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Neuhaus, Jean-Marc
Nom
Neuhaus, Jean-Marc
Affiliation principale
Fonction
Professeur ordinaire
Email
jean-marc.neuhaus@unine.ch
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Résultat de la recherche
21 Résultats
Voici les éléments 1 - 10 sur 21
- PublicationAccès libreIdentification of genes expressed during the compatible interaction of grapevine with Plasmopara viticola through suppression subtractive hybridization (SSH)(2011)
; ; 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. - PublicationAccès libreExpression of a glycosylated GFP as a bivalent reporter in exocytosis(2010)
; Di Sansebastiano, Gian PietroThe complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an α-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a β-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells. In all these cases glycosylated tags were added at the GFP termini. Because of the position of the tag and depending on the sorting and accumulation site of these modified GFP, there is always a risk of processing and degradation, and this protein design cannot be considered ideal. Here, we describe the development of three different GFPs in which the glycosylation site is internally localized at positions 80, 133, or 172 in the internal sequence. The best glycosylation site was at position 133. This glycosylated GFPgl133 appears to be protected from undesired processing of the glycosylation site and represents a bivalent reporter for biochemical and microscopic studies. After experimental validation, we can conclude that amino acid 133 is an effective glycosylation site and that the GFPgl133 is a powerful tool for in vivo investigations in plant cell biology. - PublicationAccès libreOptimisation and comparison of transient expression methods to express the green fluorescent protein in the obligate biotrophic oomycete Plasmopara viticola(2008)
; Grape downy mildew is caused by Plasmopara viti-cola, an obligate biotrophic oomycete and a major path-ogen of grapevine. Studying obligate biotrophic patho-gens is difficult as they cannot grow without their host. We therefore attempted to develop a method where the pathogen could be visualized and quantified in planta without killing the host plant. To this end P. viticola was transformed with the marker gene gfp coding for the green fluorescent protein. Various transformation methods, namely electroporation, particle bombard-ment and transformation with Agrobacterium tume-faciens were applied. Although some methods yielded positive transformation events, no stable strain of P. viticola expressing gfp could be generated. Using the electroporation method, we obtained transient P. viti-cola transformants expressing gfp over 4 generations. In contrast, particle bombardment failed in transform-ing P. viticola. Transformation with A. tumefaciens had a low efficiency, only some structures were fluorescent and fluorescence was never observed in the subsequent generations. - PublicationAccès libreThe knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens(2008)
; - PublicationAccès librePharmaceutical Proteins in Plants: A Strategic Genetic Engineering Approach for the Production of Tuberculosis Antigens(2008)
; - PublicationAccès libreBeta-aminobutyric acid-induced resistance in grapevine against downy mildew: involvement of pterostilbene(Springer, 2008)
; 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. - PublicationAccès libreArabidopsisµA-adaptin interacts with the tyrosine motif of the vacuolar sorting receptor VSR-PS1(2004)
; - PublicationAccès libreVacuolar system distribution in Arabidopsis tissues, visualized using GFP fusion proteins(2003-06-01)
; Di Sansebastiano, Gian-PietroGreen fluorescent protein (GFP) allows the direct visualization of gene expression and the subcellular localization of fusion proteins in living cells. The localization of different GFP fusion proteins in the secretory system was studied in stably transformed Arabidopsis plants cv. Wassilewskaja. Secreted GFP (SGFP) and GFP retained in the ER (GFP-KDEL) confirmed patterns already known, but two vacuolar GFPs (GFP-Chi and Aleu-GFP) labelled the Arabidopsis vacuolar system for the first time, the organization of which appears to depend on cell differentiation. GFP stability in the vacuoles may depend on pH or degradation, but these vacuolar markers can, nevertheless, be used as a tool for physiological studies making these plants suitable for mutagenesis and gene-tagging experiments. - PublicationAccès libreExpression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris(2003)
; Ward, Thomas R.A recombinant glycosylated avidin (recGAvi) with an acidic isoelectric point was expressed and secreted by the methylotrophic yeast Pichia pastoris. The coding sequence for recGAvi was de novo synthesized based on the codon usage of P. pastoris. RecGAvi is secreted at approximately 330 mg/L of culture supernatant. RecGAvi monomer displays a molecular weight of 16.5 kDa, as assessed by ESI mass spectrometry. N-terminal amino acid sequencing indicates the presence of three additional amino acids (E-A-E), which contribute to further lowering the isoelectric point to 5.4. The data presented here demonstrate that the P. pastoris system is suitable for the production of recGAvi and that the recombinant avidin displays biotin-binding properties similar to those of the hen-egg white protein. - PublicationAccès libreThe Destination for Single-Pass Membrane Proteins Is Influenced Markedly by the Length of the Hydrophobic Domain(2002-05-02)
; ; Paris, NadineThe tonoplast was proposed as a default destination of membrane-bound proteins without specific targeting signals. To investigate the nature of this targeting, we created type I fusion proteins with green fluorescent protein followed by the transmembrane domain of the human lysosomal protein LAMP1. We varied the length of the transmembrane domain from 23 to either 20 or 17 amino acids by deletion within the hydrophobic domain. The resulting chimeras, called TM23, TM20, and TM17, were expressed either transiently or stably in tobacco. TM23 clearly accumulated in the plasmalemma, as confirmed by immunoelectron microscopy. In contrast, TM17 clearly was retained in the endoplasmic reticulum, and TM20 accumulated in small mobile structures. The nature of the TM20-labeled compartments was investigated by coexpression with a marker localized mainly in the Golgi apparatus, AtERD2, fused to a yellow fluorescent protein. The strict colocalization of both fluorescent proteins indicated that TM20 accumulated in the Golgi apparatus. To further test the default destination of type I membrane proteins, green fluorescent protein was fused to the 19–amino acid transmembrane domain of the plant vacuolar sorting receptor BP-80. The resulting chimera also accumulated in the Golgi instead of in post-Golgi compartments, where native BP-80 localized. Additionally, when the transmembrane domain of BP-80 was lengthened to 22 amino acids, the reporter escaped the Golgi and accumulated in the plasma membrane. Thus, the tonoplast apparently is not a favored default destination for type I membrane proteins in plants. Moreover, the target membrane where the chimera concentrates is not unique and depends at least in part on the length of the membrane-spanning domain.
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