Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment

Flow cytometry has become the method of choice to measure the DNA content (genome size) in plants. Ease of sample preparation, fast acquisition, and accurate measurements have made the method popular in the domains of plant cell biology, systematics, evolution, genetics and biotechnology. Although the cell wall is a problem when isolating plant cells, cytometry remains a powerful tool in plant sciences. Based on our 30-years’ experience in this field, this review will focus at first on genome size measurement using simply isolated nuclei: the good practice for acquisition, nuclei isolation, appropriate buffers, kind of tissues to use. The second part will briefly review what kind of measurements it is possible to make in plant cytometry, and for what purpose: base composition, ploidy level, cell cycle, endoreplication, seed screening, and nuclei/chromosomes sorting. We will address troubleshooting. The commonly-used mathematic tools will be discussed

Deciphering the genome structure and paleohistory of _Theobroma cacao_

We sequenced and assembled the genome of _Theobroma cacao_, an economically important tropical fruit tree crop that is the source of chocolate. The assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of them anchored on the 10 _T. cacao_ chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example flavonoid-related genes. It also provides a major source of candidate genes for _T. cacao_ disease resistance and quality improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten _T. cacao_ chromosomes were shaped from an ancestor through eleven chromosome fusions. The _T. cacao_ genome can be considered as a simple living relic of higher plant evolution

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

Applications en biologie végétale : contraintes, succès, espoirs

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Understanding the interplay between surface properties and the aspect ratio of ellipsoidal nanomaterials

International audiencePolymeric nanomaterials (NM) with nonspherical morphologies have increasingly captured the spotlight in biomedical applications. Physical deformation of nanospheres (NS) represents a versatile technique for obtaining ellipsoidal NM (E-NM) that exhibit unique properties regarding their interactions with biological systems. Despite this technique's versatility and simplicity, the film's detailed composition and the parameters controlling the efficiency of the NS’s physical deformation still need to be clarified. Herein, we detailed a step-by-step formulation of a film composed of polyvinyl alcohol (PVA), and we investigated the impact of NS surface properties on the efficiency of the physical stretching. To reach those objectives, we designed two morphologies (NS and E-NM), and for each morphology, the surface composition was modified by using three different polysaccharides (chitosan, dextran, and hyaluronan). Physicochemical analysis revealed a connection between NS surface potential, their interaction with PVA, and the aspect ratio of ellipsoidal NM. Chitosan-coated E-NM had the highest aspect ratio ( = 4.9), which decreased to 2.4 with dextran and hyaluronan coatings. Isothermal titration calorimetry demonstrated strong interaction between PVA and chitosan but little interaction with dextran and hyaluronan. Finally, the impact of NM morphology and surface properties on phagocytosis showed that E-NM exhibited a reduced internalization by macrophages and reduced cytotoxicity compared to NS. Overall, in this study, we highlighted that the aspect ratio of E-NM is controlled by the strength of the interaction with PVA with the polysaccharide on the NS surface and revealed the role of NM morphology in reducing cytotoxicity and escaping internalization by macrophages

Vanilla: a challenging genus with regards to the development of genomic resources

18International audienceThe development of enabling genomic resources for Vanilla would dramatically advance international efforts to improve the genetic foundation of this important global commodity for vanillagrowing countries worldwide. Currently, global production of Vanilla planifolia rests on a precarious genetic foundation that lacks natural resistance to disease-causing pathogens (such as Fusarium) and environmental fluctuations. This low genetic diversity of cultivated vanilla leaves it highly vulnerable to disease, climatic change, and other environmental stresses, placing the entire vanilla industry potentially at risk. However, Vanilla is a challenging genus with regards to development of genetic resources: not only do Vanilla species have large genomes (approx. Cx = 2.5 Gb), but they are also characterized, like some orchids from various sections, by strict partial endoreplication (SPE) cycles, unknown in any other plant family. We will present how we took these major constraints into account in the definition of genome sequencing strategies for this genu