Voici les éléments 1 - 10 sur 105
  • Publication
    Accès libre
    The functions of RMR proteins in the "Physcomitrella patens" secretory pathway
    (2017)
    Fahr, Noémie
    ;
    Chez les plantes, les vacuoles occupent un grand nombre de fonctions, allant du maintien de la pression de turgescence et de la rigidité cellulaire en passant par le stockage ou la dégradation de diverses molécules. Deux types de vacuoles ayant un pH distinct peuvent coexister au sein d’une même cellule. Les vacuoles acides sont considérées comme étant des homologues aux lysosomes présents dans les cellules animales, tandis que les vacuoles neutres sont impliquées dans le stockage de protéines et de métabolites secondaires. L’adressage des protéines à la vacuole lytique a été largement étudié et les récepteurs vacuolaires impliqués, les VSRs, sont des protéines bien caractérisées. À l’opposé, les connaissances sur l’adressage des protéines à la vacuole neutre ou de stockage sont moindres. Les protéines RMR sont très probablement les récepteurs vacuolaires impliqués, bien que la délétion des cinq gènes RMR chez Physcomitrella patens n’ait conduit à aucun phénotype visible. Ce travail a donc pour objectif l’élucidation du rôle des protéines RMR chez la mousse.
    Dans le premier chapitre de cette thèse nous avons regroupé les principales données concernant le système sécrétoire et endomembranaire chez les plantes, et nous avons également documenté comment les protéines sont adressées aux vacuoles.
    Dans le second chapitre, nous avons étudié le système sécrétoire de la mousse en développant une bibliothèque de marqueurs fluorescents. Différents mécanismes cellulaires semblent conservés entre les mousses et les plantes à fleurs.
    Dans le troisième chapitre, nous nous sommes intéressés à la caractérisation des simples et quintuple knock-out mutants RMR. Nous avons finalement obtenu un phénotype de tri vacuolaire: un défaut d’adressage est observé avec le marqueur fluorescent Citrine-Card dans les simples, triple et quintuple KO mutants. Le signal fluorescent a été détecté dans le réticulum endoplasmique chez les mutants, tandis que la fluorescence est observée dans la vacuole centrale chez le WT. Cela montre que l’adressage à la vacuole d’une protéine comportant ce ctVSD est dépendant des RMRs.
    Dans la dernière partie de ce travail, nous avons identifié des partenaires interagissant très probablement avec la partie cytosolique de PpRMR2 par des analyses de GST pull-down et de spectrométrie de masse., Plant vacuoles play a wide range of functions within the cell, from the maintenance of turgor pressure and rigidity, to the storage or degradation of various molecules. Two types of vacuoles with distinct pH can coexist in the same single cell. Acidic vacuoles can be considered as homologues to animal lysosomes while neutral vacuoles are involved in proteins and secondary metabolites storage. Targeting of proteins to the lytic vacuole has been extensively studied and the vacuolar receptors involved, the VSRs, are now well characterized in higher plants. However, less is known about the traffic of proteins to the neutral/storage vacuole. RMR proteins are thought to be vacuolar receptors for the neutral/storage vacuole. However, the complete deletion of the five RMR genes in Physcomitrella patens did not lead to any developmental phenotype. This work aimed to investigate the role of RMR proteins in the moss.
    In the first chapter, we review the plant secretory pathway system and how proteins are targeted to vacuoles.
    In the second chapter, we studied the moss secretory pathway by developing a fluorescent reporter library. Several mechanisms seem to be conserved between the moss and the flowering plants.
    In the third chapter, we focused on the characterization of the single and quintuple knock-out RMR mutants. We finally obtained a trafficking phenotype: the fluorescent reporter Citrine-Card was mistargeted in the single, triple and quintuple KO mutants. Fluorescent signal was detected in endoplasmic reticulum in the mutants, while it was observed in the central vacuole in WT. Trafficking to vacuole of a protein carrying this ctVSD was RMR-dependent.
    In the last part of this thesis, we identified some putative binding partners of the cytosolic part of PpRMR2 by GST pull-down assay and mass spectrometry analysis.
  • Publication
    Accès libre
    Beta-aminobutyric acid-induced resistance in grapevine against downy mildew: involvement of pterostilbene
    (Springer, 2017)
    Slaughter, Ana R.
    ;
    Hamiduzzaman, Mollah Md.
    ;
    Katia Gindro
    ;
    ;
    BABA, a non-protein amino acid, was used to induce resistance in grapevine against downy mildew. BABA-induced resistance was observed in the susceptible cv. Chasselas as well as in the resistant cv. Solaris. Following BABA treatment, sporulation of Plasmopara viticola was strongly reduced and the accumulation of stilbenes increased with time following infection. Induction of trans-piceide, trans-resveratrol and, more importantly, of trans-ɛ- and trans-δ-viniferin and trans-pterostilbene was observed in BABA-primed Chasselas. On the other hand, induction of trans-resveratrol, trans δ-viniferin and trans-pterostilbene was observed in BABA-primed Solaris. The accumulation of stilbenes in BABA-primed Solaris was much higher than that found in BABA-primed Chasselas. Furthermore, BABA-treatment of Solaris led to a rapid increase in transcript levels of three genes involved in the phenylpropanoid pathway: phenylalanine ammonia lyase, cinnamate-4-hydroxylase and stilbene synthase. BABA-primed Chasselas showed increased transcript levels for cinnamate-4-hydroxylase and stilbene synthase. Here we show that pre-treatment of a susceptible grapevine cultivar with BABA prior to infection with P. viticola primed the accumulation of specific phytoalexins that are undetectable in non-BABA-primed plants. As a result, the susceptible cultivar became more resistant to downy mildew.
  • Publication
    Métadonnées seulement
    Two glycosylated vacuolar GFPs are new markers for ER-to-vacuole sorting
    (2013)
    Stigliano, Egidio
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    Faraco, Marianna
    ;
    ;
    Montefusco, Anna
    ;
    Dalessandro, Giuseppe
    ;
    Piro, Gabriella
    ;
    Di Sansebastiano, Gian-Pietro
    Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus.
  • Publication
    Métadonnées seulement
    Secretory pathway research: the more experimental systems the better
    (2012)
    Denecke, J.
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    Aniento, F.
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    Frigerio, L.
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    Hawes, C.
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    Hwang, I.
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    Mathur, J.
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    ;
    Robinson, D.
    Transient gene expression, in plant protoplasts or specific plant tissues, is a key technique in plant molecular cell biology, aimed at exploring gene products and their modifications to examine functional subdomains, their interactions with other biomolecules, and their subcellular localization. Here, we highlight some of the major advantages and potential pitfalls of the most commonly used transient gene expression models and illustrate how ectopic expression and the use of dominant mutants can provide insights into protein function.
  • Publication
    Accès libre
    Study of moss vacuoles and functional characterization of the putative vacuolar receptors: the RMR proteins
    (2012)
    Ayachi, Sanaa
    ;
    The vacuolar system of plants is a key element of plant growth and development, it fulfils many other functions. Plant cell can have more than two different vacuolar sorting systems: the lytic and the (seed) protein storage or vegetative storage vacuoles. Soluble vacuolar proteins are sorted through the secretory pathway to these vacuoles by three different routes, depending on different types of Vacuolar Sorting Determinants (VSD) and involving several types of receptors and vesicles. The AtRMR proteins has been identified in cellular structures associated with the seed storage vacuole pathway (Jiang et al. 2000). Based on its localisation and homology to a known vacuolar receptor, it has been hypothesised to be a receptor protein for the C-terminal type of VSD (ct-VSD) involved in sorting to the storage vacuole. The genome of Physcomitrella patens contains five genes coding for RMR proteins.
    My work hypothesis is that the vacuolar system of higher plants has evolved from simple ancestors, which might have been preserved in lower plants. This evolution is reflected in the gene families involved in vacuole biogenesis. In a first part, we established the moss P. patens as a model system for the study of the secretory pathways. In a second part, we performed a comparative study of the plant-specific aspects of the vacuolar system. And finally in a third part, we tried to establish the functional role of PpRMR genes by the analysis of the complete RMR deletion mutants. Several strategies were considered to investigate a putative disorder due to RMRs loss of function. So far, no phenotype was detected in the mutants. Nevertheless the absence of the RMR family gene seems not to be necessary for moss development.
  • Publication
    Métadonnées seulement
    Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium
    (2011)
    Sriranganadane, D.
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    Reichard, U.
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    Salamin, K.
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    Fratti, M.
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    Jousson, O.
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    Waridel, P.
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    Quadroni, M.
    ;
    ;
    Monod, M.
    In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepDelta mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.
  • Publication
    Métadonnées seulement
    Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH
    (2011)
    Sriranganadane, D.
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    Waridel, P.
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    Salamin, K.
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    Feuermann, M.
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    Mignon, B.
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    Staib, P.
    ;
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    Quadroni, M.
    ;
    Monod, M.
    The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.
  • Publication
    Métadonnées seulement
    Plant cell vacuoles
    (Basingstoke: Macmillan Publishers, 2011) ;
    Martinoia, E.
  • Publication
    Accès libre
    Identification of genes expressed during the compatible interaction of grapevine with Plasmopara viticola through suppression subtractive hybridization (SSH)
    (2011)
    Legay, Guillaume
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    Marouf, Elaheh
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    Berger, Dave
    ;
    ; ;
    Slaughter, Ana R.
    Grapevine (Vitis vinifera) is the most widely cultivated and economically important fruit crop, but is susceptible to a large number of diseases. Downy mildew, caused by the obligate biotrophic oomycete pathogen Plasmopara viticola, is a common disease present in all regions where vines are cultivated. We used suppression subtractive hybridization (SSH) to generate two cDNA libraries enriched for transcripts induced and repressed, respectively, in the susceptible grapevine cultivar Chasselas 24 h after inoculation with P. viticola. Differential screening on glass slide microarrays yielded over 800 putative genes that were up-regulated in response to P. viticola infection and over 200 that were down-regulated. One hundred and ninety four of these, were sequenced, identified and functionally categorised. Transcript abundance of twelve genes over a 48 h time course was examined by reverse transcriptase quantitative real-time PCR (RT-qPCR). Ten of these genes were induced/enhanced by P. viticola challenge, confirming the results of the SSH. The vast majority of the genes identified are related to defence. Interestingly, many genes involved in photosynthesis were down-regulated.
  • Publication
    Accès libre
    Localization and interaction of AtRMR receptors in the plant secretory pathway
    (2011)
    Occhialini, Alessandro
    ;
    Durant les dernières années, il a été démontré que de nombreuses protéines vacuolaires sont triés vers leur destination finale par des récepteurs vacuolaires. Par conséquent, dans cette étude je me suis concentré sur les protéines RMR (récepteur membranaire Ring-H2), une nouvelle famille de récepteurs putatifs, composées de six gènes chez Arabidopsis thaliana (AtRMR), probablement impliquées dans le transport des protéines vers les vacuoles (Jiang et al., 2000 ; Park et al., 2005; Park et al., 2007; Hinz et al., 2007). Ces récepteurs ont été identifiés grâce à leur homologie avec le domaine PA (domaine associé aux protéases) présents dans les récepteurs vacuolaires VSR qui sont bien connus pour jouer un rôle de triage de certaines protéines vacuolaires (Paris et al. , 2002).
    On toutefois, les RMRs sont beaucoup moins connus que les VSRs. Dans la présente étude, je me suis concentré sur la localisation de différents RMRs présents dans les cellules végétales. Par ailleurs, j'ai étudié la possible dimérisation (homo-dimérisation et hétéro-dimérisation) entre les différents types de récepteurs AtRMR.
    Pour la localisation, j'ai généré différents vecteurs d'expression pour les plantes portant différentes protéines fluorescentes fusionnées à différent types de AtRMR. Par la suite, ces protéines de fusion ont été localisées dans les cellules en utilisant le microscope confocal. La localisation a été effectuée chez des plantes d'Arabidopsis thaliana transgéniques et chez des feuilles de Nicotiana benthamiana transformées par agroinfiltration. Dans ces expériences, AtRMR1 et AtRMR2 ont montré des localisations subcellulaires différentes. AtRMR1 est localisé dans TGN, tandis que AtRMR2 est localisé dans la membrane du Reticulum Endoplasmique (ER). Cette différente localisation est due à la présence d'un signal putatif de localisation présent dans la séquence linker de AtRMR1. En fait, quand cette séquence est placée sur AtRMR2, elle est capable de relocaliser la protéine dans le Réseau Trans-Golgien (TGN).
    Enfin, pour tester l'éventuelle dimérisation entre différents types de AtRMR, j'ai développé une technique de Complémentation Bimoléculaire Fluorescente (BiFC). En utilisant cette technique, j'ai démontré que AtRMR1 peut former des homo-dimères et peut interagir avec AtRMR2 en formant des hétéro-dimères. De plus, homo-et hétéro-dimères montrent la même localisation dans le TGN. Ce résultat a démontré que AtRMR2 peut quitter l’ER sous forme d’hétéro-dimère grâce à la présence du signal de localisation dans la séquence linker de AtRMR1. Par ailleurs, en utilisant des mutants de délétion de différents domaines, j'ai démontré que le domaine transmembranaire et la séquence linker sont probablement les domaines impliqués dans l'interaction protéine-protéine., In the last few years, it was demonstrated that many vacuolar proteins are sorted to their final destination by cargo receptors. Therefore in this study I focused on RMR proteins (Receptor Membrane Ring-H2), a new family of putative receptors, composed of six genes in Arabidopsis thaliana (AtRMR), probably involved in protein transport to vacuoles (Jiang et al.,, 2000; Park et al., 2005; Park et al., 2007; Hinz et al., 2007). These receptors were identified by their homology to the PA domain (Protease Associated Domain) present in the Vacuolar Sorting Receptors (VSR) that are well known to bind and sort vacuolar proteins (Paris et al., 2002).
    Much less is known about these proteins than about VSRs. In the present study I focused on the localization of the different members present in plant cells. Moreover I studied the possible dimerization (homo end/or hetero) between the different types of AtRMR receptors.
    For the localization, I have generated different plant expression vectors carrying different fluorescent protein reporters fused to AtRMRs to use in a confocal microscope experiment. The localization was performed in Arabidopsis thaliana transgenic plants and Nicotiana benthamiana leaves transformed by agro-infiltration. In these experiments AtRMR1 and AtRMR2 showed different subcellular localizations. AtRMR1 localizes in TGN while AtRMR2 localizes in the membrane of ER. This different localization is due by the presence of a putative localization signal present in the sequence linker of AtRMR1. In fact this sequence, when is placed on AtRMR2, is able to relocate the protein in the TGN.
    To test the possible AtRMR-AtRMR dimerization I developed Bimolecular Fluorescence Complementation (BiFC) reporters. Using this technique I demonstrated that AtRMR1 can make homodimers and can interact with AtRMR2 making heterodimers. Moreover homo- and heterodimers showed the same localization in the TGN. This result demonstrated that AtRMR2 can exit from the ER as a heterodimer thanks to the presence of the localization signal in the sequence linker of AtRMR1. Moreover using AtRMR deletion mutants I demonstrated that the transmembrane domain and the sequence linker are probably the domains involved in protein-protein interaction.