Cette recherche a été menée en vertu de l&#39;USDA-ARS CRIS projet 5325-42000-037-00D et avec des résultats de CRADA 58-3K95-7-1198 et TFCA 58-5325-8-419. Les auteurs tiennent à remercier Suterra pour le don de l&#39;(Z, Z) -11,13-hexadecadienal, B. Higbee des discussions productives, et J. Baker pour l&#39;assistance technique.

<ol>
	<li>Bruce, T. J. A., Wadhams, L. J., Woodcock, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+host+location:+a+volatile+situation.">Insect host location: a volatile situation.</a> Trends in Plant Sci. 10, 1360-1385 (2005).</li><li>Unsicker, S. B., Kunert, G., Gershenzon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+perfumes:+the+role+of+vegetative+volatiles+in+plant+defense+against+herbivores.">Protective perfumes: the role of vegetative volatiles in plant defense against herbivores.</a> Curr. Opin. Plant Biol. 12, 479-485 (2009).</li><li>Norin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiochemicals+for+insect+pest+management.">Semiochemicals for insect pest management.</a> Pure Appl. Chem. 79, 2129-2136 (2007).</li><li>Beck, J. J., Merrill, G. B., Higbee, B. S., Light, D. M., Gee, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+seasonal+study+of+the+volatile+production+of+almonds+(Prunus+dulcis)+var.+'nonpareil'+and+relationship+to+navel+orangeworm.">In situ seasonal study of the volatile production of almonds (Prunus dulcis) var. 'nonpareil' and relationship to navel orangeworm.</a> J. Agric. Food Chem. 57, 3749-3753 (2009).</li><li>Beck, J. J., Higbee, B. S., Gee, W. S., Dragull, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+orchard+volatiles+from+California+almonds.">Ambient orchard volatiles from California almonds.</a> Phytochem. Lett. 4, 199-202 (2011).</li><li>Coffelt, J. A., Vick, K. W., Sonnet, P. E., Doolittle, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+identification,+and+synthesis+of+a+female+sex+pheromone+of+the+navel+orangeworm,+Amyelois+transitella+(Lepidoptera:+Pyralidae).">Isolation identification, and synthesis of a female sex pheromone of the navel orangeworm, Amyelois transitella (Lepidoptera: Pyralidae).</a> J. Chem. Ecol. 5, 955-933 (1979).</li><li>Leal, W. S., Parra-Pedrazzoli, A. L., Kaissling, K. -. E., Morgan, T. I., Zalom, F. G., Pesak, D. J., Dundulis, E. A., Burks, C. S., Higbee, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+pheromone+chemistry+in+the+navel+orangeworm:+novel+sex+attractants+and+a+behavioral+antagonist.">Unusual pheromone chemistry in the navel orangeworm: novel sex attractants and a behavioral antagonist.</a> Naturwissenschaften. 92, 139-146 (2005).</li><li>Kanno, H., Kuenen, L. P. S., Klingler, K. A., Millar, J. G., Carde, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attractiveness+of+a+four-component+pheromone+blend+to+male+navel+orangeworm+moths.">Attractiveness of a four-component pheromone blend to male navel orangeworm moths.</a> J. Chem. Ecol. 36, 584-591 (2010).</li><li>Takacs, S., Gries, G., Gries, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiochemical-mediated+location+of+host+habitat+by+Apanteles+carpatus+(Say)+(Hymenoptera:+Braconidae),+a+parasitoid+of+cloths+moth+larvae.">Semiochemical-mediated location of host habitat by Apanteles carpatus (Say) (Hymenoptera: Braconidae), a parasitoid of cloths moth larvae.</a> J. Chem. Ecol. 23, 459-472 (1997).</li><li>Karimifar, N., Gries, R., Khaskin, G., Gries, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+food+semiochemicals+attract+omnivorous+German+cockroaches,+Blattella+germanica.">General food semiochemicals attract omnivorous German cockroaches, Blattella germanica.</a> J. Agric. Food Chem. 59, 1330-1337 (2011).</li><li>Molyneux, R. J., Schieberle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+identification:+a+Journal+of+Agricultural+and+Food+Chemistry+perspective.">Compound identification: a Journal of Agricultural and Food Chemistry perspective.</a> J. Agric. Food Chem. 55, 4625-4629 (2007).</li><li>Marion-Poll, F., Thiery, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+EAG+responses+to+host-plant+volatiles+delivered+by+a+gas+chromatograph.">Dynamics of EAG responses to host-plant volatiles delivered by a gas chromatograph.</a> Entomol. Exp. Appl. 80, 120-123 (1996).</li><li>Beck, J. J., Higbee, B. S., Merrill, G. B., Roitman, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+volatile+emissions+from+undamaged+and+mechanically+damaged+almonds.">Comparison of volatile emissions from undamaged and mechanically damaged almonds.</a> J. Sci. Food Agric. 88, 1363-1368 (2008).</li><li>Lucas, P., Renou, M., Tellier, F., Hammoud, A., Audemard, H., Descoins, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+and+field+activity+of+halogenated+analogs+of+(E,E)-8-10-dodecadien-1-ol,+the+main+pheromone+component+in+codling+moth+(Cydia+pomonella+L.).">Electrophysiology and field activity of halogenated analogs of (E,E)-8-10-dodecadien-1-ol, the main pheromone component in codling moth (Cydia pomonella L.).</a> J. Chem. Ecol. 20, 489-503 (1994).</li><li>Rodriguez-Saona, C., Poland, T. M., Miller, J. R., Stelinski, L. L., Grant, G. G., de Groot, P., Buchan, L., MacDonald, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+electrophysiological+responses+of+the+emerald+ash+borer,+Agrilus+planipennis,+to+induced+volatiles+of+Manchurian+ash,+Fraxinus+mandshurica.">Behavioral and electrophysiological responses of the emerald ash borer, Agrilus planipennis, to induced volatiles of Manchurian ash, Fraxinus mandshurica.</a> Chemoecology. 16, 75-86 (2006).</li><li>Burks, C. S., Brandl, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+abundance+of+navel+orangeworm+(Leipidoptera:+Pyralidae)+in+figs+and+effect+of+peripheral+aerosol+dispensers+on+sexual+communication.">Seasonal abundance of navel orangeworm (Leipidoptera: Pyralidae) in figs and effect of peripheral aerosol dispensers on sexual communication.</a> J. Insect Sci. 4, 1-8 (2004).</li></ol>

L&#39;auteur a une recherche de coopération existantes et d&#39;un accord de développement avec la société Paramount agriculture, une société ayant des liens avec Suterra LLC.

Utilisation des enregistrements électroantennogramme comme un bio-essai pour déterminer les réponses chimioréception d&#39;un insecte cible est assez commun et de nombreuses études utilisant des EAG comme un détecteur pour les effluents d&#39;une chromatographie en phase gazeuse (GC-EAD) peut être trouvé dans la littérature. 9,10 La méthode illustrée fournira un dépistage rapide de quantités équivalentes de composants volatils avec des réplications d&#39;assignation d&#39;confiant de la réacti...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Acétophénone </td><td> Alfa-Aesar </td><td> A12727 </td><td> Femme contrôle positif </td></tr><tr><td> (Z, Z) -11,13-Hexadecadienal </td><td> Suterra </td><td></td><td> Homme de contrôle positif </td></tr><tr><td> α-humulène </td><td> Aldrich </td><td> 53675 </td><td> Sesquiterpène </td></tr><tr><td> 2-undécanone </td><td> Aldrich </td><td> U1303 </td><td> Dérivé d&#39;acide gras </td></tr><tr><td> 2-phényl éthanol </td><td> Aldrich </td><td> 77861 </td><td> Benzénoïde </td></tr><tr><td> Pentane </td><td> EMD </td><td> PX0167-1 </td><td> Solvant </td></tr><tr><td> Contrôleur d&#39;acquisition 4-Channel </td><td> Syntech </td><td> JeCAD-4 </td><td></td></tr><tr><td> EAG sonde, pré-amplificateur </td><td> Syntech </td><td> Type de PRG-2 </td><td></td></tr><tr><td> Support d&#39;antenne pour </td><td> Syntech </td><td> Pour PRG-2 </td><td> Électrode fourche </td></tr><tr><td> Contrôleur de stimulation </td><td> Syntech </td><td> CS-55 </td><td> Le débit d&#39;air et de bouffées </td></tr><tr><td> Gel électrode Spectra </td><td> Parker </td><td> 12-02 </td><td></td></tr><tr><td> Disques biologiques sur </td><td> Whatman </td><td> 2017-006 </td><td> 6 mm </td></tr><tr><td> Pipettes Pasteur </td><td> VWR </td><td> 14673-010 </td><td> 5 ¾ &quot;(14,6 cm) </td></tr><tr><td> Parafilm M </td><td> Bemis </td><td> PM-992 </td><td></td></tr></tbody></table>

electroantennographic bioassay as a screening tool for host plant volatiles

Volatiles des plantes jouent un rôle important dans les interactions plantes-insectes. Les insectes herbivores utilisent volatiles végétales, connues sous le nom kairomones, afin de localiser leur plante hôte. 1,2 Quand une plante hôte est un élément important des dommages agronomiques alimentation des produits de base par des insectes ravageurs peuvent causer de graves pertes économiques aux producteurs. En conséquence, kairomones peut être utilisé comme appâts pour attirer ou confondre ces insectes et, par conséquent, offrent une alternative écologique aux pesticides pour lutter contre les insectes. 3 Malheureusement, les plantes peuvent émettre un grand nombre volatiles avec des compositions différentes et des ratios d&#39;émissions dépendent de la phénologie de la marchandise ou l&#39;heure de la journée. Cela rend l&#39;identification de composants biologiquement actifs ou des mélanges de composants volatils un processus ardu. Pour aider à identifier les composants bioactifs des émissions volatiles de la plante hôte, nous comptons les Electroantennographie en laboratoire de dépistage bioessai (EAG). EAG est un outil efficace pour évaluer et enred électrophysiologique des réponses olfactives d&#39;un insecte via leurs récepteurs antennaires. Le processus de sélection EAG peut aider à réduire le nombre de volatiles testés pour identifier les composants bioactifs prometteurs. Cependant, les essais biologiques EAG seulement fournir des informations sur l&#39;activation des récepteurs. Il ne fournit pas d&#39;informations sur le type de comportement de l&#39;insecte le composé provoque, ce qui pourrait être aussi un attractif, le type de répulsif ou l&#39;autre de la réponse comportementale. Volatiles obtenir une réponse significative par EAG, par rapport à un contrôle positif approprié, sont généralement prises à des essais complémentaires pour les réponses comportementales de l&#39;insecte ravageur. Le dispositif expérimental présenté détaille la méthodologie employée pour dépister à base d&#39;amandes volatiles de la plante hôte 4,5 par la mesure des réponses électrophysiologiques antennaires d&#39;un adulte orangeworm insecte ravageur du nombril (Amyelois transitella) à des composants individuels et des mélanges simples de composants via EAG biologique. La méthode utilise deux exantennes circoncis placé à travers un support &quot;fork&quot; de l&#39;électrode. Le protocole a démontré ici présente un rapide, à haut débit méthode standardisée pour volatiles de dépistage. Chaque volatile est à un ensemble, quantité constante d&#39;uniformiser le niveau du stimulus et donc de permettre des réponses antennaires à être représentatives de la chemoreceptivity relative. Le contrôle négatif permet d&#39;éliminer la réponse électrophysiologique à la fois la force du solvant résiduel et mécanique de la bouffée. Le contrôle positif (dans ce acétophénone par exemple) est un composé unique qui a suscité une réponse cohérente à partir du nombril orangeworm mâle et femelle (NOW) papillon. Une norme écomone supplémentaire qui fournit la réponse cohérente et est utilisé pour des études de dosage biologique avec le mâle MAINTENANT papillon est (Z, Z) -11,13-hexdecadienal, un composant aldéhyde de la phéromone sexuelle femelle produite en 6-8.

Watch this Scientific Journal Video about Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles at JoVE.com

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Une méthode pour rapidement volatiles écran d&#39;accueil de plantes par la mesure de la réponse électrophysiologique de orangeworm nombril adulte (<em&gt; Amyelois transitella</em&gt;) Antennes à des composants individuels et des mélanges par analyse électroantennographique est démontrée.

Essais biologiques électroantennographique comme outil de dépistage pour les substances volatiles de la plante hôte

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

electroantennographic-bioassay-as-screening-tool-for-host-plant

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13.6K vues.  Agricultural Research Service. Une méthode pour rapidement volatiles écran d'accueil de plantes par la mesure de la réponse électrophysiologique de orangeworm nombril adulte ( Amyelois transitella) Antennes à des composants individuels et des mélanges par analyse électroantennographique est démontrée.

Vidéo: Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

Un β-glucuronidase (GUS) dosage basé mort cellulaire

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

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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 ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

Fluorescence microscopie dépistage et séquençage de prochaine génération: des outils utiles pour l&#39;identification des gènes impliqués dans des organites intégrité

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilisation de la séquence d&#39;arrêt SecM comme un outil pour isoler les polypeptides Ribosome Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

ARN catalyseur en tant que reporter pour le criblage de médicaments contre l&#39;ARN Édition dans trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

Un bioessai simple pour l&#39;évaluation des facteurs de croissance endothélial vasculaire

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

assistée par laser microdissection (LAM) comme outil de profilage transcriptionnel des types cellulaires individuels

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

Un test de concurrence plaque comme une rapide évaluation préliminaire de la Suppression de la maladie

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify&#160;molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts.
Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is&#160;carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.

Le séquençage par insertion de transposons comme outil pour élucider les facteurs de colonisation bactérienne dans un symbiote Burkholderia gladioli de coléoptères Lagria villosa

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological ...

Detecting Virus and Salivary Proteins of a Leafhopper Vector in the Plant Host

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran insects that have piercing-sucking mouthparts. During viral transmission, the insect saliva bridges the virus-vector-host because the saliva vectors viruses, and the insect proteins, trigger or suppress the immune response of plants from insects into plant hosts. The identification and functional analyses of salivary proteins are becoming a new area of focus in the research field of arbovirus-host interactions. This protocol provides a system to detect proteins in the saliva of leafhoppers using the plant host. The leafhopper vector Nephotettix cincticeps infected with rice dwarf virus (RDV) serves as an example. The vitellogenin and major outer capsid protein P8 of RDV vectored by the saliva of N. cincticeps can be detected simultaneously in the rice plant that N. cincticeps feeds on. This method is applicable for testing the salivary proteins that are transiently retained in the plant host after insect feeding. It is believed that this system of detection will benefit the study of hemipteran-virus-plant or hemipteran-plant interactions.

This protocol demonstrates how to use the plant host to detect salivary proteins of leafhopper and plant viral proteins released by leafhopper vectors.

Détection de virus et de protéines salivaires d’un vecteur cicadelle dans la plante hôte

Insect vectors horizontally transmit many plant viruses of agricultural importance. More than one-half of plant viruses are transmitted by hemipteran ...