Rhodococcus fascians, a plant pathogenic actinomycete with a very broad host range, causes leafy galls and other malformations. The plant hormone, group, the cytokinins has been implicated in the alteration of host morphology. The aim of this project was to gain insight into the interaction of the cytokinin biosynthetic, isopentenyltransferase (IPT), cytokinin activating ( LOG (The Lonely Guy)) and the cytokinin metabolic, cytokinin oxidase/dehydrogenase (CKX) gene families of both Pisum sativum and R. fascians during infection of the plant. R. fascians colonisation and infection of pea were examined using scanning electron microscopy (SEM) and light microscopy. The expression of genes related to cytokinin biosynthesis, activation and metabolism were isolated and assessed with polymerase chain reaction (PCR) and real time-quantitative PCR (RT-qPCR) analysis. Primers were designed to discriminate between pea genes and R. fascians genes. In addition, the response of the pea cotyledons to R. fascians was measured through chlorophyll estimation and the expression of the transporter genes, sucrose transporter (SUT) and amino acid permease (AAP) which were assayed through RT-qPCR. The pea response regulators were monitored as an indirect measure of the level of endogenous cytokinins in pea. Two R. fascians strains, the avirulent strain 589 and the virulent strain 602, were selected for this project based on their virulence and similar growth rate under identical conditions. The virulence of R. fascians virulent strain 602 was also confirmed through Koch's postulates. The phenotypic alterations in the pea infected with the virulent strain 602 included stunted growth, multiple shoots, small leaves, thickened primary roots and reduced secondary root growth. Delayed senescence of shoots and dark green, intact cotyledons were also observed. Microscopic analyses revealed epiphytic colonisation by both the avirulent strain 589 and the virulent strain 602 in pea cotyledons, roots, shoots and leaves and endophytic colonisation in the seed coat from the time of seed inoculation to 45 days post inoculation (dpi). The expression of R. fascians genes was relatively high at 5 and 9 dpi in pea cotyledons and at 15 and 25 dpi in roots and shoots of pea infected with the virulent strain 602. The expression of RfIPT, RfLOG and RfCKX was not detected both in the control pea and the pea infected with the avirulent strain 589. The cytokinin biosynthesis, metabolism and response regulator (RR) multi-gene families of PsIPTs, PsLOGs, PsCKXs and PsRRs revealed differential and tissue-specific expression patterns. The expression of PsIPTs and PsLOGs was induced immediately after inoculation with the R. fascians virulent strain 602 in the cotyledon but not in roots and shoots, and the expression level reduced at later growth stages. The PsCKXs and PsRRs expression level increased with the growth of the host infected with the virulent strain 602. In pea infected with the avirulent strain 589 the expression of PsIPTs, PsLOGs and PsCKXs gene family members generally increased after 25 dpi in cotyledons, roots and shoots, whereas PsRRs expression was low at all time points. The up-regulation of PsIPTs and PsLOGs immediately after inoculation in cotyledons and at 15 dpi in roots and shoots by R. fascians virulent strain leads to elevated cytokinins which is reflected by the up-regulation of PsRRs. The plant responds to elevated cytokinin by producing phenotypic changes including shoot malformations. The plant activates its cytokinin homeostasis mechanism due to change in cytokinin level which is indicated by up-regulation of PsCKXs. Generally, the expression of the PsRRs was also up-regulated over time following infection by the R. fascians virulent strain. This indicates the presence of biologically active cytokinins in the host which maintain the symptoms. The outcome due to the avirulent strains indicates that, even though PsIPTs and PsLOGs are up-regulated at later growth stages (25 to 35 dpi), expression of PsCKX gene families were varied (either up-regulated or down regulated after 25 dpi). However, PsRRs expression was down-regulated suggesting low cytokinins levels in tissues which may be due to the activation of homeostatic mechanisms of the plant to reduce the level of biologically active cytokinins. The chlorophyll content increased in cotyledons infected with the virulent strain 602 and PsSUTs and PsAAPs expression pattern in pea cotyledon and shoot infected with the virulent strain 602 indicates that R. fascians converts the infected tissue into a sink for their establishment and growth.

Rhodococcus fascians is a Gram-positive phytopathogenic bacterium which induces the development of leafy galls, local amplifications of multiple buds, on most infected plants. This process is linked to the production of phytohormones along with the presence of essential virulence-associated genes like the plasmid loci att and fas and the chromosomal gene vicA. However, the presence of these genes is not sufficient to ensure the infection phenotype development, indicating that other genes play a role in R. fascians pathogenicity. In this work, we studied the metabolic modifications occurring when the bacterium interacts with its host using a proteomic approach. A comparison between virulent and avirulent strains showed variations in the expression of catalases. In the virulent strain, besides the transitory induction of the att locus expression, the bacterium changes its metabolism from the Krebs cycle to the glyoxylate shunt, a process which is frequently observed in bacteria confronted to a hostile environment. The expression of the shunt-specific enzyme isocitrate lyase increased, while expression of fumarate hydratase and pyruvate dehydrogenase decreased. Hence, we focused on the link between the glyoxylate shunt and virulence. A screening of a R. fascians mutant library based on the capacity of bacteria to use acetate as the sole carbon source, a metabolic pathway depending on the glyoxylate shunt, resulted in the identification of a new gene essential for R. fascians pathogenicity. This gene encodes a glycosyl transferase, an enzyme known to be involved in the bacterial cell wall biosynthesis but possibly also implicated in cytokinin secretion. A mutant in this gene harboured an altered colony phenotype and could not induce malformations on infected plants. Accordingly, our results were integrated in the leafy gall pathology model recently presented by Stes et al. (2011). Finally, the several questions that are raised by this work, allowed us to suggest further research perspectives in order to unveil a little more of the R. fascians mysterious ways to interact with the plant./Rhodococcus fascians est une bactérie Gram-positive phytopathogène qui induit le développement de galles feuillées, des amplifications locales de multiples bourgeons, sur la plupart des plantes infectées. Ce processus est lié à la production de phytohormones ainsi qu’à la présence de gènes essentiels associés à la virulence tels que les loci plasmidiques att et fas et le gène chromosomique vicA. Cependant, la présence de ces gènes ne suffit pas à garantir le développement du phénotype d’infection, indiquant que d’autres gènes jouent un rôle dans la pathogénicité de R. fascians. Dans ce travail, nous avons étudié les modifications métaboliques qui se produisent lorsque la bactérie interagit avec son hôte par une approche protéomique. Une comparaison entre les souches virulente et avirulente a mis en évidence des variations d’expression au niveau des catalases. Dans la souche virulente, outre l’induction transitoire de l’expression du locus att, la bactérie change son métabolisme pour passer du cycle de Krebs au shunt du glyoxylate, un processus fréquemment observé chez les bactéries confrontées à un environnement hostile. L’expression de l’isocitrate lyase, enzyme spécifique au shunt, augmente, tandis que celle de la fumarate hydratase et de la pyruvate déhydrogénase diminue. Nous nous sommes donc intéressés au lien entre le shunt du glyoxylate et la virulence. Le screening d’une banque de mutants de R. fascians basé sur la capacité de la bactérie à utiliser l’acétate comme seule source de carbone, une voie métabolique dépendant du shunt du glyoxylate, a permis d’identifier un nouveau gène essentiel pour la pathogénicité de R. fascians. Ce gène code pour une glycosyl transferase, une enzyme impliquée dans la biosynthèse de la paroi bactérienne mais également dans la sécrétion des cytokinines. Un mutant dans ce gène présente un phénotype de colonie altéré et ne peut induire de malformations chez les plantes infectées. Finalement, nos résultats et les pistes d’interprétations que nous avons émisent nous permettent de compléter le modèle de l’interaction R. fascians-plante proposé récemment par Stes et al. (2011). Des perspectives de recherches visant une meilleure compréhension de ce pathosystème sont proposées. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Plants are continuously confronted with a multitude attack either abiotic but also biotic in nature. Interestingly, despite the abundance of bacteria that plant has to face, only few are able to induce death or disease in the host plant. It is therefore likely that, in addition to secondary metabolites with antimicrobial properties, plants also synthesize secondary metabolites which are able to inhibit the expression of virulence genes in bacteria without affecting either growth or viability, which allows plants to host willingly or not bacterial populations. This work focuses on the identification of such metabolites in Malagasy plants (genera Dalbergia and Combretum) and the demonstration of their inhibitory effect on the expression of virulence genes in two different pathosystems: Rhodococcus fascians (a phytopathogen) and Pseudomonas aeruginosa (an opportunistic pathogen). Thus, two metabolites were isolated using a combination of chromatographic techniques coupled with tests that evaluate the expression of certain genes involved in the virulence mechanisms of these bacteria. The first is a new prenylated isoflavanone, named perbergin, isolated from the bark extract of D. pervillei. It was shown that the perbergin target attR gene expression, encoding a LysR-type transcriptional regulator that plays a key role in regulating the expression of virulence genes of R. fascians and the transition from an epiphytic to a pathogenic lifestyle. Therefore, we have also shown that the expression of all virulence genes known to date in R. fascians is also affected while the expression of genes involved in epiphytic fitness of the bacteria is not altered. In addition, the application of perbergin at the time of infection of plants susceptible to R. fascians shows that this molecule reduces in vivo the virulence of R. fascians, highlighting the potential of perbergin as an anti-infective agent. The second is a flavonoid known as catechin, isolated from the bark extract of C. albiflorum. Catechin significantly inhibits the expression of genes that regulate the mechanism of quorum sensing in P. aeruginosa such as lasI, LasR, rhlI and rhlR but also lasB and rhlA which expression depends on quorum sensing. Therefore, the production of virulence factors such as pyocyanin and elastase is significantly affected. Because of the limited number of our arsenal of antibiotics and their increasing ineffectiveness, the identification of these compounds create a path to an alternative in the fight against pathogenic bacteria and multidrug resistance of pathogenic bacteria to antibiotics. Our results also demonstrate the richness of Malagasy plants as (re)sources of new therapeutic molecules and the importance of widening the range of bacterial targets to be investigated to develop new strategies to fight within the endless war that we are waging against bacteria pathogens.<p><p>Les plantes sont continuellement confrontées à une multitude d’attaques qu’elles soient de nature abiotique ou surtout biotique. Il est intéressant de noter que malgré la multitude de bactéries auxquelles les plantes doivent faire face, seules quelques unes sont capables d’induire la mort ou une maladie chez la plante hôte. Il est dès lors fort probable que, outre les métabolites secondaires ayant des propriétés antimicrobiennes, les plantes synthétisent également des métabolites secondaires capables d’inhiber l’expression des gènes de virulence chez les bactéries sans toutefois affecter ni leur croissance ni leur viabilité, ce qui permet aux plantes de contenir les populations bactériennes qu’elles hébergent de gré ou de force. Ce travail porte sur l’identification de ce type de métabolites dans des plantes malgaches (genres Dalbergia et Combretum) et la démonstration de leurs effets inhibiteurs sur l’expression de gènes de virulence chez deux pathosystèmes différents: Rhodococcus fascians (un phytopathogène) et Pseudomonas aeruginosa (un pathogène opportuniste). Ainsi, deux métabolites ont été isolés en utilisant une combinaison de techniques chromatographiques couplées avec des tests qui évaluent l’expression de certains gènes impliqués dans les mécanismes de virulence de ces bactéries. Le premier est un nouvel isoflavanone prénylé, nommé perbergine, isolé à partir de l’extrait d’écorces de D. pervillei. Il a été montré que la perbergine cible l’expression du gène attR, codant un régulateur transcriptionnel de type LysR qui joue un rôle clé dans la régulation de l’expression des gènes de virulence de R. fascians et qui assure la transition entre un mode de vie épiphyte et le mode pathogène. En conséquence, nous avons également montré que l’expression de l’ensemble des gènes de virulence connu à ce jour chez R. fascians est également affectée alors que l’expression de gènes impliqués dans l’aptitude épiphyte de la bactérie n’est pas altérée. Par ailleurs, l’application de perbergine au moment de l’infection de plantes sensibles à R. fascians montre que cette molécule atténue la virulence de R. fascians in vivo, mettant en exergue le potentiel de la perbergine comme agent anti-infectieux. Le deuxième est un flavonoïde, connu sous le nom de catéchine, isolé de l’extrait d’écorces de C. albiflorum. La catéchine inhibe significativement l’expression des gènes régulateurs du mécanisme du quorum sensing chez P. aeruginosa tels que lasI, lasR, rhlI et rhlR et également lasB et rhlA dont l’expression dépend du quorum sensing. En conséquence, la production des facteurs de virulence tels que la pyocyanine et l’élastase est significativement affectée. Compte tenu de l’appauvrissement de notre arsenal d’antibiotiques et de leur inefficacité croissante, l’identification de ces composés ouvre une voie alternative de lutte contre les bactéries pathogènes et la multirésistance des bactéries pathogènes aux antibiotiques. Nos résultats démontrent également la richesse des plantes malgaches comme (res)sources de nouvelles molécules thérapeutiques et l’importance d’élargir le champ des cibles bactériennes à investiguer pour développer de nouvelles stratégies de lutte dans la guerre sans fin que nous menons contre les bactéries pathogènes. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.