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Douet, Véronique

Nom
Douet, Véronique
Affiliation principale
Email
Veronique.Douet@unine.ch
Identifiants
https://libra.unine.ch/handle/123456789/335

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  • Publication
    Accès libre
    The Novel Chloroplast Outer Membrane Kinase KOC1 Is a Required Component of the Plastid Protein Import Machinery
    (2017)
    Zufferey, Mónica
    ;
    Montandon, Cyrille
    ;
    Douet, Véronique 
    ;
    Demarsy, Emilie
    ;
    Agne, Birgit
    ;
    Baginsky, Sacha
    ;
    Kessler, Félix 
    The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo. However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis.
  • Publication
    Accès libre
    Identification of Plastoglobules as a Site of Carotenoid Cleavage
    (2016)
    Rottet, Sarah
    ;
    Devillers, Julie
    ;
    Glauser, Gaétan
    ;
    Douet, Véronique 
    ;
    Besagni, Céline
    ;
    Kessler, Félix 
    Carotenoids play an essential role in light harvesting and protection from excess light. During chloroplast senescence carotenoids are released from their binding proteins and are eventually metabolized. Carotenoid cleavage dioxygenase 4 (CCD4) is involved in carotenoid breakdown in senescing leaf and desiccating seed, and is part of the proteome of plastoglobules (PG), which are thylakoid-associated lipid droplets. Here, we demonstrate that CCD4 is functionally active in PG. Leaves of Arabidopsis thaliana ccd4 mutants constitutively expressing CCD4 fused to yellow fluorescent protein showed strong fluorescence in PG and reduced carotenoid levels upon dark- induced senescence. Lipidome-wide analysis indicated that ß-carotene, lutein, and violaxanthin were the principle substrates of CCD4 in vivo and were cleaved in senescing chloroplasts. Moreover, carotenoids were shown to accumulate in PG of ccd4 mutant plants during senescence, indicating translocation of carotenoids to PG prior to degradation.
  • Publication
    Métadonnées seulement
  • Publication
    Accès libre
    Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants
    (2013)
    Shanmugabalaji, Venkatasalam
    ;
    Besagni, Céline
    ;
    Piller, Lucia Eugeni
    ;
    Douet, Véronique 
    ;
    Ruf, Stephanie
    ;
    Bock, Ralph
    ;
    Kessler, Félix 
    Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C- terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35–HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35–HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes.
  • Publication
    Métadonnées seulement
    Identification of host bloodmeal source and Borrelia burgdorferi sensu lato in field-collected Ixodes ricinus ticks in Chaumont (Switzerland)
    (2007)
    Cadenas, Francisca Moran
    ;
    Rais, Olivier 
    ;
    Humair, Pierre-François
    ;
    Douet, Véronique 
    ;
    Moret, Jacqueline
    ;
    Gern, Lise 
    To evaluate the importance of vertebrate species as tick hosts and as reservoir hosts in two endemic areas for Lyme borreliosis in Switzerland, we applied molecular methods for the analysis of bloodmeal source and Borrelia infection in questing Ixodes ricinus L. ticks. In total, 1,326 questing ticks were simultaneously analyzed for Borrelia and for blood meal remnants by using reverse line blot. An overall infection prevalence of 19.0% was recorded for Borrelia sp., with similar rates in both sites. Using a newly developed method for the analysis of bloodmeal targeting the 12S rDNA mitochondrial gene, identification of host DNA from field-collected ticks was possible in 43.6% of cases. Success of host identification at the genus and species level reached 72%. In one site, host identification success reached its maximum in spring (93% in May), decreasing in summer (20% in July) and rising in autumn (73% in October). In the other site, identification rate in ticks remained low from April to July and increased in autumn reaching 68% in October and November. The most prevalent identified host DNA was artiodactyls in both sites. Red squirrel DNA was significantly more frequently detected in ticks collected in one site, whereas insectivore DNA was more frequent in ticks in the other site. DNA from more than one vertebrate host was detected in 19.5% of nymphs and 18.9% of adults. Host DNA was identified in 48.4% of the Borrelia infected ticks. Although DNA from all Borrelia species was found in at least some ticks with DNA from mammals and some ticks with DNA from birds, our results confirm a general association of B. afzelii and B. burgdorferi sensu stricto with rodents, and B. valaisiana and B. garinii with birds.
  • Publication
    Métadonnées seulement
    Ixodes ricinus density and infection prevalence of Borrelia burgdorferi sensu lato along a north-facing altitudinal gradient in the Rhône Valley (Switzerland)
    (2007)
    Burri, Caroline
    ;
    Cadenas, Francisca Moran
    ;
    Douet, Véronique 
    ;
    Moret, Jacqueline
    ;
    Gern, Lise 
    Questing Ixodes ricinus ticks were sampled monthly along a north-facing altitudinal gradient in the canton of Valais, Switzerland, from March 2004 to February 2005. Tick density and infection with Borrelia burgdorferi sensu lato were monitored. Ticks were collected by flagging vegetation at three different altitudes (750 m, 880 m, and 1020 m above sea level). Ticks were examined for Borrelia by polymerase chain reaction (PCR) followed by reverse line blot. At the three altitudes, questing tick activity was not observed under 10 degrees C and was reduced when saturation deficit was higher than 5 mm Hg, most questing tick activity was occurred between 2 mm Hg and 7 mm Hg. Tick density and peak tick density were highest at 1020 m. High saturation deficits at the lowest altitudes appear to impair the tick population. The prevalence of B. burgdorferi infection in nymphs and adults decreased with altitude. The prevalence of infection was higher in adult ticks (47%) than in nymphs (29%). Four B. burgdorferi sensu lato genospecies were detected: B. afzelii (40%), B. garinii (22%), B. valaisiana (12%) and B. burgdorferi sensu stricto (6%). Mixed infections were detected in 13% of infected ticks.
  • Publication
    Métadonnées seulement
    Phenology of Ixodes ricinus and infection with Borrelia burgdorferi sensu lato along a North- and South-facing altitudinal gradient on Chaumont Mountain, Switzerland
    (2007)
    Cadenas, Francisca Moran
    ;
    Rais, Olivier 
    ;
    Jouda, Fatima
    ;
    Douet, Véronique 
    ;
    Humair, Pierre-François
    ;
    Moret, Jacqueline
    ;
    Gern, Lise 
    Questing Ixodes ricinus L. ticks were collected monthly from 2003 to 2005 on the north- and south-facing slopes of Chaumont Mountain in Neuchatel, Switzerland, at altitudes varying from 620 to 1,070 in. On the south-facing slope, questing tick density was higher than on the north-facing slope, and it decreased with altitude. Density tended to increase with altitude on the north-facing slope. Saturation deficit values higher than 10 mmHg and lasting for >2 mo were often recorded on the south-facing slope, explaining seasonal patterns of questing tick activity. The overall prevalence of Borrelia burgdorferi sensu lato was 22.4%, and prevalence differed according to exposure and among years. No difference was noticed between nymphs and adults. Four Borrelia species were identified. Mixed infections were detected in 52 ticks, B. garinii and B. valaisiana (n = 21) and B. afzelii and B. burgdorferi s.s. (n = 20) were the most frequent associations observed. The density of infected ticks varied from 3.6 to 78.7 infected nymphs per 1 00 m(2) and from 0.6 to 16.9 infected adults per 100 m(2), both slopes combined. The study on the south-facing slope was a follow-up of a previous study carried out at the same location during 1999-2001. Comparison of climatic data between the two periods showed a marked increase in saturation deficit. Substantial differences in density and phenology of ticks also were observed. At high elevations, ticks were significantly more abundant during the current study. This can be explained by rising temperatures recorded during summer at altitude, reaching values similar to those registered in the first study beneath. At the lowest altitude, adults were significantly less abundant, probably due to long-lasting high saturation deficits that impaired nymphal survival. The density of Borrelia-infected ticks was higher than in the previous study.
  • Publication
    Métadonnées seulement
    Molecular identification of bloodmeal source in Ixodes ricinus ticks using 12S rDNA as a genetic marker
    (2007)
    Humair, Pierre-François
    ;
    Douet, Véronique 
    ;
    Cadenas, Francisca Moran
    ;
    Schouls, Leo M
    ;
    Van de Pol, Ingrid
    ;
    Gern, Lise 
    We developed an efficient molecular method for the identification of the bloodmeal sources in the tick Ixodes ricinus (L.), the European vector of the agents of Lyme borreliosis and tick-borne encephalitis. A approximate to 145-bp orthologous fragment of the vertebrate mitochondrial 12S rDNA was used as a molecular marker to discriminate host vertebrate species. The method consists of a single run polymerase chain reaction amplification of the 12S rDNA molecular marker by using nondegenerate primers followed by a reverse line blot hybridization assay by using specific oligonucleotide probes. The palette of probes allowed us to distinguish major groups of host vertebrates (e.g., mammals, small rodents, artiodactyls, birds, lizards) and to identify the bloodmeal sources at the genus or species level. External primers were designed and used to sequence the 12S rDNA molecular marker of a broad range of known or potential host vertebrate species (n = 60), including mammal (n = 28), bird (n = 31), and reptile (n = 1) species. The use of this technique coupled with known methods for identification of tick-borne pathogens (e.g., Borrelia burgdorferi sensu lato) allowed us to determine the source of infective bloodmeal and to identify reservoir species. The present method was successfully used to identify the source of bloodmeals in all feeding I. ricinus ticks and in half of questing field-collected L ricinus ticks. Moreover, the bloodmeal source was identified in 65% of ticks infected with B. burgdorferi sensu lato. Further development of this technique may be envisaged for the detection of other vector-borne patbogens and their reservoir hosts.