Cette production et les travaux expérimentaux présentés ici reflètent l&#39;aide dévouée et enthousiaste de Reynaldo Pareja aimablement pris en charge par Lina Aguirre, Gilberto Córdoba, William Mera, Ximena Bonilla, et Darío Viveros. Nous remercions également John Miles et trois lecteurs anonymes pour les commentaires qui nous ont aidés à améliorer la vidéo et du manuscrit.

1. Plantes <ol><li> Boutures simple à partir de plantes matures sont utilisés comme unités expérimentales. Les boutures sont taillés à 10 cm pour assurer l&#39;uniformité du matériel de plantation. </li><li> Pour éviter la contamination par des agents pathogènes des plantes, les boutures sont lavés dans une solution d&#39;hypochlorite de sodium à 3% pendant 2 min, puis rincée avec de l&#39;eau du robinet thoughroughly. </li><li> Chaque bouture est plantée dans ca. 36 g de sol stérile dans un tube cylindrique en PVC (5,3 cm de diamètre extérieur de 6,5 cm de hauteur) scellé à l&#39;extrémité inférieure avec une tasse de styromousse et coiffé d&#39;une douille en PVC. La coupe est maintenu en place par un anneau en mousse placée dans l&#39;ouverture centrale de la douille. </li><li> Les plantes sont fécondées et arrosés au besoin pour maintenir l&#39;humidité du sol suffisante. </li><li> Les plantes poussent pendant un mois avant d&#39;être contestée par les insectes, ce qui permet le développement des racines superficielles, qui servent de sites d&#39;alimentation pour les nymphes cercope racine xylème-alimentation. </li><li> Tubes en PVC avec des boutures enracinées sont inversées pour les huit d avant l&#39;infestation de stimuler davantage la croissance des racines superficielles, partiellement tenu compte des différences inhérentes à l&#39;architecture des racines. Cette étape nécessite une source de lumière artificielle, qui nous fournissent pour 24 h par jour. Pour éviter échappe confondants d&#39;herbivores à la résistance des herbivores, des plantes individuelles réplique avec peu ou pas de racines superficielles après ce traitement d&#39;inversion sont éliminés de l&#39;expérience. Cette élimination doit être rares, jamais conduisant à l&#39;élimination du génotype qu&#39;ils représentent. </li></ol> 2. Insectes <ol><li> Cercopes adultes sont recueillies sur le terrain, identifiés à l&#39;espèce, et introduit dans des cages de ponte où ils sont fournis feuillage des brachiariagrasses sensibles à nourrir. Les femelles pondent leurs œufs dans le sol contenue dans un bac peu profond, placé sur le fond des cages. </li><li> Après plusieurs jours, le sol est en suspension dans l&#39;eau et passé à travers une série de tamis (42, 60 et 150 mesh) pour collecter les oeufs. Suspendre le matériel tamisée dans une solution saline à 30% des causes les oeufs matures à flotter, laissant derrière lui des œufs immatures et toute matière organique résiduelle. </li><li> Pour éviter la contamination par des pathogènes d&#39;insectes, les œufs sont désinfectés dans une solution d&#39;hypochlorite de sodium à 0,5% pendant 5 min, puis rincée avec de l&#39;eau distillée. </li><li> Chaque usine de test (unité expérimentale) est infesté à la surface du sol avec six oeufs matures. Seuls les oeufs matures à proximité d&#39;incubation sont sélectionnés pour les infestations. Ils peuvent être identifiés par la présence de deux points rouges dans leur partie antérieure, correspondant aux yeux de l&#39;insecte; traces roses dans leur partie postérieure, correspondant à l&#39;abdomen de l&#39;insecte, et un opercule en entier. </li><li> L&#39;éclosion réussie est confirmée quatre jours plus tard, à quel point tout oeuf non éclos est remplacé par un nouveau-né cercope de notre colonie. </li><li> Test de plantes sont disposées en blocs complets randomisés avec six répétitions par génotype. </li></ol> 3. Évaluations <ol><li> L&#39;expérience est évaluée ca. 30 jours après l&#39;infestation (horaires exacts varient selon les espèces d&#39;insectes étudiés). </li><li> Les dommages sont notés sur une échelle de 1 à 5 visuels, où 1 correspond à aucun dommage visible et 5 correspond à une usine de mort (figure 1). Notation des dommages plante fournit une estimation de la tolérance. </li></ol><img src="/files/ftp_upload/3047/3047fig1.jpg" alt="Figure 1" /> Ampleur des dommages figure 1. Utilisé pour évaluer la tolérance au cercope herbivores dans les Brachiaria spp. 1 = aucun dommage détectable; 2 = 0-25% du feuillage nécrotiques; 3 = 25-50% du feuillage nécrotiques; 4 = 50-75% du feuillage nécrotiques; 5 = 75-100% du feuillage nécrotiques. <ol start="3"><li> La survie des insectes est évaluée par le comptage des insectes d&#39;atteindre leur stade soit final ou au stade adulte (figure 2). De notation pour la survie des insectes fournit une estimation de l&#39;antibiose. </li></ol><img src="/files/ftp_upload/3047/3047fig2.jpg" alt="Figure 2" /> Figure 2. Cercope le développement du premier stade (à gauche) à l&#39;adulte (à droite). Seuls les insectes d&#39;atteindre au moins leur stade final (flèches) à la date de l&#39;évaluation sont considérés comme des «survivants». 4. Les résultats représentatifs Les résultats d&#39;un test hybrides représentant Brachiaria sélection pour la résistance à l&#39;reducta cercope Aeneolamia sont présentés dans la figure 3. <img src="/files/ftp_upload/3047/3047fig3.jpg" alt="Figure 3" /> Figure 3. Résultats du dépistage hybrides Brachiaria pour la résistance à cercope Aeneolamia reducta. Nous considérons que les plantes résistantes si leur réponse à l&#39;herbivorie tombe dans la moitié inférieure de ce complot. Augmente Antibiose de droite à gauche et à la tolérance augmente de haut en bas. Les flèches indiquent à nos chèques résistants.

<ol>
	<li>Painter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect resistance in crop plants. , (1951).</li><li>Kogan, M., Ortman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antixenosis-a+new+term+proposed+to+define+Painter's+'Nonpreference'+modality+of+resistance.">Antixenosis-a new term proposed to define Painter's 'Nonpreference' modality of resistance.</a> Bull. Entomol. Soc. Am. 24, 175-176 (1978).</li><li>Kennedy, G., Gould, F., Deponti, O., Stinner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological,+agricultural,+genetic,+and+commercial+considerations+in+the+deployment+of+insect-resistant+germplasm.">Ecological, agricultural, genetic, and commercial considerations in the deployment of insect-resistant germplasm.</a> Environ. Entomol. 16, 327-338 (1987).</li><li>Holmann, F., Peck, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+damage+of+grassland+spittlebugs+(Homoptera+Cercopidae)+in+Colombia:+a+first+approximation+of+impact+on+animal+production+in+Brachiaria+decumbens.">Economic damage of grassland spittlebugs (Homoptera Cercopidae) in Colombia: a first approximation of impact on animal production in Brachiaria decumbens.</a> Neotrop. Entomol. 31, 275-284 (2002).</li><li>Miles, J. W., Cardona, C., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+selection+in+a+synthetic+brachiariagrass+population+improves+resistance+to+three+spittlebug+species.">Recurrent selection in a synthetic brachiariagrass population improves resistance to three spittlebug species.</a> Crop Sci. 46, 1088-1093 (2006).</li><li>Ferrufino, A., Lapointe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+plant+resistance+in+Brachiaria+grasses+to+the+spittlebug+Zulia+colombiana.">Host plant resistance in Brachiaria grasses to the spittlebug Zulia colombiana.</a> Entomol. Exp. Appl. 51, 155-162 (1989).</li><li>Lapointe, S. L., Serrano, M. S., Arango, G. L., Sotelo, G., Cordoba, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+to+spittlebugs+(Homoptera:+Cercopidae)+in+accessions+of+Brachiaria+spp.">Antibiosis to spittlebugs (Homoptera: Cercopidae) in accessions of Brachiaria spp.</a> J. Econ. Entomol. 82, 1764-1766 (1992).</li><li>Cardona, C., Miles, J. W., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+methodology+for+massive+screening+of+Brachiaria+spp.+genotypes+for+resistance+to+Aeneolamia+varia+(Homoptera:+Cercopidae).">An improved methodology for massive screening of Brachiaria spp. genotypes for resistance to Aeneolamia varia (Homoptera: Cercopidae).</a> J. Econ. Entomol. 92, 490-496 (1999).</li><li>Cardona, C., Fory, P., Sotelo, G., Pabon, A., Diaz, G., Miles, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+and+tolerance+to+five+species+of+spittlebug+(Homoptera:+Cercopidae)+in+Brachiaria+spp.:+implications+for+breeding+for+resistance.">Antibiosis and tolerance to five species of spittlebug (Homoptera: Cercopidae) in Brachiaria spp.: implications for breeding for resistance.</a> J. Econ. Entomol. 97, 635-645 (2004).</li><li>Smith, C., Khan, Z., Pathak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques for evaluating insect resistance in crop plants. , (1994).</li><li>Lapointe, S., Sotelo, G., Arango, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+for+rearing+spittlebugs+(Homoptera:+Cercopidae).">Improved technique for rearing spittlebugs (Homoptera: Cercopidae).</a> J. Econ. Entomol. 82, 1764-1766 (1989).</li><li>Lopez, F., Cardona, C., Miles, J. W., Sotelo, G., Montoya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+resistance+to+adult+spittlebugs+(Hemiptera:+Cercopidae)+in+Brachiaria+spp.:+methods+and+categories+of+resistance.">Screening for resistance to adult spittlebugs (Hemiptera: Cercopidae) in Brachiaria spp.: methods and categories of resistance.</a> J. Econ. Entomol. 102, 1309-1316 (2009).</li></ol>

Discrimination entre les mécanismes de résistance des herbivores peut éclairer les efforts d&#39;amélioration des plantes 3. Résistance repose sur antibiose forte pourrait, dans certaines circonstances, de créer des pressions de sélection pour les biotypes de ravageurs plus agressif. D&#39;autre part, la résistance basée sur la tolérance pourrait permettre les populations de ravageurs à augmenter jusqu&#39;à la tolérance est finalement débordé. Sélection pour la résistance durable, exige donc un examen attentif de ce que les mécanismes de résistance conférerait plus de stabilité pour une culture particulière / système de lutte [voir Kennedy et al. (1987) pour une discussion approfondie sur le sujet] 3. Cette vidéo de démonstration s&#39;appuie sur plusieurs études antérieures visant à estimer les contributions relatives de l&#39;antibiose et de tolérance à la résistance dans le cercope brachiariagrasses 6-9. En contraste avec les tests à choix ou des expériences de terrain, aucun des tests à choix s&#39;assurer que toutes les plantes reçoivent la pression des herbivores même, fournissant ainsi de neutraliser les différences résultant de comportements herbivores (par exemple, la préférence d&#39;hôte, agrégation). Pour cette raison, cependant, pas le choix des tests ne sont pas adaptées pour évaluer la résistance des antixénose. Artificiellement l&#39;élevage des colonies d&#39;insectes est la méthode préférée pour obtenir des insectes de test pour des essais de criblage pour la résistance 10. Colonies de fournir une source fiable et uniforme de cercopes d&#39;un âge connu pour des expériences à réaliser à tout moment de l&#39;année 11. Il peut cependant être nécessaire d&#39;effectuer périodiquement infuser individus sauvages dans la colonie afin de s&#39;assurer que la colonie ne s&#39;écarte pas de populations génétiquement domaine pertinent 10. Pour évaluer l&#39;aptitude de ces herbivores artificiellement élevés, et de surveiller d&#39;éventuels changements dans leur agressivité au cours du temps, il est important que toutes les projections incluent des contrôles appropriés de niveaux de résistance connue. Plusieurs paramètres doivent être définis avec soin à la conception d&#39;un test de dépistage de résistance approprié. Quelques paramètres clés que nous avons considéré la conception de notre test incluent l&#39;âge de la plante hôte, l&#39;espèce et le stade de développement de l&#39;cercopes, le niveau d&#39;infestation et la durée de la plante-insecte de contact 8,9,12. Plusieurs cycles de l&#39;expérimentation et le raffinement du protocole peut être nécessaire pour atteindre à une rapide, rentable, fiable, test qui prédit correctement la résistance des herbivores dans le domaine.

characterizing herbivore resistance mechanisms: spittlebugs on brachiaria spp. as an example

Les plantes peuvent résister aux dommages herbivore par trois mécanismes principaux: antixénose, antibiose et de tolérance 1. Antixénose est le degré auquel la plante est évité lorsque l&#39;herbivore est capable de sélectionner d&#39;autres plantes 2. Antibiose est le degré auquel la plante affecte l&#39;aptitude de l&#39;herbivore se nourrissant de ce 1. La tolérance est le degré auquel la plante peut résister ou réparer les dommages causés par les herbivores, sans compromettre la croissance de l&#39;herbivore et la reproduction 1. La durabilité de la résistance des herbivores dans un milieu agricole dépend dans une large mesure sur le mécanisme de résistance favorisée lors des efforts d&#39;amélioration des plantes 3. Nous démontrons une expérience non-choix visant à estimer les contributions relatives de l&#39;antibiose et de tolérance à la résistance dans le cercope Brachiaria spp. Plusieurs espèces de graminées d&#39;Afrique de l&#39;Brachiaria genre sont de fourrage précieux et plantes de pâturage dans la région néotropicale, mais ils peuvent être sérieusement remis en question par plusieurs espèces indigènes de cercopes (Hemiptera: Cercopidae) 4. Afin d&#39;évaluer leur résistance aux cercopes, les plantes sont propagées végétativement- par boutures de tiges et de laisser pousser pendant environ un mois, permettant la croissance des racines superficielles sur lesquelles cercopes peuvent se nourrir. À ce moment, chaque plante test est individuelle a contesté avec six oeufs d&#39;incubation cercope proximité. Les infestations sont autorisés à progresser pendant un mois avant d&#39;évaluer les dommages et la survie des insectes. Notation des dommages plante fournit une estimation de la tolérance tout en marquant la survie des insectes fournit une estimation de l&#39;antibiose. Ce protocole a facilité notre objectif d&#39;amélioration des plantes pour améliorer la résistance au cercope brachiariagrases commerciale 5.

Watch this Scientific Journal Video about Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example at JoVE.com

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Cette vidéo explique les mécanismes de résistance de la plante hôte à l&#39;herbivorie et démontre un test non-choix que les estimations de la contribution relative de l&#39;antibiose et de la tolérance au cercope résistance<em&gt; Brachiaria</em&gt; Spp.

Caractériser les mécanismes de résistance Herbivores: cercopes sur Brachiaria Spp. comme un exemple

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

characterizing-herbivore-resistance-mechanisms-spittlebugs-on

Research

JoVE Journal

Biology

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

13.7K vues.  CIAT. Cette vidéo explique les mécanismes de résistance de la plante hôte à l'herbivorie et démontre un test non-choix que les estimations de la contribution relative de l'antibiose et de la tolérance au cercope résistance Brachiaria Spp.

Vidéo: Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Les études génétiques des protéines humaines réparation de l&#39;ADN utilisant de la levure comme système modèle

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Recherche

Biologie

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

Herbivores induite Volatiles Blueberry et intra-usine de signalisation

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

La mesure de la perméabilité à l&#39;eau Coefficient osmotique (P<sub&gt; F</sub&gt;) Des cellules sphériques: protoplastes de plantes isolées comme un exemple

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La technique d&#39;infiltration-centrifugation pour l&#39;extraction de apoplastique Fluid feuilles des plantes Utilisation<em&gt; Phaseolus vulgaris</em&gt;, Un exemple

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

L’imagerie FITC-dextran comme Reporter pour l’exocytose régulée

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

Étalonnage et utilisation des performances de microcoil MRM démontrés sur les racines de la truncatule Medicago à 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

Explorer les épitranscriptomes m6A et m5C lors d’une infection virale : un exemple avec le VIH

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Caractérisation des propriétés mécaniques de la paroi cellulaire primaire dans les organes vivants de plantes à l’aide de la microscopie à force atomique

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

Méthode d’électroporation pour transformer Rickettsia spp. avec un vecteur navette exprimant une protéine fluorescente dans des lignées cellulaires de tiques

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

Étude du mécanisme de transport du zinc dans les cellules de mammifères avec le dosage de FluoZin-3

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Pleins feux sur l’auteur : Explorer la régulation cellulaire du zinc grâce à la fonctionnalité ZnT1 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Caractérisation des transporteurs de zinc chez les mammifères à l’aide d’un test in vitro de transport du zinc

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...