A simple and efficient method for the long-term preservation of plant cell suspension cultures

<p>Abstract</p> <p>Background</p> <p>The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority.</p> <p>Results</p> <p>Sycamore (<it>Acer pseudoplatanus</it>) and Arabidopsis cell were preserved over six months as suspensions cultures in a phosphate-free nutrient medium at 5°C. The cell recovery monitored via gas exchange measurements and metabolic profiling using <it>in vitro </it>and <it>in vivo </it>¹³C- and ³¹P-NMR took a couple of hours, and cell growth restarted without appreciable delay. No measurable cell death was observed.</p> <p>Conclusion</p> <p>We provide a simple method to preserve physiologically homogenous plant cell cultures without subculture over several months. The protocol based on the blockage of cell growth and low culture temperature is robust for heterotrophic and semi-autotrophic cells and should be adjustable to cell lines other than those utilised in this study. It requires no specialized equipment and is suitable for routine laboratory use.</p

Metabolic processes sustaining the reviviscence of lichen Xanthoria elegans (Link) in high mountain environments.

International audienceTo survive in high mountain environments lichens must adapt themselves to alternating periods of desiccation and hydration. Respiration and photosynthesis of the foliaceous lichen, Xanthoria elegans, in the dehydrated state were below the threshold of CO2-detection by infrared gas analysis. Following hydration, respiration totally recovered within seconds and photosynthesis within minutes. In order to identify metabolic processes that may contribute to the quick and efficient reactivation of lichen physiological processes, we analysed the metabolite profile of lichen thalli step by step during hydration/dehydration cycles, using 31P- and 13C-NMR. It appeared that the recovery of respiration was prepared during dehydration by the accumulation of a reserve of gluconate 6-P (glcn-6-P) and by the preservation of nucleotide pools, whereas glycolytic and photosynthetic intermediates like glucose 6-P and ribulose 1,5-diphosphate were absent. The large pools of polyols present in both X. elegans photo- and mycobiont are likely to contribute to the protection of cell constituents like nucleotides, proteins, and membrane lipids, and to preserve the integrity of intracellular structures during desiccation. Our data indicate that glcn-6-P accumulated due to activation of the oxidative pentose phosphate pathway, in response to a need for reducing power (NADPH) during the dehydration-triggered down-regulation of cell metabolism. On the contrary, glcn-6-P was metabolised immediately after hydration, supplying respiration with substrates during the replenishment of pools of glycolytic and photosynthetic intermediates. Finally, the high net photosynthetic activity of wet X. elegans thalli at low temperature may help this alpine lichen to take advantage of brief hydration opportunities such as ice melting, thus favouring its growth in harsh high mountain climates

Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by Sclerotinia sclerotiorum.

International audienceInteractions between the necrotrophic fungus Sclerotinia sclerotiorum and one of its hosts, Helianthus annuus L., were analyzed during fungal colonization of plant tissues. Metabolomic analysis, based on (13)C- and (31)P-NMR spectroscopy, was used to draw up the profiles of soluble metabolites of the two partners before interaction, and to trace the fate of metabolites specific of each partner during colonization. In sunflower cotyledons, the main soluble carbohydrates were glucose, fructose, sucrose and glutamate. In S. sclerotiorum extracts, glucose, trehalose and mannitol were the predominant soluble carbon stores. During infection, a decline in sugars and amino acids was observed in the plant and fungus total content. Sucrose and fructose, initially present almost exclusively in plant, were reduced by 85%. We used a biochemical approach to correlate the disappearance of sucrose with the expression and the activity of fungal invertase. The expression of two hexose transporters, Sshxt1 and Sshxt2, was enhanced during infection. A database search for hexose transporters homologues in the S. sclerotiorum genome revealed a multigenic sugar transport system. Furthermore, the composition of the pool of reserve sugars and polyols during infection was investigated. Whereas mannitol was produced in vitro and accumulated in planta, glycerol was exclusively produced in infected tissues and increased during colonization. The hypothesis that the induction of glycerol synthesis in S. sclerotiorum exerts a positive effect on osmotic protection of fungal cells and favors fungal growth in plant tissues is discussed. Taken together, our data revealed the importance of carbon-nutrient exchanges during the necrotrophic pathogenesis of S. sclerotiorum

A Survey of the Humoral Immune Response of Cancer Patients to a Panel of Human Tumor Antigens

Evidence is growing for both humoral and cellular immune recognition of human tumor antigens. Antibodies with specificity for antigens initially recognized by cytotoxic T lymphocytes (CTLs), e.g., MAGE and tyrosinase, have been detected in melanoma patient sera, and CTLs with specificity for NY-ESO-1, a cancer-testis (CT) antigen initially identified by autologous antibody, have recently been identified. To establish a screening system for the humoral response to autoimmunogenic tumor antigens, an enzyme-linked immunosorbent assay (ELISA) was developed using recombinant NY-ESO-1, MAGE-1, MAGE-3, SSX2, Melan-A, and tyrosinase proteins. A survey of sera from 234 cancer patients showed antibodies to NY-ESO-1 in 19 patients, to MAGE-1 in 3, to MAGE-3 in 2, and to SSX2 in 1 patient. No reactivity to these antigens was found in sera from 70 normal individuals. The frequency of NY-ESO-1 antibody was 9.4% in melanoma patients and 12.5% in ovarian cancer patients. Comparison of tumor NY-ESO-1 phenotype and NY-ESO-1 antibody response in 62 stage IV melanoma patients showed that all patients with NY-ESO-1+ antibody had NY-ESO-1+ tumors, and no patients with NY-ESO-1− tumors had NY-ESO-1 antibody. As the proportion of melanomas expressing NY-ESO-1 is 20–40% and only patients with NY-ESO-1+ tumors have antibody, this would suggest that a high percentage of patients with NY-ESO-1+ tumors develop an antibody response to NY-ESO-1

Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. 15NH4 labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized 15N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while 15N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt

Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea–specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.Fil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Amselem, Joelle. Institut National de la Recherche Agronomique; FranciaFil: Cuomo, Christina A.. Broad Institute of MIT and Harvard; Estados UnidosFil: Jan, A. L. van Kan. Wageningen University; Países BajosFil: Viaud, Muriel. Institut National de la Recherche Agronomique; FranciaFil: Benito, Ernesto P.. Universidad de Salamanca; EspañaFil: Couloux, Arnaud. Centre National de Séquençage. Genoscope; FranciaFil: Coutinho, Pedro M.. Centre National de la Recherche Scientifique; FranciaFil: Vries, Ronald P. de. Microbiology and Kluyver Centre for Genomics of Industrial Fermentations; Países Bajos. Fungal Biodiversity Centre; Países BajosFil: Dyer, Paul S.. The University Of Nottingham; Reino UnidoFil: Fillinger, Sabine. Institut National de la Recherche Agronomique; FranciaFil: Fournier, Elisabeth. Institut National de la Recherche Agronomique; Francia. Centre de coopération internationale en recherche agronomique pour le développement; FranciaFil: Gout, Lilian. Institut National de la Recherche Agronomique; FranciaFil: Hahn, Matthias. University Of Kaiserlautern; AlemaniaFil: Kohn, Linda. University Of Toronto; CanadáFil: Lapalu, Nicolas. Institut National de la Recherche Agronomique; FranciaFil: Plummer, Kim M.. la Trobe University; AustraliaFil: Pradier, Jean-Marc. Institut National de la Recherche Agronomique; FranciaFil: Quévillon, Emmanuel. Institut National de la Recherche Agronomique; Francia. Centre National de la Recherche Scientifique; FranciaFil: Sharon, Amir. Tel Aviv University. Department of Molecular Biology and Ecology of Plants; IsraelFil: Simon, Adeline. Institut National de la Recherche Agronomique; FranciaFil: Tudzynski, Bettina. Institut für Biologie und Biotechnologie der Pflanzen; AlemaniaFil: Tudzynski, Paul. Institut für Biologie und Biotechnologie der Pflanzen; AlemaniaFil: Wincker, Patrick. Centre National de Séquençage. Genoscope; FranciaFil: Andrew, Marion. University Of Toronto; CanadáFil: Anthouard, Véronique. Centre National de Séquençage. Genoscope; FranciaFil: Beever, Ross E.. Landcare Research; Nueva ZelandaFil: Beffa, Rolland. Centre National de la Recherche Scientifique; FranciaFil: Benoit, Isabelle . Microbiology and Kluyver Centre for Genomics of Industrial Fermentations; Países BajosFil: Bouzid, Ourdia. Microbiology and Kluyver Centre for Genomics of Industrial Fermentations; Países Bajo

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

DMTs and Covid-19 severity in MS: a pooled analysis from Italy and France

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR&nbsp;=&nbsp;2.05, 95%CI&nbsp;=&nbsp;1.39–3.02, p&nbsp;&lt;&nbsp;0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR&nbsp;=&nbsp;0.42, 95%CI&nbsp;=&nbsp;0.18–0.99, p&nbsp;=&nbsp;0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon