UPA-box (up-regulated por AvrBs3), sugerindo que estes genes poderiam ser transativados por PthA em citros. De fato, ensaios de retardamento de mobilidade eletroforética (electrophoretic mobility shift assay ou EMSA), demonstraram a ligação específica de PthA2 e 4 ao TATA-box encontrado na região promotora do gene que codifica uma proteínas relacionada à patogênese (pathogenesis-related proteins ou PR). Este resultado corrobora com a hipótese de que os efetores TAL atuam como proteínas ligadoras de elementos TATA. Finalmente, experimentos de co-imunoprecipitação de cromatina (ChIP) e cotransformação demonstraram, ainda que em resultados preliminares, que particularmente PthA4 é capaz de transativar pr5 in planta. Embora o cancro cítrico ainda não seja completamente entendido a nível molecular, os dados aqui apresentados sugerem fortemente a ação de PthAs como fatores de transcrição, bem como aponta candidatos à regulação positiva intimamente associados aos processos de hipertrofia e hiperplasia. Além disso, as regiões promotoras aqui isoladas podem ajudar no desenvolvimento de novas estratégias para a geração de plantas resistentes à cancrose / Abstract: Citrus canker is a result of a compatible interaction between Xanthomonas axonopodis pv. citri and Citrus spp. There is no cure for citrus canker, and the disease is easily spread and difficult to be managed. The scenario is threatening since the disease dramatically diminishes the quality of fruits in infected plants leading to great economic losses for the world citrus producers. The main citrus canker symptoms known as hypertrophy (cell enlargement) and hyperplasia (cell division) are PthA-dependent. PthA is an effector protein from X. citri which belongs to the TAL effectors (transcription activatorlike) family. The closest homologue of PthA is AvrBs3 from Xanthomonas campestris pv. vesicatoria, a TAL effector that acts as a transcriptional factor to modulate host transcription to the pathogen's benefit. Similarity shared by these two proteins is around 97%, suggesting that PthA plays a similar role in the citrus host. Through a number of microarray experiments, we investigate the gene transcription in sweet orange (Citrus sinensis) in response to the transient expression of PthA from X. citri or PthC from X. aurantifolii, pathotype C, a bacteria that causes citrus canker in Mexican lime but in orange trigger a hypersensitive response in sweet orange. We observed that PthCs down-regulated various auxin signaling genes and induced the expression of genes involved in defense and gene silencing. On the other hand, PthAs induces several genes implicated in canker development such as cell division and elongation, cell-wall synthesis and remodeling, synthesis, mobilization and signaling of auxin and gibberellin. Promoter regions of PthA-induced genes were isolated and shown to have predicted PthA and PthC binding sites at or near their putative TATA boxes. Moreover, competition gel shift assays confirmed that PthA4 shows preferential binding to the TATA box of the pathogenesis-related (pr5) gene promoter, supporting the idea that TAL effectors may act as general TATA-binding proteins. Finally, both chromatin immunoprecipitation (ChIP) and co-transformation assays demonstrated however as preliminary results, that PthA4 is able to transactivate pr5 in planta. Albeit the molecular mechanism by which citrus canker develop remains elusive at the molecular level, we provided data supporting the notion that PthA acts as a transcriptional factor, as well as identified PthA-induced genes associated with hypertrophy and hyperplasia. Furthermore, the promoter regions isolated here might be useful to obtain citrus plants resistant to the canker bacteria / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Salmonella est un pathogène intracellulaire dont la virulence dépend de la fonction de deux systèmes de sécrétion du type trois (T3SS). Les T3SSs sont responsables pour la transduction de protéines effectrices dans le cytoplasme de la cellule hôte afin d'initier l'invasion de la cellule et de former la vie intracellulaire de Salmonella. Plusieurs effecteurs forment la SCV et induisent un réseau de tubules qui est impliqué dans la stabilisation de la SCV. Il consiste de trois différents genres de tubules. Nous avons pu montrer que les protéines effectrices SseF et SseG sont responsables pour la formation d'un genre de ces tubules, les LAMP-1 negative tubules (LNTs). Leur fonction est importante puisque des souches de Salmonella qui induisent que des LNTs et ne pas d'autres tubules sont apte de créer une SCV stable. Ceci améliore la réplication et virulence in vivo comparé à des souches qui ne peuvent pas induire des tubules. En utilisant les LNTs comme modèle, nous avons essayé de comprendre la contribution des tubules à la formation de la SCV et aussi leurs interaction avec les endosomes tardives et les lysosomes (LE/lys). Nous avons découvert une contribution essentielle de la petite GTPase Arl8B à la fusion de tubules avec les LE/lys. Ainsi, le knockdown d'Arl8B réduit la capacité de reproduction de Salmonella dans la cellule hôte. Nous avons pu démontrer qu'une interaction entre l'effecteur SifA et Arl8B est responsable pour ces observations. / Salmonella is an intracellular pathogen, whose virulence relies on the function of two type three secretion systems (T3SSs). The T3SSs are responsible for the delivery of effector proteins into the host cell cytoplasm in order to mediate invasion of the cell and to shape Salmonella's intracellular life.Salmonella's intracellular survival and replication depends on its niche, the Salmonella containing vacuole (SCV), a compartment that is derived from host plasma membrane. Several effectors shape the SCV and give rise to a tubular network, which is implicated in the SCV's stabilization and consists of three different kinds of tubules. We were able to show that the effector proteins SseF and SseG play in concert to form one kind of tubules, the recently discovered LAMP-1-negative tubules (LNTs). Their function is important to Salmonella, as strains having only LNTs but none of the other tubules are able to create a stable SCV, which leads to better replication and virulence in vivo compared to a strain that lacks in tubule formation. Starting from these LNTs as working model, we tried to understand the contribution of tubules to the formation of the SCV and their interactions with the late endosomal / lysosomal compartment (LE/lys). We deciphered the small GTPase Arl8B to play an essential role in the fusion of tubules with LE/lys. Thereby, the knockdown of Arl8B reduced Salmonella's capability to replicate within host cells. We were able to show that an interaction between the effector SifA and Arl8B was responsible for our observations.

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012

No description available.

Il a été établi que la majorité des plantes vasculaires abritent des micro-organismes endophytes bactériens et fongiques, qui peuvent coloniser les tissus végétaux et former des associations allant du mutualisme à la pathogénèse. Les symbioses végétales mutualistes les plus communes impliquent les champignons endo-mycorhiziens arbusculaires (AMF). Ces champignons s’associent aux racines des plantes et leur permettent d’améliorer leur nutrition minérale, tandis qu’ils bénéficient des composés produits par l'hôte. Toutefois, les plantes de la famille Ericaceae s’engagent plutôt dans des associations mutualistes avec les champignons mycorhiziens éricoïdes (ErMF). Ces derniers sont morphologiquement et taxonomiquement mal définis, en apparence distribués aléatoirement parmi les espèces issues des grandes divisions taxonomiques des Ascomycota et Basidiomycota. En raison de cette incohérence taxonomique et de l'absence d'une histoire évolutive explicative, la diversité réelle de ces champignons est mal caractérisée. De ce fait, ce projet vise à étudier le microbiote associé à la plante Ericaceae Vaccinium macrocarpon Aït (canneberge), axant la recherche sur les angles morphologiques, génomiques et transcriptomiques des champignons de type ErMF et autres endophytes capables de contrôler la croissance des agents phytopathogènes et de stimuler la croissance des plantes. Notre première démarche présentée dans le chapitre 2 s’est focalisée sur la caractérisation du microbiote endophyte bactérien et fongique de la canneberge, une plante vivace principalement produite en Amérique du Nord, notamment au Québec. Nous avons isolé et identifié 180 micro-organismes à partir de plantes de cultivars variés, collectées de champs différents, et avons démontré l'existence d'une variabilité dans le microbiote selon les tissus, les cultivars, et même entre les champs d'une même ferme. Parmi les endophytes d’intérêt identifiés, l’isolat fongique Lachnum sp. EC5 a stimulé la croissance des cultivars de canneberge Stevens et Mullica Queen et a formé des structures intracellulaires similaires à celles des ErMF à l’intérieur des cellules racinaires de la canneberge. De plus, l’isolat EB37 identifié Bacillus velezensis s’est révélé être un puissant agent antifongique, montrant cependant une tolérance particulière au champignon Lachnum sp. EC5, lors des tests de confrontation. Ce volet sera détaillé avec plus de précision dans le chapitre 4. Le chapitre 3 a porté sur l’analyse génomique comparative de l’isolat fongique Lachnum sp. EC5 avec plusieurs espèces de champignons Leotiomycetes ErMF, saprophytes et pathogènes. Nous avons analysé le sécrétome protéique prédit de ces champignons et mis en évidence que les gènes codant pour les enzymes de dégradation des parois végétales ne sont pas corrélés au mode de vie fongique (mycorhizien, pathogène ou saprophyte). A l’inverse, 10 protéines effectrices de Lachnum sp. EC5 prédites pour cibler spécifiquement un compartiment intracellulaire chez les cellules végétales ont des similarités avec celles d’espèces mutualistes comme Meliniomyces variabilis et Oidiodendron maius. Aussi, la protéine effectrice putative Zn-MP, prédite pour cibler, potentiellement, les chloroplastes végétaux nous permet de proposer un rôle dans le renforcement de l’immunité végétale. Le chapitre 4 s’est intéressé aux mécanismes de régulation d'expression de gènes induits lors de l’interaction entre le champignon Lachnum sp. EC5 et la bactérie B. velezensis EB37. Ces mécanismes ont été comparés à ceux activés chez la bactérie en présence de champignons pathogènes. Nous avons démontré une physiologique cellulaire bactérienne distincte en présence de Lachnum sp. EC5, dénotée par une faible expression des gènes induits lors du stress nutritif associé aux processus de sporulation, de formation du biofilm, de secretion de CAZymes et de lipopeptides. Nous avons suggéré que la sous-régulation de ces mécanismes serait essentiellement explicable par une disponibilité plus importante en glucose ou en d’autres sources de carbone préférentielles pour la bactérie. En réponse, le champignon Lachnum sp. EC5 a vécu différents changements morphologiques. Il aurait détoxifié ses environnements intra et extra-cellulaires et surexprimé sa voie de production de carbone dépendante du cycle du glyoxylate, générant ainsi des conditions favorisant un contact physique entre les deux micro-organismes. En conclusion, nous avons argumenté et documenté que la définition des ErMF basée uniquement sur des critères morphologiques est mal adaptée à catégoriser ces champignons. Notre approche multidisciplinaire a mis en évidence la diversité du microbiote de la canneberge, a étendu la notion d’ErMF à d'autres champignons jusqu'ici exclus de ce groupe, et a souligné l'importance des associations interspécifiques sur l’interaction ErMF-plantes. Ces avancées permettront d’améliorer nos connaissances sur le microbiote des plantes éricacées contribuant, au développement de solutions environnementales éco-responsables pour l’industrie de la canneberge. / It has been established that the majority of vascular plants harbour bacterial and fungal endophytes that colonize plant tissues, and thus form associations that range from mutualism to pathogenesis. Mycorrhizal fungi are a particular class of endophytes that associate with plant roots and enhance plant mineral uptake. The most common type of mutualistic plant symbiosis involves arbuscular mycorrhizal fungi (AMF), whereas plants of the Ericaceae family instead engage in mutualistic associations with ericoid mycorrhizal fungi (ErMF). The ErMF group, in its current definition, includes both ascomycete and basidiomycete species, yet is morphologically, taxonomically and evolutionarily poorly defined, which implies that the group’s true diversity is not well understood. The objective of this project is to complement morphological information with genomic and transcriptomic data to better understand the role of ErMF in 1) controlling the negative effects of pathogenic infections, and 2) the potential plant growth stimulation for the Ericaceous plant Vaccinium macrocarpon Ait. Our first approach presented in Chapter 2 focused on the characterization of the bacterial and fungal endo-symbiotic microbiota of the Ericaceous plant, Vaccinium macrocarpon Ait (cranberry), a perennial plant mainly in North America, particularly in Quebec. We isolated ~180 distinct bacterial and fungal endophytes collected from roots, stems, and leaves of cranberry plants cultivated in Quebec, Canada. We show that the cranberry microbiome varies substantially between tissues, cultivars, and across fields of the same farm. Among the isolated endophytes, the fungus Lachnum sp. EC5 was found to promote the growth of cranberry cultivars Stevens and Mullica Queen, and to form intracellular structures resembling those other ErMF inside the cortical root cells. In addition, the bacterium Bacillus velezensis (EB37) has been found to be a potent antifungal agent. Interestingly, a confrontation test between EB37 and the fungus Lachnum sp. EC5 revealed a mutual tolerance, which we will describe later in chapter 4. In chapter 3, our project focused on the comparative genomic analysis of the fungus Lachnum sp. EC5 with several Leotiomycete ErMF, saprophytes and pathogens. We analyzed fungal secretomes and demonstrated that genes encoding plant cell wall degradation enzymes are conserved between the tested fungi which suggests that such proteins are not indicative of a particular fungal lifestyle. On the other hand, 10 effector proteins identified in Lachnum sp. EC5 were also only found in mutualistic fungi, such as Meliniomyces variabilis, Oidiodendron maius and have been reported to target the plant intracellular compartments. Also, the identification of the putative effector protein Zn-MP, specific to Lachnum sp. EC5 and predicted to target plant chloroplasts, suggest a role in the reinforcement of plant immunity. Chapter 4 focuses on the patterns of gene expression regulation induced in the biocontrol bacterium B. velezensis EB37 in interaction with the potentially mutualistic fungus Lachnum sp. EC5. These mechanisms were then compared to those activated when the bacterium is in the presence of pathogenic/saprophytic fungi. We demonstrated that in co-culture with Lachnum sp. EC5, EB37expresses fewer genes related to stress, and fewer related to the stationary phase which often involves production of bacterial biofilms and lipopeptides, such as mycosubtilin. We suggest that the lessened response to stress is related to an increased availability of glucose or other preferential sources of carbons for the bacterium. Conversely, Lachnum sp. EC5 in the presence of EB37 underwent morphological changes by a higher lateral branching., detoxified its external and internal environment by expressing both a catalase activity and efflux pumps, and overexpressed its glyoxylate cycle-dependent carbon production pathway, and thus promoting favourable conditions for close physical contact with the bacterium. In conclusion, we demonstrated that the morphological-based definitions are poorly adapted to the categorization of ErMF fungi. Our multidisciplinary approach highlighted the diversity of the cranberry microbiota, extended the notion of ErMF to other fungi hitherto excluded from this fungal group and underlined the importance of interspecific associations on the ErMF-plant interaction. These advances enhance our understanding of the Ericaceous plant microbiota and contributes to the development of sustainable solutions for the cranberry industry.