Das Bakterium Bradyrhizobium japonicum ist wie andere Rhizobien in der Lage mit Pflanzen der Familie Fabales (Leguminosen) eine Symbiose einzugehen. Im symbiontischen Zustand fixieren die Mikrosymbionten atmosphärischen, molekularen Stickstoff und stellen diesen den Pflanzen in verwertbarer Form zur Verfügung. Im Gegenzug erhalten die Bakterien von den Pflanzen verschiedene Verbindungen als Kohlenstoff- und Energiequelle. Dem geht ein komplexer Signalaustausch voraus um die gegenseitige Erkennung der Partner und die Spezies-spezifische Symbiose zu ermöglichen. Auf Seite der Bakterien erfolgt die Reaktion auf die Gegenwart pflanzlicher Signalmoleküle. In B. japonicum induziert das Flavonoid Genistein die Transkription einer Reihe von Genen. Durch die Expression der nod-Gene erfolgt eine Synthese von Lipochitooligosacchariden. Diese sogenannten Nodulationsfaktoren rufen wiederum eine Reaktion in der Wirtspflanze hervor. Um die zugrunde liegende Genexpression und deren Regulationsmechanismen zu erforschen, standen bis dato nur Methoden für die Untersuchung einzelner Gene zur Verfügung. Die erstmals 1995 publizierte Microarraytechnologie eröffnete die Möglichkeit zu einem Zeitpunkt die Gentranskription eines gesamten Organismus zu untersuchen (Schena et al. 1995). Um mit dieser Technologie die Gentranskription bei B. japonicum zu untersuchen, wurde ein auf PCR-Produkten basierender Microarray hergestellt. Grundlage für die Synthese genspezifischer PCR-Produkte war die symbiontische Region von B. japonicum und andere zu diesem Zeitpunkt bekannte Gensequenzen (Göttfert et al. 2001). Nach der 2002 erfolgten Veröffentlichung des Genoms von B. japonicum erfolgte ein Abgleich der bisher verwendeten Sequenzen mit der vollständigen Sequenzinformation (Kaneko et al. 2002a). Nach Synthese der PCR-Produkte und deren Kontrolle durch Sequenzierung erfolgte die Herstellung des Microarrays unter Einsatz eines Microarrayspotters. Geeignete Techniken der cDNA-Markierung und die Microarray-Hybridisierung wurden mit Total-RNA von B. japonicum-Kulturen ausgetestet und etabliert. Eine differentielle Expremierung war eine Stunde nach Genistein- bzw. Methanolgabe zum Kulturmedium nicht nachweisbar. Zum darauffolgenden Zeitpunkt konnte die bekannte Induktion der nod-Gene durch dass Flavonoid Genistein beobachtet werden. Diese Induktion fiel in den verbleibenden zwei Zeitpunkten stark ab. Dieser Abfall der Induktionsrate ließ sich nicht mit dem Genisteingehalt im Kulturmedium erklären, da dieser Zeitraum konstant blieb. Vier Stunden nach Genisteingabe war die Induktion der Gene nolA und nodD2, deren Produkte sind an der negativen Regulation der nod-Gene beteiligt, nachweisbar. Dies wäre eine mögliche Erklärung für den Abfall der Induktion der nod-Gene. Im gleichen untersuchten Zeitraum wurde die Transkription verschiedener Gene der Stickstofffixierung und Denitrifikation durch das Flavonoid Genistein induziert. Auch für die bekannten Regulatoren dieser Gene war eine Induktion nachweisbar. Nach bisherigen Arbeiten war die Expression dieser Gene auf mikroaerobe und anaerobe Zustände, wie z.B. dem symbiontischen Stadium, beschränkt. Eine Induktion durch Flavonoide ist bisher nicht beobachtet worden. Um die Verwendbarkeit des Microarrays auch für den symbiontischen Zustand von B. japonicum zu testen, wurden Versuche mit Wurzelknöllchen von Sojabohne (Glycine max) durchgeführt. Dafür wurden Keimlinge der Sojabohne (G. max) mit B. japonicum inokuliert und die Total-RNA der entstehenden Wurzelknöllchen isoliert. Jedoch war eine Kreuzhybridisierung mit pflanzlicher Total-RNA zu beobachten. Der auf PCR-Produkten basierende Microarray ist für die Untersuchung des symbiontischen Stadiums von B. japonicum nicht einsetzbar. Im Vergleich dazu wurde ein auf Oligomeren basierender, kommerzieller Microarray für diesen Versuch getestet. Die Kreuzhybridisierung mit pflanzlicher Total-RNA war auf die Oligomere für die 16S rDNA und 23S rDNA reduziert.

Fifteen mutants of Sinorhizobium (Rhizobium ) meliloti unable to utilize acetate as a sale carbon source (Ace-) were characterized in this study. Merodiploid complementation tests showed that nine of these mutations were in loci distinct from previously described gluconeogenic loci. The chromosomal locations of the mutations were determined, and complementing clones were isolated from the cosmid library of S. meliloti genomic DNA. The mutants were placed into four groups (I--IV) based on genetic linkage in phage co-transduction. None of the mutations were in glyoxylate shunt enzyme-encoding genes. Nucleotide sequence analysis of ace mutants from Groups III and IV showed mutations in genes encoding acetyl-CoA synthetase ( acsB) and anaerobic coproporphyrinogen III oxidase (hemN ) respectively. Cell extracts of the hemN mutant exhibited double the isocitrate lyase levels of the wild type. The acsB mutant lacked acetyl-CoA synthetase activity and had an interesting growth phenotype; it was able to grow on low concentrations of acetate only. (Abstract shortened by UMI.)

This study was undertaken to characterize the role and pathway for assimilation of the intracellular carbon storage compound, poly-beta-hydroxybutyrate (PHB), in Rhizobium (Sinorhizobium) meliloti. Mutants unable to utilize the degradation intermediates, 3-hydroxybutyrate (HB) and/or acetoacetate (AA) were characterized. A mutant unable to utilize HB (Hbu-) while retaining the ability to utilize AA was found to be deficient in 3-hydroxybutyrate dehydrogenase (Bdh) activity. The bdhA mutant showed no symbiotic defects in association with alfalfa plants. However, when co-inoculated with the wild type, the mutant showed significantly reduced competitiveness. A more severe competition defect was observed for a PHB synthesis mutant (phaC). Both these mutants also showed reduced competitiveness when subjected to multiple cycles of subculturing through alternating carbon-rich and carbon-poor media, with the phaC mutant showing a greater loss in competitiveness. The results indicate that the ability to efficiently deposit and utilize cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability. / The gene encoding Bdh (bdhA) was isolated and sequenced. Two transcription start sites, S1 and S2 were identified but no known consensus promoter sequences were identified upstream of either start site. A sigma 54 consensus binding sequence was found to be located between S1 and S2 but no corresponding transcript was detected. Transcriptional bdhA-lacZ fusion studies indicated that gene expression was growth-phase associated. The bdhA gene from Rhizobium sp. NGR234 was also isolated and characterized and found to be highly homologous to the R. meliloti bdhA sequence. Unlike R. meliloti , NGR234 is able to accumulate PHB during symbiosis. An NGR234 bdhA mutant showed symbiotic defects on Leucaena but not on Tephrosia, Macroptilium or Vigna host plants, indicating that the phenotype was host-dependent. / Mutations that suppress the Hbu- phenotype without restoring Bdh activity were identified, indicating the existence of a Bdh-independent pathway for HB utilization. These mutations mapped to the age-1 locus, which causes enhanced growth rate on HB and AA minimal media. Introduction of plasmid-borne multiple copies of a gene encoding acetoacetyl-CoA synthetase (acsA) into the bdhA mutant also results in suppression of the Hbu- phenotype. A possible mechanism of suppression involving direct activation of HB to 3-hydroxybutyryl-CoA, followed by reduction to acetoacetyl-CoA by the NADP-acetoacetyl-CoA reductase (encoded by phaB) was investigated. A strain carrying the triple mutations, age-1::Tn5-Tp, bdhA ::Tn5 and phaB::OSmSp retained the ability to utilize HB, indicating that the bypass mechanism does not involve NADP-acetoacetyl-CoA reductase. / The phaB mutant does not accumulate PHB or utilize HB or AA. Furthermore, colonies of the phaB and phaC mutants exhibit non-mucoid phenotype on yeast extract mannitol agar. The observation that a R. meliloti exoS null mutant is also Hbu- provides further support for a link between PHB and exopolysaccharide synthesis. Since ExoS is a positive regulator of succinoglycan biosynthesis it is hypothesized that regulation of succinoglycan synthesis by ExoS requires PHB synthesis.

Thesis (M.S.)--State University of New York at Binghamton, Biological Sciences Department, 2007. / Includes bibliographical references.

Thesis--Leyden. / In Periodical Room.

Made available in DSpace on 2016-12-08T15:50:03Z (GMT). No. of bitstreams: 1 PGMS12DA008.pdf: 1237418 bytes, checksum: f369aa1152db6b98aced7001e1799850 (MD5) Previous issue date: 2012-03-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Currently the common bean (Phaseolus vulgaris L.) occupies an area of over 4 million ha in Brazil, being cultivated in various management systems and technological level. The state of Santa Catarina has to feature family farms into small holdings, where the bean crop is conducted in small plots for subsistence and low technology. Under these conditions it is normal to use Creole bean genotypes, where the reproduction of seeds is carried by the producer and the use of technologies such as rhizobial inoculation and fertilizer application is rarely performed in order to achieve maximum crop yield potential. The characterization of landraces of common bean associated with the efficient use of nitrogen-fixing microorganisms (N) and mycorrhizal associations can be of great value, since most likely to contain alleles for local adaptation, disease resistance and tolerance to major climatic adversities. The objectives of this study were to evaluate the occurrence and diversity of rhizobacteria on roots of bean plants grown on small farms in West and Mid-western state of Santa Catarina and evaluate the response in nutrition and gene expression of bean genotypes submitted to inoculation with divergent strains of rhizobacteria and AMF (Glomus clarum nicol. & Schenck). The analysis of occurrence and genetic and physiological characterization of rhizobacteria in bean roots showed a high density of rhizobacteria and the isolation and physiological characterization of strains was observed in the potential auxin production and biological nitrogen fixation. The identification of the strains showed the presence of the rhizobacteria Pseudomonas sp. Rhizobium sp. Pseudomonas moraviensis and Stenotrophomonas maltophilia. The sequencing of the strains allowed us to observe that there is a predominance of Pseudomonas sp.: 13 out of 15 strains identified. In assessing the effect of bean genotypes, phosphorus and inoculation with Glomus clarum nutrition and gene expression, the results show that the dose effect of P provided in shoot dry weight, the maximum value of dry mass observed with 400 mg P kg-1. The effect of genotype was observed in P and P accumulated in the harvest at 28 days after sowing, and the genotype BAF55 was superior to genotype commercial BAF115. The P present effect on mycorrhizal colonization in two harvests. In the harvest at 28 days after sowing, the genotype inoculated with commercial BAF115 G. clarum and 100 mg P kg-1 was 123% increase in root colonization in comparison with the treatment without application of P. In genotype BAF55 conditions of inoculation with G. clarum and indigenous AMF was observed that the treatment without P application showed the lowest values of mycorrhizal colonization. At the time of harvest to 48 days after sowing, the genotype BAF55 inoculation with G. clarum increased colonization in relation to the status of indigenous AMF. The analysis of gene expression PVPT gene demonstrated that there is an effect of the genotypes studied, rates of P and inoculation with G. clarum. Was observed in gene expression of the gene PVPT that genotype BAF55 responded with both inoculation with G. clarum as with the native AMF population, while the genotype trade BAF115 (Valente) answered only in the AMF treatment with native soil. In both genotypes was possible to observe the response of gene expression occurred in a dose of 100 mg P kg-1, and this response is associated with the native soil and mycorrhizal inoculation with non G. clarum. In the study with co-inoculation observed positive effect of inoculation with rhizobacteria and G. clarum dry matter yield, P and N uptake of shoots in bean plants. The best treatment was observed co-inoculation G. clarum and strain of Pseudomonas moraviensis Fe55, which obtained the highest values of dry matter, N and P accumulated in shoots and dry weight of nodules. A strain Fe55 of Pseudomonas moraviensis performed better than strain Ai27 the parameters analyzed in this trial / Atualmente a cultura do feijoeiro (Phaseolus vulgaris L.) ocupa uma área de mais de 4 milhões de ha no Brasil, sendo cultivada em diversos sistemas de manejo e grau tecnológico. O estado de Santa Catarina tem com característica a agricultura familiar em pequenas propriedades, onde a cultura do feijoeiro é conduzida em pequenas áreas para subsistência e com baixa tecnologia. Nestas condições é normal o uso de genótipos crioulos de feijoeiro, onde a reprodução das sementes é realizada pelo próprio produtor e o uso de tecnologias como a inoculação com rizóbios e a aplicação de fertilizantes é raramente realizada de forma a obter o máximo potencial produtivo da cultura. A caracterização de acessos crioulos de feijoeiro associada ao uso eficiente de microrganismos fixadores de nitrogênio (N) e associações com FMAs pode ser de grande valor, já que apresentam maior probabilidade de conter alelos para adaptação local, resistência a doenças e tolerância as principais adversidades edafoclimáticas. Os objetivos deste estudo foram avaliar a ocorrência e a diversidade de rizobactérias em raízes de feijoeiro cultivados em pequenas propriedades, no Oeste e Meio-oeste do estado de Santa Catarina e avaliar a resposta na nutrição e expressão gênica de genótipos crioulos de feijoeiro submetidos à inoculação com estirpes divergentes de rizobactérias e FMA (Glomus clarum nicol. & schenck). A análise de ocorrência e caracterização fisiológica e genética de rizobactérias em raízes de feijoeiro mostrou uma alta densidade de rizobactérias, com o isolamento e caracterização fisiológica das estirpes foi observado o potencial de produção de auxina e fixação biológica de nitrogênio. A identificação das estirpes demonstrou a presença de rizobactérias do gênero Pseudomonas sp.; Rhizobium sp.; Pseudomonas moraviensis e Stenotrophomonas maltophilia. O seqüenciamento das estirpes permitiu contatar um predomínio do gênero Pseudomonas sp.: 13 de um total de 15 estirpes identificadas. Na avaliação do efeito dos genótipos de feijoeiro, doses de fósforo e inoculação com Glomus clarum na nutrição e expressão gênica, os resultados demonstram que a dose de P proporcionou efeito na massa seca da parte aérea, sendo o valor máximo de massa seca observado com a dose de 400 mg de P kg-1. O efeito do genótipo foi observado no teor de P e P acumulado, sendo que o genótipo crioulo BAF55 foi superior ao genótipo comercial BAF115. As doses de P proporcionaram efeito na colonização micorrízica nas duas épocas de coleta (28 e 48 dias após a semeadura). Na coleta aos 28 dias, no genótipo comercial BAF115 com inoculação de G. clarum e dose de 100 mg P kg-1 houve elevação de 123% na colonização micorrízica em comparação com o tratamento sem aplicação de P. No genótipo crioulo BAF55 nas condições de inoculação com G. clarum e FMAs nativos foi possível observar que o tratamento sem aplicação de P apresentou os menores valores de colonização micorrízica. Na época de coleta aos 48 dias, no genótipo BAF55 a inoculação com G. clarum aumentou a colonização em relação à condição de FMAs nativos. A análise da expressão genética demonstrou que existe efeito dos genótipos analisados, doses de P e inoculação com G. clarum no gene PVPT. Foi observado na expressão gênica do gene PVPT que o genótipo crioulo BAF55 respondeu tanto com a inoculação com G. clarum quanto com a população de FMAs nativas, enquanto que o genótipo comercial BAF115 (Valente) respondeu somente no tratamento com FMAs nativos do solo. Em ambos os genótipos foi possível observar a resposta da expressão genética ocorreu na dose de 100 mg de P kg-1, sendo que esta resposta está associada aos FMAs nativos do solo e não inoculação com G. clarum. No estudo com coinoculação foi observado efeito positivo da inoculação com rizobactérias e G. clarum na massa seca, P e N acumulado da parte aérea em plantas de feijoeiro. O melhor tratamento observado foi a coinoculação de G. clarum e a estirpe Fe55 de Pseudomonas moraviensis, que obteve os maiores valores de massa seca, P e N acumulado na parte aérea e na massa seca dos nódulos. A estirpe Fe55 de Pseudomonas moraviensis apresentou melhor desempenho que a estirpe Ai27 nos parâmetros analisados neste ensaio

Os efeitos de Rhizobium sp. ICB503 no desenvolvimento de plantas de cana-de-açúcar foram observados em experimentos realizados em duas estações climáticas diferentes. Plantas mantidas em estufa por dois meses foram e submetidas aos tratamentos: B: inoculadas com bactéria: BN inoculadas com bactéria e tratadas com NO3-; N tratadas com NO3- e controle C, não inoculadas e não tratadas com NO3-. Mensalmente analisaram-se parâmetros de crescimento e fisiológicos. O maior crescimento das plantas ocorreu no verão. A presença da bactéria (B e BN) aumentou a biomassa total, área foliar, assimilação de CO2 e %N nas folhas. O comprimento radicular e a composição de aminoácidos do xilema não foram influenciados pelo microrganismo. A bactéria estabeleceu-se endofíticamente nas raízes. O tratamento BN apresentou os maiores valores fisiológicos e de crescimento. Este resultado sugere que a aplicação de adubos nitrogenados juntamente com bactérias poderia acarretar em maior produtividade dos cultivos de cana-de-açúcar. / The effects of Rhizobium sp. ICB503 on the development of sugarcane plants were observed in experiments carried out in two different seasons. Sugarcane plants were submeted to the treatments: B, inoculated with bacteria , BN inoculated with bacteria and treated with nitrate; N treated with NO3-, and control C, uninoculated and untreated with nitrate (NO3-) Plants were maintained for two months in greenhouse. Monthly, several growth and physiological parameters were analyzed. Better results were obtained in summer. The presence of the bacteria (B and BN) increased the total plant biomass, leaf area, CO2 assimilation and percentage of nitrogen in leaves. However, root length and amino acids composition of xylem were not influenced. The bacterium was able to establish endophytically in roots. The treatment BN showed the best results suggesting that the application of nitrogen fertilizers along with bacteria could increase the productivity in sugarcane fields.

Les endosymbioses racinaires sont des associations bénéfiques établies entre les racines des plantes et des micro-organismes du sol. Ces symbioses ont un intérêt agronomique et écologique puisque les plantes fournissent à leurs partenaires microbiens une niche écologique et des sucres issus de la photosynthèse et en retour, les micro-organismes associés aux racines vont fournir à la plante des nutriments minéraux qui sont actuellement apportés dans l’agriculture conventionnelle sous forme d’engrais. Durant ma thèse, j’ai particulièrement étudié la symbiose endomycorhizienne à arbuscules (AMS). Elle implique des champignons du groupe des Gloméromycètes et plus de 80 % des plantes terrestres. Ainsi cette symbiose est la plus répandue sur terre connue à l’heure actuelle. Plusieurs étapes importantes pour l’établissement de l’AMS ont été définies. La première de ces étapes est la reconnaissance mutuelle entre le champignon endomycorhizien et la plante hôte. Le champignon est capable de percevoir les plantes par les exsudats racinaires qu’elles sécrètent dans la rhizosphère. Dans le mélange complexe de molécules que sont les exsudats racinaires, des phytohormones appelées strigolactones activent le métabolisme des champignons endomycorhizien, la ramification des leurs hyphes et la production de molécules fongiques appelée facteurs Myc. La perception des facteurs Myc par la plante active des processus permettant la colonisation des racines par le champignon. Ce dialogue moléculaire entre champignons endomycorhiziens et plantes hôtes reste toutefois méconnu. Des molécules de type Lipo-chitooligosaccharides (LCO) ou chito-oligosaccharides (CO) ont été identifiées dans les exsudats de spores ou d’hyphes de champignons et activent la voie de signalisation symbiotique chez les plantes mais leurs rôles respectifs dans l’établissement de l’AMS restent mal compris. Du côté de la plante, des récepteurs potentiels aux LCOs et aux COs sont codés par les gènes de la famille des Lysin Motif Receptor-Like Kinase (LysM-RLK) qui sont capables de lier les constituants structuraux des LCOs et des COs. Cependant aucune preuve n’avait été apportée, au commencement de ma thèse, permettant de conclure sur le rôle des LCOs, des COs, et des LysM-RLKs dans la mise en place de l’AMS. C’est ce que je me suis attachée à démontrer durant ma thèse. Pour cela, j’ai travaillé sur une dicotylédone (la tomate : Solanum lycopersicum) et sur une monocotylédone (Brachypodium distachyon, un modèle pour le blé). Pour identifier les récepteurs aux LCOs dans ces plantes et déterminer leur rôle dans l’AMS nous avons mis en place des techniques de génétique inverse. Nous avons ensuite déterminé l’affinité de ces récepteurs pour les LCOs. Ainsi, nous avons montré que la perception des LCOs dans la tomate est importante pour la mise en place de l’AMS. Par ailleurs, je me suis intéressée à la symbiose entre des bactéries du type rhizobium et des plantes principalement de la famille des légumineuses. La mise en place de cette symbiose nécessite la synthèse de LCOs par les rhizobia et leur perception par la plante via des récepteurs de la famille des LysM-RLKs. Ces similarités que la symbiose rhizobium-légumineuses partage avec l’AMS nous ont conduits à poser la question de savoir si les récepteurs de LCOs impliqués dans l’AMS (beaucoup plus ancienne que la symbiose rhizobium-légumineuse) ont été recrutés durant l’évolution pour jouer un rôle dans la symbiose rhizobium-légumineuse. J’ai pu montrer que les récepteurs de LCOs impliqués dans l’AMS chez les espèces non-légumineuses susmentionnées sont fonctionnels l’établissement de la symbiose rhizobium-légumineuse chez une légumineuse. / Root endosymbioses are beneficial associations established between plant roots and soil microorganisms. These symbioses have an agronomic and ecological interest as plants provide their microbial partners with an ecological niche and carbohydrates from photosynthesis. In return, the root-associated microorganisms provide the plant with minerals that are currently being delivered in conventional agriculture as fertilizers. During my thesis, I particularly studied the arbuscular mycorrhizal symbiosis (AMS). It involves fungi of the Glomeromycota group and more than 80 % of land plants. This is the currently known most widespread symbiosis on earth. Important steps for the AMS establishment have been defined. The first step is the mutual recognition between the endomycorrhizal fungus and the host plant. Fungi can perceive plants through the root exudates. In the complex mixture of molecules in the root exudates, phytohormones called strigolactones activate the endomycorrhizal fungal metabolism, the branching of their hyphae and the production of fungal molecules called Myc-Factors. Myc-Factors are perceived by the plant and activate a signaling pathway allowing root colonization by the fungus. However, parts of the molecular dialogue between endomycorrhizal fungi and host plants remain unknown. Lipo-chitooligosaccharide (LCO) or chito-oligosaccharides (CO) molecules have been found in exudates of fungal spores or hyphae and were shown to activate the plant symbiotic signaling pathway, however their respective roles in the AMS establishment are unclear. Putative plant receptors for LCOs and COs are encoded by genes from the Lysin Motif Receptor-Like Kinase family (LysM-RLK) which are able of binding the structural LCO and CO components. However, at the beginning of my PhD, we had no evidence allowing to conclude about the involvement of LCOs, COs, or LysM-RLKs in the AMS establishment. During my thesis, I aimed to understand the role the LCOs and their plant receptors in AMS. For this, I used on a dicotyledon (the tomato: Solanum lycopersicum) and on a monocotyledon (Brachypodium distachyon that is a model for wheat). In order to identify the LCO receptors in these two species, I used a reverse genetic approach. Then I determined these receptors affinity for various LCO structures. I showed that in tomato, LCO perception is important for AMS establishment. In addition, I have studied the symbiosis between rhizobium-type bacteria and plants of the legume family. Interestingly, the establishment of this symbiosis requires LCO synthesis by rhizobia and LCO perception by the plant via receptors of the LysM-RLK family. The fact that rhizobium-legume symbiosis shares similarities with the AMS led us to ask whether the LCO receptors involved in AMS (a much more ancient symbiosis than the rhizobium-legume symbiosis) have been recruited during evolution for a role in the rhizobium-legume symbiosis. I demonstrated that the LysM-RLKs involved in AMS in the above mentioned non-legume species are functional for the rhizobium-legumes establishment in a legume species.

En réponse à une carence en azote dans le sol, les légumineuses sont capables d’interagir avec une bactérie du sol, Rhizobium, pour former un nouvel organe spécifique: la nodosité fixatrice d’azote atmosphérique. Chez la plante modèle des légumineuses Medicago truncatula, le récepteur aux cytokinines MtCRE1 est essentiel pour cette interaction symbiotique. Cependant, trois autres récepteurs aux cytokinines CHASE Histidine Kinase (CHK) existent chez M. truncatula. Les quatre CHKs ont des profils d’expression redondants dans les étapes précoces de la formation des nodosités, et plus divergeant dans les nodosités différenciées, même si MtCRE1 est le plus exprimé. Le locus génomique du plus proche homologue de MtCRE1 chez la plante non-symbiotique Arabidopsis, AHK4, complémente l’initiation des nodosités, mais seulement partiellement les phénotypes de croissance des nodosités et de fixation d’azote. Parmi les Régulateurs de Réponse de type B agissant en aval des CHKs, RRB3 a été sélectionné pour réaliser une stratégie de délétion de domaines protéiques, révélant son rôle positif dans la nodulation. Des données transcriptomiques indiquant une régulation de MtCRE1 dans l’épiderme en réponse à un « traitement symiotique », des approches fonctionnelles ont été réalisées et ont permis d’identifier un rôle négatif des cytokinines et de la voie MtCRE1 dans l’épiderme en réponse à ces « conditions symbiotiques ».En parallèle de ces travaux, des mutants des CHKs chez le pois ont été générés. A terme, ces recherches permettront de sélectionner un génotype tolérant à différents stress biotiques et abiotiques sans affecter les symbioses bénéfiques chez une espèce d’intérêt agronomique. / Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen-fixing soil bacteria to form a new specific organ: the nitrogen-fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other CHASE Histidine Kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCRE1 has the strongest expression. Interestingly, a genomic locus of the MtCRE1 homolog from the aposymbiotic Arabidopsis plant, AHK4, rescues noduleinitiation, but only partially the nodule growth phenotype, and not the nitrogen fixation capacity. Among type-B Response Regulators acting downstream of CHKs, RRB3 has been selected to perform protein deletions, revealing a positive role RRB3 in nodulation. Transcriptomic data indicating an epidermis MtCRE1 regulation during a « symbiotic traitment », functional approaches showed a negative role of cytokinins and MtCRE1 pathway in epidermis in response to « symbiotic conditions ». chk mutants have been generated in Pisum sativum. Ultimately, the aim of these researches is to set the bases to select in legume species of agronomic interest a genotype tolerant to biotic and abiotic stresses without affecting beneficial symbioses.

No description available.