The Poplar-Poplar Rust Interaction: Insights from Genomics and Transcriptomics

Poplars are extensively cultivated worldwide, and their susceptibility to the leaf rust fungus Melampsora larici-populina leads to considerable damages in plantations. Despite a good knowledge of the poplar rust life cycle, and particularly the epidemics on poplar, the perennial status of the plant host and the obligate biotrophic lifestyle of the rust fungus are bottlenecks for molecular investigations. Following the completion of both M. larici-populina and Populus trichocarpa genome sequences, gene families involved in poplar resistance or in rust fungus virulence were investigated, allowing the identification of key genetic determinants likely controlling the outcome of the interaction. Specific expansions of resistance and defense-related genes in poplar indicate probable innovations in perennial species in relation with host-pathogen interactions. The genome of M. Larici-populina contains a strikingly high number of genes encoding small secreted proteins (SSPs) representing hundreds of candidate effectors. Transcriptome analyses of interacting partners in compatible and incompatible interactions revealed conserved set of genes involved in poplar defense reactions as well as timely regulated expression of SSP transcripts during host tissues colonisation. Ongoing functional studies of selected candidate effectors will be achieved mainly on the basis of recombinant protein purification and subsequent characterisation

Crystal Structure of Saccharomyces cerevisiae ECM4, a Xi-Class Glutathione Transferase that Reacts with Glutathionyl-(hydro)quinones

International audienceGlutathionyl-hydroquinone reductases (GHRs) belong to the recently characterized Xi-class of glutathione transferases (GSTXs) according to unique structural properties and are present in all but animal kingdoms. The GHR ScECM4 from the yeast Saccharomyces cerevisiae has been studied since 1997 when it was found to be potentially involved in cell-wall biosyn-thesis. Up to now and in spite of biological studies made on this enzyme, its physiological role remains challenging. The work here reports its crystallographic study. In addition to exhibiting the general GSTX structural features, ScECM4 shows extensions including a huge loop which contributes to the quaternary assembly. These structural extensions are probably specific to Saccharomycetaceae. Soaking of ScECM4 crystals with GS-menadione results in a structure where glutathione forms a mixed disulfide bond with the cysteine 46. Solution studies confirm that ScECM4 has reductase activity for GS-menadione in presence of glutathione. Moreover, the high resolution structures allowed us to propose new roles of conserved residues of the active site to assist the cysteine 46 during the catalytic act

Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance

Yeast Qri7 and human OSGEPL are members of the orthologous Kae1(OSGEP)/YgjD protein family, the last class of universally conserved proteins without assigned function. Phylogenetic analyses indicate that the eukaryotic Qri7(OSGEPL) proteins originated from bacterial YgjD proteins. We have recently shown that the archaeal Kae1 protein is a DNA-binding protein that exhibits apurinic endonuclease activity in vitro. We show here that the Qri7/OSGEPL proteins localize in mitochondria and are involved in mitochondrial genome maintenance in two model eukaryotic organisms, Saccharomyces cerevisiae and Caenorhabditis elegans. Furthermore, S. cerevisiae Qri7 complements the loss of the bacterial YgjD protein in Escherichia coli, suggesting that Qri7/OSGEPL and YgjD proteins have retained similar functions in modern organisms. We suggest to name members of the Kae1(OSGEP)/YgjD family UGMP, for Universal Genome Maintenance Proteins

Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A

Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability

The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Production, fonction et localisation d'Orchestine: calciprotéine spécifique de la matrice organique des structures minéralisées élaborées par le crustacé terrestre Orchestia cavimana

Rapporteurs: Pr. Jean-Louis SAFFAR, Université Paris V ; Dr. Jean-Yves SIRE DR CNRS - Paris VII ; Examinateurs: Dr. Jean DELACHAMBRE, DR CNRS - Dijon ; Dr. Frédéric MARIN, IsoTis - Bilthoven (Pays-Bas) ; Pr. Alain PUGIN, Université de Bourgogne ; Directeur de thèse: Dr. Gilles LUQUET, Université de BourgogneAs most Crustaceans, Orchestia cavimana possesses a mineralized exoskeleton which is periodically replaced. Because of the terrestrial behaviours of this animal, this molting cycle is related to calcium storage and resorption processes. Calcium storage occurs, as calcareous concretions, in diverticula of the midgut called posterior cæca. Calcareous concretions are essentially composed of amorphous calcium carbonate precipitated within a proteinaceous organic matrix composed of a soluble fraction and an insoluble one in an EDTA-buffer. Among the soluble components of the organic matrix, a previous study led to characterize a polypeptide of 23 kDa in SDS-PAGE called Orchestin. This protein, whose gene has been cloned and sequenced, is unglycosylated and binds calcium. The aim of this work was to further characterize this protein marker. The results obtained demonstrated that Orchestin is phophorylated on serine and threonine residues. In order to study the relation between these phosphorylations and the calcium-binding ability of this protein, we produced a recombinant protein devoid of post-translational modification. The comparison of the calcium-binding ability of the native, dephosphorylated (by specific phosphatases) and recombinant proteins led us to show that phosphorylations on the serine residues are of great importance in this ability. Moreover, Orchestin interacts with calcite crystal growth in an in vitro precipitation experiment. On the other hand, the recombinant protein permitted us to explain the discrepancy of molecular masses observed between the native (23 kDa) and sequence deduced (12,4 kDa) proteins. We could thus conclude to the correspondence between the gene orchestin and the protein Orchestin. Finally the recombinant protein was used to produce antibodies in order to locate Orchestin in the biomineralized structures elaborated by O. cavimana during its molting cycle. Orchestin is not only located in the non-mineralized layers of the calcareous concretions (storage structures) but also in the same layers of the calcified spherules (mineralized structures elaborated after ecdysis to resorb the stored calcium). The physical-chemical features exhibited by Orchestin strongly suggest that this calcium-binding phosphoprotein is a key-molecule in the formation of the storage and reabsorption mineralized structures cyclically elaborated by O. cavimana.Comme la plupart des Crustacés, Orchestia cavimana possède un exosquelette minéralisé qu'il renouvelle cycliquement. Du fait des mœurs terrestres de cet animal, ces cycles de mue sont associés à des processus de stockage et de résorption de calcium. Le stockage a lieu sous forme de concrétions calcaires au niveau de diverticules de l'intestin moyen appelés cæcums postérieurs. Les concrétions calcaires sont essentiellement constituées de carbonate de calcium amorphe précipité au sein d'une matrice organique comprenant une fraction protéique soluble et une autre insoluble dans un tampon contenant de l'EDTA. Des résultats précédents ont permis de mettre en évidence, parmi les constituants de la fraction soluble, une protéine acide de masse apparente en SDS-PAGE de 23 kDa qui a été appelée Orchestine. Cette protéine, dont le gène a été cloné et séquencé, n'est pas glycosylée et fixe le calcium. Le but de ce travail a été de poursuivre la caractérisation de ce marqueur protéique. Pour ce faire, nous avons montré qu'Orchestine est phosphorylée sur des résidus sérine et tyrosine. Afin d'étudier les relations entre ces phosphorylations et l'aptitude de la protéine à fixer le calcium, nous avons produit une protéine recombinante dépourvue de toute modification post-traductionnelle. La comparaison de l'aptitude à fixer le calcium des protéines native, native déphosphorylée par diverses phosphatases spécifiques, et recombinante nous a permis de conclure à l'importance fondamentale des sérines dans cette aptitude. De plus, Orchestine interfère dans la croissance in vitro de cristaux de carbonate de calcium. D'autre part, la protéine recombinante nous a permis de lever l'ambiguïté de la divergence de masse moléculaire de la protéine observée en SDS-PAGE (de 23 kDa) et de celle déduite de la séquence (de 12,4 kDa) et de conclure à la correspondance gène orchestine-protéine Orchestine. Enfin la protéine recombinante a été utilisée pour la production d'un anticorps polyclonal afin de localiser Orchestine dans les structures biominéralisées élaborées par O. cavimana lors de son cycle de mue. Orchestine semble non seulement localisée dans les couches non minéralisées des concrétions calcaires (structures de réserve du calcium) mais aussi dans celles des sphérules calciques (structures permettant la remobilisation du calcium). Les propriétés ainsi mises en évidence nous conduisent à envisager qu'Orchestine est une molécule-clé dans la formation des structures de stockage et déstockage élaborées de manière cyclique par O. cavimana

Production, fonction et localisation d'Orchestine (calciprotéine spécifique de la matrice organique des structures minéralisées élaborées par le crustacé terrestre Orchestia cavimana)

Le crustacé Orchestia cavimana, du fait de ses mœurs terrestres, stocke, sous forme de concrétions calcaires, du calcium nécessaire au durcissement de sa nouvelle cuticule dans des organes appelés cæcums postérieurs. Orchestine, protéine extraite de ces concrétions, est une protéine acide non glycosylée liant le calcium. Cette protéine interfère dans la croissance in vitro de cristaux de CaCO3 et, est phosphorylée sur des résidus sérine et tyrosine. La comparaison de l'aptitude à fixer le calcium des protéines native, native déphosphorylée, et recombinante a mis en évidence le rôle fondamental des sérines dans cette aptitude. A l'aide d'un anticorps spécifique, Orchestine a été localisée dans les couches non minéralisées des concrétions (structure de réserve du Ca2+) mais aussi dans celles des sphérules (structures de remobilisation du Ca2+) ce qui suggère qu'Orchestine serait une molécule-clé des structures minéralisées de stockage et déstockage élaborées cycliquement par ce crustacé.The crustacean Orchestia cavimana, because of its terrestrial behaviours, stores calcium, as calcareous concretions, in organs called posterior cæca in order to harden its new cuticule. Orchestin, extract from this concretions, is an acidic unglycosylated protein which binds calcium. This protein interacts with calcite crystal growth in an in vitro precipitation experiment and, is phophorylated on serine and threonine residues. The comparison of the calcium-binding ability of the native, dephosphorylated and recombinant proteins show that phosphorylations on the serine residues are fundamental for this ability. By using a specific antibody, Orchestin was localised not only in the non-mineralized layers of the calcareous concretions (Ca2+ storage structures) but also in the same layers of the calcified spherules (Ca2+ resorption structures) suggesting that Orchestin is a key-molecule of the storage and reabsorption mineralized structures elaborated cyclically by this crustacean.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF