Screening antivirals with a mCherry-expressing recombinant bovine respiratory syncytial virus: a proof of concept using cyclopamine

Bovine respiratory syncytial virus (BRSV) is a pathogenic pneumovirus and a major cause of acute respiratory infections in calves. Although different vaccines are available against BRSV, their efficiency remains limited, and no efficient and large-scale treatment exists. Here, we developed a new reverse genetics system for BRSV expressing the red fluorescent protein mCherry, based on a field strain isolated from a sick calf in Sweden. Although this recombinant fluorescent virus replicated slightly less efficiently compared to the wild type virus, both viruses were shown to be sensitive to the natural steroidal alkaloid cyclopamine, which was previously shown to inhibit human RSV replication. Our data thus point to the potential of this recombinant fluorescent BRSV as a powerful tool in preclinical drug discovery to enable high throughput compound screening

A DNA-Modified Live Vaccine Prime-Boost Strategy Broadens the T-Cell Response and Enhances the Antibody Response against the Porcine Reproductive and Respiratory Syndrome Virus.

The Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) induces reproductive disorders in sows and respiratory illnesses in growing pigs and is considered as one of the main pathogenic agents responsible for economic losses in the porcine industry worldwide. Modified live PRRSV vaccines (MLVs) are very effective vaccine types against homologous strains but they present only partial protection against heterologous viral variants. With the goal to induce broad and cross-protective immunity, we generated DNA vaccines encoding B and T antigens derived from a European subtype 1 strain that include T-cell epitope sequences known to be conserved across strains. These antigens were expressed either in a native form or in the form of vaccibodies targeted to the endocytic receptor XCR1 and CD11c expressed by different types of antigen-presenting cells (APCs). When delivered in skin with cationic nanoparticles and surface electroporation, multiple DNA vaccinations as a stand-alone regimen induced substantial antibody and T-cell responses, which were not promoted by targeting antigens to APCs. Interestingly, a DNA-MLV prime-boost strategy strongly enhanced the antibody response and broadened the T-cell responses over the one induced by MLV or DNA-only. The anti-nucleoprotein antibody response induced by the DNA-MLV prime-boost was clearly promoted by targeting the antigen to CD11c and XCR1, indicating a benefit of APC-targeting on the B-cell response. In conclusion, a DNA-MLV prime-boost strategy, by enhancing the potency and breadth of MLV vaccines, stands as a promising vaccine strategy to improve the control of PRRSV in infected herds

Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport

Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate‐dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments

Porcine Reproductive and Respiratory Syndrome Virus Type 1.3 Lena Triggers Conventional Dendritic Cells 1 Activation and T Helper 1 Immune Response Without Infecting Dendritic Cells

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an arterivirus responsible for highly contagious infection and huge economic losses in pig industry. Two species, PRRSV-1 and PRRSV-2 are distinguished, PRRSV-1 being more prevalent in Europe. PRRSV-1 can further be divided in subtypes. PRRSV-1.3 such as Lena are more pathogenic than PRRSV-1.1 such as Lelystad or Flanders13. PRRSV-1.3 viruses trigger a higher Th1 response than PRRSV-1.1, although the role of the cellular immune response in PRRSV clearance remains ill defined. The pathogenicity as well as the T cell response inductions may be differentially impacted according to the capacity of the virus strain to infect and/or activate DCs. However, the interactions of PRRSV with in vivo-differentiated-DC subtypes such as conventional DC1 (cDC1), cDC2, and monocyte-derived DCs (moDC) have not been thoroughly investigated. Here, DC subpopulations from Lena in vivo infected pigs were analyzed for viral genome detection. This experiment demonstrates that cDC1, cDC2, and moDC are not infected in vivo by Lena. Analysis of DC cytokines production revealed that cDC1 are clearly activated in vivo by Lena. In vitro comparison of 3 Europeans strains revealed no infection of the cDC1 and cDC2 and no or little infection of moDC with Lena, whereas the two PRRSV-1.1 strains infect none of the 3 DC subtypes. In vitro investigation of T helper polarization and cytokines production demonstrate that Lena induces a higher Th1 polarization and IFNγ secretion than FL13 and LV. Altogether, this work suggests an activation of cDC1 by Lena associated with a Th1 immune response polarization

Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus

Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning

Species-wide variation in shoot nitrate concentration, and genetic loci controlling nitrate, phosphorus and potassium accumulation in Brassica napus L

Large nitrogen, phosphorus and potassium fertiliser inputs are used in many crop systems. Identifying genetic loci controlling nutrient accumulation may be useful in crop breeding strategies to increase fertiliser use efficiency and reduce financial and environmental costs. Here, variation in leaf nitrate concentration across a diversity population of 383 genotypes of Brassica napus was characterised. Genetic loci controlling variation leaf nitrate, phosphorus and potassium concentrations, were identified through Associative Transcriptomics using single nucleotide polymorphism (SNP) markers and gene expression markers (GEMs). Leaf nitrate concentration varied over 8-fold across the diversity population. A total of 455 SNP markers were associated with leaf nitrate concentration after false-discovery-rate (FDR) correction. In linkage disequilibrium of highly associated markers are a number of known nitrate transporters and sensors, including a gene thought to mediate expression of the major nitrate transporter NRT1.1. Several genes influencing root and root-hair development co-localise with chromosomal regions associated with leaf P concentration. Orthologues of three ABC-transporters involved in suberin synthesis in roots also co-localise with association peaks for both leaf nitrate and phosphorus. Allelic variation at nearby, highly associated SNPs confers large variation in leaf nitrate and phosphorus concentration. A total of five GEMs associate with leaf K concentration after FDR correction including a GEM that corresponds to an auxin-response family protein. Candidate loci, genes and favourable alleles identified here may prove useful in marker-assisted selection strategies to improve fertiliser use efficiency in B. napus

NRT1.1-dependent nitrate signaling pathways in Arabidopsis thaliana.

Les plantes sont capables de percevoir dans leur environnement la disponibilité en nitrate (NO3-), un macro-nutriment essentiel. Chez Arabidopsis thaliana, le transporteur de NO3- NRT1.1 constitue un système de perception qui active de nombreuses réponses au NO3-, notamment la régulation de l'expression de gènes et le développement des racines latérales. Dans ce dernier cas, un mécanisme de transduction du signal a été proposé. Celui-ci met en jeu une activité de transport d'auxine par NRT1.1 qui est inhibée par le NO3-. Cependant, le(s) mécanisme(s) moléculaire(s) permettant à NRT1.1 de contrôler un large panel de réponses au NO3- reste(nt) largement inconnu(s). L'objectif de ce travail était donc d'approfondir nos connaissances sur les voies de signalisation du NO3- dépendantes de NRT1.1. Grâce à l'analyse de mutants et de lignées transgéniques exprimant des versions de NRT1.1 présentant des mutations ponctuelles, nous avons pu découpler certaines des réponses NRT1.1-dépendantes et montré que cette protéine peut percevoir/transduire le signal NO3- au travers d'au moins trois mécanismes distincts, possédant des bases structurales différentes au sein de la protéine. D'autre part, ce travail a permis de valider l'hypothèse selon laquelle NRT1.1, en intervenant comme transporteur d'auxine, contrôle directement le développement des racines latérales, et ce indépendamment des autres transporteurs d'auxine qui y sont exprimés. Enfin, nous avons montré qu'en plus de sa régulation transcriptionnelle déjà connue, NRT1.1 est soumis à une puissante et complexe régulation post-transcriptionnelle. En effet, le transcrit NRT1.1 est stabilisé en présence de NO3- dans la racine alors que l'accumulation de la protéine NRT1.1 est réprimée par le NO3- spécifiquement au niveau des primordia de racines latérales. Les résultats obtenus au cours de ce travail ont permis d'élaborer un modèle cohérent du rôle de signalisation joué par NRT1.1, et ouvrent de nombreuses perspectives pour comprendre comment, chez les plantes, un « transcepteur » (transporteur/senseur) peut contrôler une vaste gamme de réponses adaptatives aux facteurs de l'environnement.Plants are able to sense the external availability of nitrate (NO3-), a major macro-nutrient. In Arabidopsis thaliana, the NO3- transporter NRT1.1 acts as a sensor that triggers many different adaptive responses, including the regulation of gene expression and lateral root development. In the latter case, a transduction mechanism that involves a NO3--inhibited auxin transport activity dependent of NRT1 has been proposed. However, the molecular mechanism(s) allowing NRT1.1 to control such a large palette of NO3- responses is still largely unknown. Thus the aim of this work was to better understand and characterize the NRT1.1-dependent NO3- signaling pathway(s). Using mutants and transgenic lines expressing point mutated forms of NRT1.1, we uncoupled several of the NRT1.1-dependent responses and thus demonstrated that NR1.1 can sense/transduce NO3- signal through at least three distinct mechanisms at the protein level. This work also largely confirmed the hypothesis that NRT1.1 directly controls lateral root development through its auxin transport activity regardless of the other auxin transporters expressed in lateral root primordia. Finally, we showed that, besides the already well characterized transcriptional NO3--dependent regulation of NRT1.1, this gene is also subjected to complex post-transcriptional regulations. Indeed, on the one hand, NRT1.1 mRNA is stabilized by NO3- in roots whereas, on the other hand, protein accumulation is specifically repressed by NO3- in lateral root primordia. Altogether, these results allowed us to build a comprehensive model of the complex NRT1.1 signaling and open many perspectives to understand how plant “transceptors” (transporter/sensor) can monitor a large variety of adaptive responses to environmental factors

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Nitrate sensing and signaling in plants.

International audienceNitrate (NO(3)(-)) is a major nutrient for plants, taken up by their roots from the soil. Plants are able to sense NO(3)(-) in their environment, allowing them to quickly respond to the dramatic fluctuations of its availability. Significant advances have been made during the recent period concerning the molecular mechanisms of NO(3)(-) sensing and signaling in the model plant Arabidopsis thaliana. The striking action of NO(3)(-) as a signal regulating genome expression has been unraveled. Note worthily, NO(3)(-) sensing systems have been identified. These correspond to membrane transporters also ensuring the uptake of NO(3)(-) into root cells, thus generalizing the nutrient 'transceptor' (transporter/receptor) concept defined in yeast. Furthermore, components of the downstream transduction cascades, such as transcription factors or kinases, have also been isolated. A breakthrough arising from this improved knowledge is a better understanding of the integration of NO(3)(-) and hormone signaling pathways, that explains the extraordinary developmental plasticity of plants in response to NO(3)(-)