,
, Superfrost diagnostic slides 10-wells 8 mm (Zuzi, ref: 30503101)
,
, Stock solution paraformaldehyde (PFA) 16% (EMS, ref: 15710). Storage at -20°C
, Phosphate-buffered saline (PBS) 0.01 M : NaCl 137 mM, KCl 47 mM, KH2PO4 1.5 mM and Na2HPO4, p.2
, Bovine serum albumine (BSA
, AURION, ref: 900.011) for blocking non-specific binding sites. Storage at +4°C
, SIGMA, ref: P1379), vol.20
, Primary monoclonal antibody (PlantProbes, www.plantprobes.net/). Storage at +4°C
, A wet chamber for maintaining air humidity (Fig. 1., see Note 2)
, Secondary antibody: Anti-rat IgG coupled with FITC (Fluorescein isothiocyanate; SIGMA, ref: F6258). Storage at +4°C
, Ultrathin tweezers
, Microscope slides 18-wells-Poly-L-Lysine sterile (IBIDI, ref: 81824)
,
, Stock solution paraformaldehyde (PFA) 16% (EMS, ref: 15710). Storage in -20°C
, PIPES (Alfa Aesar, ref: A16090)
,
, Phosphate-buffered saline (PBS) 0.01 M : NaCl 137 mM, KCl 47 mM, KH2PO4 1.5 mM and Na2HPO4, p.2
, Bovine serum albumine (BSA
, AURION, ref: 900.011) for blocking non-specific binding sites. Storage at +4°C
, Primary monoclonal antibody (PlantProbes www.plantprobes.net/). Storage at +4°C
, A wet chamber for maintaining air humidity (Fig. 1.,see Note 2)
, Secondary antibody: Anti-rat IgG coupled with TRITC (Tetramethylrhodamine; SIGMA, ref: T5778). Storage at -20°C
, Citifluor (Agar scientific, ref: AF2 R1320)
, Extraction buffer: 50 mM Tris-base (adjust pH 8 with HCl), 10 mM Na2EDTA(H2O)2, 2 mM Na2S2O5, 1% Triton X-100, vol.15
, Radial gel diffusion :AGPs detection / semi-quantification
, Radial gel diffusion : 1% (w/v) agarose gel containing 0.15 M NaCl, 0.02% (w/v) NaNO3, 10 µg/mL ?-D-GlcY
, Gum Arabic (Gum Acacia, Fisher scientific
, AGPs quantification 1. Rocket gel : 1% (w/v) agarose gel containing 25 mM Tris-base
, Gel tray 18x15 cm (see Note 17)
,
, Gum Arabic (Gum Acacia, Fisher scientific, ref: G/1050/53)
, 1% (w/v) NaCl
, Running buffer 1: 25 mM Tris-base (adjust pH 8.3 with HCl), 200 mM glycine (see Note 16)
, Isoelectric focusing electrophoresis system (IEF)
, Agarose gel electrophoresis: detection of AGP sub-populations
, Agarose gel : 1% (w/v) agarose gel containing 90 mM Tris-base (adjust pH 8.3 with HCl), 90 mM boric acid, vol.2
, Gel tray 18x15 cm and the comb (see Note 17)
,
, Agarose gel electrophoresis system
, 8 2D electrophoresis: characterization of AGP sub-populations and quantification
, Agarose gel : 1% (w/v) agarose gel containing 90 mM Tris-base (adjust pH 8.3 with HCl), 90 mM boric acid, vol.2
, Gel tray 18x15 cm and the comb (see Note 17)
,
, Loading buffer: 32% (v/v) glycerol, 2% (w/v) Bromophenol blue
,
, Agarose gel electrophoresis system
, Rocket gel : 1% (w/v) agarose gel containing 25 mM Tris-base (adjust pH 8.3 with HCl), 200 mM glycine, 20 µg/mL ?-D-GlcY
, Running buffer 1: 25 mM Tris-base
, Isoelectric focusing electrophoresis system (IEF)
, place them onto a 10 wells superfrost diagnostic slide (Fig. 2B.) and fix them by adding 20 µL of 4% PFA (Paraformaldehyde) (Fig. 2C.) for 30 to 40 minutes at room temperature (RT). The liquid is removed gently by placing, Cut root tips using a scalpel (Fig. 2A.)
, Wash briefly at RT with PBS 1X (see Note 5)
, Incubate for 30 to 45 minutes at RT with 3% (w/v) BSA diluted in PBS 1X (see Note 6)
, RT with PBS 1X and then with PBS 1X + 0.1% (v/v) Tween, vol.20
, Incubate overnight (O/N) with primary antibody at +4°C in a wet chamber. Primary antibody is diluted at 1:5 with PBST (see Note 8)
, Wash twice briefly at RT with PBST
, Incubate 2 hours at RT in darkness with the secondary antibody diluted at 1:30 in PBST, in a wet chamber. The second antibody is an Anti-rat IgG coupled with FITC (see Notes, vol.9
, Observe with a confocal laser-scanning microscope (Excitation : 488 nm ; Emission : 500-535 nm). Results are shown in Fig, vol.3
, Pisum sativum) The immunolabeling of "thick" root tips protocol provides a good preservation of BCs and mucilage despite several washing steps (see Note 11), This method has been adapted from
, place it onto a sterile 18-wells microscope slide Poly-L-Lysine (Fig. 4B.), and fix with 20 µL of 4% PFA diluted in 50 mM PIPES, pH 7 and 1 mM CaCl2 (Fig. 4C.) for 40 minutes at RT. The liquid is gently removed by placing
, Wash 10 minutes 4 times at RT with PBS 1X containing 1% (w/v) BSA (see Notes, vol.5
, N with the primary antibody at +4°C in a wet chamber
, Incubate 2 hours at +25°C in darkness with the secondary antibody diluted at 1:50 in PBS 1X containing 1% (w/v) BSA in a wet chamber. The second antibody is an Antirat IgG coupled with TRITC (see Notes, vol.9
, Finally wash at RT with PBS 1X for 10 minutes
, Observe with a confocal laser-scanning microscope (Excitation : 550 nm ; Emission : 560-600 nm). Results are shown in Fig, vol.5
, Grind 10 g of plant material with liquid nitrogen using a mortar and a pestle
, Freeze the ground material then freeze-dry it (see Note 19)
, Mix 1 g of the freeze-dried material with 40 mL of extraction buffer in 50 mL corning tube and incubate at +4?C O/N under agitation
, Centrifuge for 10 minutes at +4?C, 14000.
, Centrifuge for 10 minutes at +4?C, 14000.
, Carefully remove the supernatant and suspend the pellet in 40 mL of 50 mM Tris-base
, Centrifuge for 10 minutes at +4?C, 14000.
, Collect the supernatant and resuspend the pellet in 20 mL of 50 mM Tris-base previously adjusted to pH 8 with HCl
, Centrifuge for 10 minutes at +4?C, 14000.
, After dialysis, freeze and freeze-dry the solution
, their structural heterogeneity, binding of AGPs to ?-D-GlcY can vary depending on the sub-populations present in the sample, and therefore impacts purification quality
, Precipitate the AGPs in 2 mL Eppendorf tube by adding an equal volume of 2 mg/mL ?-D-GlcY to the solubilized AGPs solution (see Note 18)
, Incubate at +4?C for 48 hours
, Centrifuge for 90 minutes at RT, 10000×g
, Recover the pellet containing ?-D-GlcY /AGPs complex
, Add 1 mL of 1% (w/v) NaCl to the pellet and vortex
, Centrifuge for 10 minutes at RT, 10000×g
, Centrifuge for 10 minutes at RT, 10000×g
, Dissolve the pellet by adding 500 µL of pure DMSO and vortex (see Note 23)
, Add 10% to 30% (w/v) sodium hydrosulfite and vortex. Then, add ultra-pure water until the color becomes clear yellow (see Note 24)
, Desalt the clear yellow solution with a size exclusion chromatography (PD-10 desalting columns)
, Arabinogalactan-proteins: structure, biosynthesis, and function, Annual Review of Plant Physiology, vol.34, pp.47-70, 1983.
, Arabinogalactan-proteins from Nicotiana alata and Pyrus communis contain glycosylphosphatidylinositol membrane anchors, Proceedings of the National Academy of Sciences, vol.95, pp.7921-7926, 1998.
, Presence of a glycosylphosphatidylinositol lipid anchor on rose arabinogalactan proteins, Journal of biological chemistry, vol.274, pp.14724-14733, 1999.
, Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function, Frontiers in Plant Science, vol.5, p.499, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01842174
, Arabinogalactan-proteins: structure, expression and function, Cellular and Molecular Life Sciences CMLS, vol.58, pp.1399-1417, 2001.
, The Biology of Arabinogalactan Proteins, Annual Review of Plant Biology, vol.58, pp.137-161, 2007.
, Arabinogalactan-Proteins: Key Regulators at the Cell Surface?, Plant Physiology, vol.153, pp.403-419, 2010.
, Root Border-Like Cells of Arabidopsis. Microscopical Characterization and Role in the Interaction with Rhizobacteria, Plant Physiology, vol.138, pp.998-1008, 2005.
, Effect of Arabinogalactan Proteins from the Root Caps of Pea and Brassica napus on Aphanomyces euteiches Zoospore Chemotaxis and Germination, Plant Physiology, vol.159, pp.1658-1670, 2012.
URL : https://hal.archives-ouvertes.fr/hal-01848269
, Arabinogalactan proteins in root-microbe interactions, Trends in Plant Science, vol.18, pp.440-449, 2013.
URL : https://hal.archives-ouvertes.fr/hal-01843944
, Root border cells and secretions as critical elements in plant host defense, Current Opinion in Plant Biology, vol.16, pp.489-495, 2013.
URL : https://hal.archives-ouvertes.fr/hal-01843945
, Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source, Molecular Plant-Microbe Interactions, vol.14, pp.775-784, 2001.
, Root exudate of Solanum tuberosum is enriched in galactose-containing molecules and impacts the growth of Pectobacterium atrosepticum, Annals of Botany. mcw128, 2016.
URL : https://hal.archives-ouvertes.fr/hal-01837958
, The Organization Pattern of Root Border-Like Cells of Arabidopsis Is Dependent on Cell Wall Homogalacturonan, Plant Physiology, vol.150, pp.1411-1421, 2009.
URL : https://hal.archives-ouvertes.fr/hal-00433500
, Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot, Planta, vol.198, pp.452-459, 1996.
, Characterization of carbohydrate structural features recognized by anti-arabinogalactan-protein monoclonal antibodies, Glycobiology, vol.6, pp.131-139, 1996.
, High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles, Glycoconjugate Journal, vol.25, pp.37-48, 2008.
, A set of cell surface glycoproteins forms an early marker of cell position, but not cell type, in the root apical meristem of Daucus carota L, Development, vol.106, pp.47-56, 1989.
, Patterns of expression of the JIM4 arabinogalactan-protein epitope in cell cultures and during somatic embryogenesis in Daucus carota L, Planta, vol.180, pp.285-292, 1990.
, Developmentally-regulated epitopes of cell surface arabinogalactan-proteins and their relation to root tissue pattern formation, The Plant Journal, vol.1, pp.317-326, 1991.
, A family of abundant plasma membrane-associated glycoproteins related to the arabinogalactan proteins is unique to flowering plants, The Journal of cell biology, vol.108, pp.1967-1977, 1989.
, Organ-Specific Arabinogalactan-Proteins of Lycopersicon peruvianum(Mill) Demonstrated by Crossed Electrophoresis, Plant Physiology, vol.80, pp.786-789, 1986.
, Quantification of Arabinogalactan-Protein in Plant Extracts by Single Radial Gel Diffusion, Analytical Biochemistry, vol.148, pp.446-450, 1985.
, Structural analysis of the carbohydrate moiety of arabinogalactan-proteins from stigmas and styles of Nicotiana alata, Carbohydrate Research, vol.277, pp.67-85, 1995.
, A role for arabinogalactan-proteins in root epidermal cell expansion, Planta, vol.203, pp.289-294, 1997.
, Identification and partial characterization of proteins and proteoglycans encrusting the secondary cell walls of flax fibres, Planta, vol.211, pp.256-264, 2000.
, Extraction and Detection of Arabinogalactan Proteins, The Plant Cell Wall: Methods and Protocols, pp.245-254, 2011.
, Precipitation of arabic acid and some seed polysaccharides by glycosylphenylazo dyes, Biochemical Journal, vol.105, p.1, 1967.
, A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of beta-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana, The Plant Journal, vol.9, pp.919-925, 1996.
, ) ?-Galactosyl Yariv Reagent Binds to the -1,3-Galactan of Arabinogalactan Proteins, Plant Physiology, vol.161, pp.1117-1126, 2013.
, Annexes ~ 222 ~
, The Yariv reagent: Behaviour in different solvents and interaction with a gum arabic arabinogalactanprotein, Carbohydrate Polymers, vol.106, pp.460-468, 2014.
, Monoclonal Antibodies, Carbohydrate-Binding Modules, and the Detection of Polysaccharides in Plant Cell Walls, 2011.
, The Plant Cell Wall: Methods and Protocols, pp.103-113
, Deciphering the Responses of Root Border-Like Cells of Arabidopsis and Flax to Pathogen-Derived Elicitors, Plant Physiology, vol.163, pp.1584-1597, 2013.
URL : https://hal.archives-ouvertes.fr/hal-01973870
,
,
, Super photo mais je pourrais l'améliorer avant de leur montrer?
, Ca ne serait pas plus joli en rouge ?
,
, Il faudrait que j'arrive à superposer ces 2 images? (overlay)
, Il faudrait que j'arrive à superposer ces 56 images ! (stack)
,
, une partie de l'image qui m'intéresse
, 17 ? ou une vidéo avec mes images prises à différents temps, encore mieux !
,
, Je ne pourrais pas utiliser ces images pour « quantifier » mon marquage ?
, Maintenant je dois mesurer les éléments de mon image?
,
,
,
,
, Je dois absolument mettre une échelle ! Insertion de l'échelle avec Image J, calibration d'image et détermination de l'échelle Plusieurs cas se présentent : Cas n° 1 : je dispose de l'image au format
, Réaliser une vidéo ou une animation gif peut paraître très compliqué. Mais ce n'est pas le cas. En fait, avec ImageJ c'est relativement simple
, mettez les images qui vont composer votre vidéo/animation sous forme d'un stack. Pour une vidéo, allez dans File
, Animated Gif? Une fenêtre s'ouvre vous proposant plusieurs options dont voici les plus importantes : ? Name : Nom du fichier ? Set Global Lookup Table Options : Type de coloration pour l'animation. Mettez "Load from Current Image, Pour une animation Gif, allez dans File, Save as
, Set delay in milliseconds : Intervalle de temps entre 2 images définissant ainsi la vitesse de lecture ? Number of loop? : Nombre de répétitions de l'animation
, Journal of Phytopathology, vol.158, p.585593, 2010.
, Frontiers in Plant Science, vol.3, p.93, 2012.
, Biochemistry Insights, vol.8, issue.S2, p.113, 2015.
, Plant Physiology, vol.163, p.15841587, 2013.
, Plant Molecular Biology, vol.64, issue.8, p.439451, 2007.
, , vol.106, p.1469914704, 2009.
, Journal of Biological Chemistry, vol.280, p.11421148, 2005.
, Frontiers in Plant Science, vol.3, p.93, 2012.
, New Phytologist, vol.187, p.449460, 2010.
, Toutefois, leur mode d'action dans la réponse immunitaire végétale n'est pas encore bien connu et reste à élucider. Les extensines interviennent dans le renforcement de la paroi, un des premiers remparts cellulaires contre les pathogènes, en se liant entre elles de manière intra-et intermoléculaire. Ce « cross-linking » est catalysé par des enzymes peroxydases spécifiques et nécessite une correcte conformation des extensines, laquelle est conférée par leur partie glycosylée. Dans ce projet de thèse, nous avons donc entrepris d'étudier l'impact de la glycosylation des extensines sur la défense racinaire et tenté de caractériser, de manière préliminaire, des peroxydases potentiellement impliquées dans le « cross-linking » chez Arabidopsis thaliana. Des techniques d'immunocytochimie réalisées sur une sélection de mutants affectés dans la glycosylation des extensines ont révélé une modulation de la distribution des extensines dans la racine d'A. thaliana en réponse à une élicitation avec un peptide bactérien, la flagelline 22. L'un des résultats majeurs de cette étude a été de montrer l'importance de l'arabinosylation des extensines dans la colonisation de la racine par l'oomycète pathogène Phytophthora parasitica. Ainsi, l'ensemble de ces résultats nous a permis d'élaborer un modèle proposant d, The Plant Journal, 61, 9921000. Zoospores Résumé Les extensines sont des glycoprotéines pariétales appartenant à la famille des HRGPs (Hydroxy prolin-rich glycoproteins) impliquées dans plusieurs fonctions telles que la croissance, le développement et la défense des plantes contre les pathogènes, 2010.
, Mots-clés : extensines, arabinosylation, défense, racine, paroi, glycosylation, éliciteur, Arabidopsis thaliana