<ol><li> Nous devons d&#39;abord préparer le bouchon à vis 2ml flacons pour l&#39;extraction: Ajouter un tampon d&#39;extraction 400ul (1-propanol: H2O: HCl concentré 2:1:0.002 vol / vol / vol) et 10 pi de la norme interne respective (10ng/μl ) dans un flacon. Gel dans l&#39;azote liquide, puis ajouter les perles en céramique. </li><li> Ajouter de la matière végétale (entre 50 et 200mg) alors les flacons sont toujours positionnés dans l&#39;azote liquide. Notons que le poids de la matière végétale ajoutée doit être estimé avant tout autre traitement pour permettre à plus tard pour la quantification exacte, qui est basé sur le poids frais. </li><li> Assurez-vous que tout l&#39;azote liquide est évaporé avant à fermeture hermétique du flacon avec un bouchon. Notez que la PAC doit avoir un joint en caoutchouc pour empêcher les déversements de solvants et donc, composés extraits lors de l&#39;homogénéisation. </li><li> Homogénéiser le tissu dans un homogénéisateur Precellys FastPrep ou meuleuse billes à une vitesse de 6000 (pour les Precellys) pendant 30 sec. </li><li> Retirez les flacons d&#39;homogénéisateur individuellement, ouvrir avec précaution le bouchon et ajoutez 1 ml de dichlorométhane à chaque échantillon. Fermer hermétiquement les flacons à nouveau et homogénéiser à nouveau 10-15sec à 6000 (Precellys). </li><li> Transfert des flacons d&#39;une centrifugeuse de table et de séparer la phase organique de la phase aqueuse à 10.000xg pour 60sec. </li><li> Après séparation des phases de transfert de la couche inférieure verte (phase organique, contient les hormones et les normes internes) de flacons individuels dans un flacon en verre 4ml propre. Eviter le transfert d&#39;eau (phase supérieure). Alternativement, l&#39;acétate d&#39;éthyle peut être utilisé au lieu du dichlorométhane. Dans ce cas, la phase ethyacetate sera la couche supérieure (vert) et il est maintenant plus facile à enlever et transféré dans un flacon en verre 4ml frais. Comme avant, éviter le transfert de l&#39;eau. </li><li> Soufflez le solvant avec de l&#39;air grâce à un collecteur d&#39;environ 10-25 min (dépend de l&#39;échantillon); vérifiez avec une pointe d&#39;air unique si les échantillons sont secs (avec soin). Ne pas trop sécher les échantillons, cela pourrait causer des pertes de composés. </li><li> Une fois les échantillons sont séchés, nous pouvons commencer la méthylation de l&#39;acide carboxylique contenant des composés dans nos échantillons. Tout d&#39;abord, 100 ul d&#39;un mélange diethyether / méthanol (9:1 vol / vol) sont ajoutés à chaque flacon suivie par 4μl d&#39;une solution 2M triméthylsilyldiazométhane (dans l&#39;hexane, Sigma Aldrich). Les flacons sont immédiatement fermé avec un bouchon à vis open-top équipé d&#39;un septum en silicone de téflon. Agiter doucement et incuber à température ambiante pendant 25 à 30min. </li><li> Après avoir terminé l&#39;étape 9, tourner sur un bloc de chauffage et de mettre à la température désirée de collecte. La volatilité des composés méthylés dépend de leur taille, mais aussi sur les autres groupes, plus polaires tels que = O,-OH, et-NH. Pour l&#39;acide jasmonique et esters méthyliques d&#39;acide salicylique, une collection de températures autour de 80 ° C sont suffisantes, alors que, par exemple d&#39;autres composés comme les acides gras C18, jasmonique conjugués d&#39;acide acide aminé, coronatine, et 12-oxo-phytodienoic ester méthylique d&#39;acide nécessitent de température entre 180-200 ° C efficacement volatiliser. </li><li> Après une incubation de 30min de la réaction de méthylation doit être arrêté afin d&#39;éviter indésirables réaction secondaire. Cela se fait par addition de 4 pi d&#39;un mélange acide acétique-2M solution (dans l&#39;hexane) pour chaque flacon. Après la fermeture des flacons avec des bouchons et un vortex bref, les échantillons sont laissés à incuber pendant encore 30 min. </li><li> Les filtres utilisés pour cette extraction en phase vapeur sont faites à la main et dans la forme présentée n&#39;est pas disponible commercialement. Ils sont constitués d&#39;un tube extérieur en Téflon (environ 7-8cm de long), dans laquelle d&#39;un côté un tube de verre bien ajusté d&#39;environ 4cm de longueur est inséré. De l&#39;autre côté du tube de téflon d&#39;un disque en acier inoxydable maille est poussé dans le tube jusqu&#39;à ce qu&#39;il atteigne le tube de verre intérieure et sur qu&#39;environ 20 à 30 mg d&#39;adsorbant (soit SuperQ 80/100 (interrompu) ou HayeSep ® Q80/100) sont ajoutés. Un autre disque inox maille d&#39;acier est insérée et, enfin, un bout de verre est poussé dans le tube en Téflon, ce soin en appuyant sur le disque en acier inoxydable maille sur l&#39;absorbant. Un portrait détaillé d&#39;un filtre est montré dans la figure 1. <img src="/files/ftp_upload/1127/1127fig1.jpg" alt="Figure 1" /> Figure 1. </li><li> Pendant le temps d&#39;incubation de l&#39;étape 11, les filtres pour la collecte de l&#39;méthylé et donc, les hormones végétales volatiles, doivent être lavés à l&#39;aide 200ul d&#39;abord le méthanol, puis 2 lavages avec du dichlorométhane 200ul chacun. Solvant restant dans les filtres est alors emportée par l&#39;air. Les filtres peuvent maintenant être utilisés pour le piégeage des hormones volatiles par extraction en phase vapeur. </li><li> Après 30min d&#39;incubation, les cloisons en caoutchouc du flacon 4ml bouchons sont coupées avec un scalpel. Pour recueillir les hormones végétales par extraction en phase vapeur des filtres nettoyés sont connectés à des lignes vide individuelles à un débit d&#39;environ 800ml/min. Les filtres sont ensuite insérées dans le flacon à travers le septum coupé. Une petite pointe de pipette est également inséré pour agir comme une entrée d&#39;air. Durant cette partie initiale de la procédure des flacons avec les filtres insérés doivent être tenusdans une position verticale pour éviter tout du liquide à l&#39;intérieur du flacon pour entrer en contact avec l&#39;extrémité du filtre, ce qui pourrait contaminer le filtre et ensuite aussi la GC / MS. Les flacons sont ensuite tenu la main jusqu&#39;à ce que tout le liquide soit évaporé à travers le filtre. Puis les flacons avec les filtres ci-joints sont placés sur le bloc de chauffage et de la chaleur évaporée composés recueillis pour 3min. Les principaux composants du système ainsi que la configuration sont représentés dans la figure 1. Après cette dernière étape d&#39;extraction en phase vapeur des flacons avec les filtres encore attaché sont placés sur une grille et laisser refroidir. </li><li> Quand les filtres ont atteint la température ambiante de plus, ils sont élues avec 150 ml de dichlorométhane dans un 1,5 ml GC-flacon équipé d&#39;un insert. Alter souffler le solvant restant dans le filtre les flacons sont bouchés et analysé par GC / MS. </li></ol> Un chromatographe en phase gazeuse équipé d&#39;un injecteur split / splitless (mode splitless à 250 ° C, le volume d&#39;injection 1μ) interfacé à un spectromètre de masse fonctionnant en mode d&#39;ionisation chimique (CI) doit être utilisé pour l&#39;analyse). Les composés sont séparés sur HP-1MS colonne (30m x 0.25mm x 025μm) qui s&#39;est tenue à 40 ° C pendant 1min après l&#39;injection, puis la température programmée à 15 ° C / min à 250 ° C (pour 10min), avec l&#39;hélium comme le transporteur gaz (0.7ml/min débit constant). Pour l&#39;analyse MS-fondé à la fois, un quadropole (par exemple Agilent HP5973) et un piège à ions à base spectromètre de masse (par exemple Varian Saturn 2200) peuvent être utilisés. Comme un gaz CI soit l&#39;isobutane (livré dans des réservoirs à gaz) ou le méthanol (vapeurs d&#39;un réservoir de liquide sont utilisés) peut être utilisé, cependant, dans chaque cas, les fragments de la réaction d&#39;ionisation doit être vérifié avant l&#39;sélectionnée ions count (CTI) des programmes sont créés. Nous avons utilisé le programme suivant pour un Varian GC 3900/Saturn système MS 2200 et le méthanol comme réactif IC: 5.00-11.30min ions (M +1) + 153 à 158 (salicylate de méthyle 153, le méthyl-2 H 5 salicylate); 11.30-13.30min ions (M +1) + 207 à 228 (jasmonate de méthyle 207 et 225, 209 et méthyl dihydrojasmonate 227); 13,30 16.30min-ions (M +1) + 237 à 300 (méthyl acide abscissique 261, méthyl-2 H 6-acide abscissique 267 (acide abscissique produit principalement [MH 2 O 1] + ions). Tous les composés doivent être identifiés par comparaison avec d&#39;authentiques normes disponibles dans le commerce. Quantification des JA, SA, et ABA est fait en corrélant la surface du pic (extrait des ions) avec la surface du pic (ions extraits) de la norme internes respectives et est également basée sur le poids frais de la matière végétale utilisée.

<ol>
	<li>Schmelz, E. A., Engelberth, J., Tumlinson, J. H., Alborn, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+vapor+phase+extraction+in+metabolic+profiling+of+phytohormones+and+other+metabolites.">The use of vapor phase extraction in metabolic profiling of phytohormones and other metabolites.</a> Plant Journal. 39, 790-808 (2004).</li><li>Engelberth, J., Alborn, H. T., Schmelz, E. A., Tumlinson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+signals+prime+plants+against+insect+herbivore+attack.">Airborne signals prime plants against insect herbivore attack.</a> PNAS. 101, 1781-1785 (2004).</li><li>Schmelz, E. A., Engelberth, J., Alborn, H. T., O&amp;acute, D. o. n. n. e. l., Sammons, P., Toshima, M., H, J. H. T. u. m. l. i. n. s. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+phytohormones,+phytotoxins,+and+volatile+organic+compounds+in+plants.">Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants.</a> PNAS. 100, 10552-10557 (2003).</li><li>Engelberth, J., Schmelz, E. A., Alborn, H., Cardoza, Y. J., Huang, J., Tumlinson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quantification+of+Jasmonic+acid+and+Salicylic+acid+by+Vapor+Phase+Extraction+and+Gas+Chromatography+-+Chemical+Ionization-Mass+Spectrometry.">Simultaneous Quantification of Jasmonic acid and Salicylic acid by Vapor Phase Extraction and Gas Chromatography - Chemical Ionization-Mass Spectrometry.</a> Analytical Biochemistry. 312, 242-250 (2003).</li></ol>

La méthode présentée ici a été prouvée à travailler de manière fiable pour un large éventail de composés végétaux de signalisation condition que la taille et la nature chimique du composé ne l&#39;empêche pas d&#39;être extraite, méthylé, et vaporisé. En outre, la procédure de méthylation comme il est décrit ici n&#39;est pas le seul moyen de dérivation. Silyllation et l&#39;acétylation des méthodes alternatives et des protocoles peuvent être facilement trouvés en ligne. <p class="jove_cont...

monitoring plant hormones during stress responses

Les hormones végétales et des composés apparentés de signalisation jouent un rôle important dans la régulation des réponses des plantes à divers stimuli environnementaux et les contraintes. Parmi les contraintes les plus sévères sont insectes herbivores, une infection pathogène, et la sécheresse. Pour chacune de ces contraintes un ensemble spécifique d&#39;hormones et / ou de leurs combinaisons sont connues pour affiner les réponses, assurant ainsi la survie de la plante. Les principales hormones impliquées dans la régulation de ces réponses sont l&#39;acide jasmonique (JA), l&#39;acide salicylique (SA), et l&#39;acide abscissique (ABA). Pour mieux comprendre le rôle des hormones individuelles ainsi que leur interaction potentielle lors de ces réponses, il est nécessaire de surveiller les changements dans leur abondance dans un temporel aussi bien que de façon spatiale. Pour la quantification facile, sensible et reproductible de ces signaux et d&#39;autres composés que nous avons développé une méthode basée sur l&#39;extraction en phase vapeur et la chromatographie gazeuse / spectrométrie de masse (GC / MS) (1, 2, 3, 4). Après l&#39;extraction de ces composés à partir des tissus végétaux par aqueuse acide 1-propanol mélangé avec de l&#39;acide dichlorométhane contenant des composés carboxyliques sont méthylés, volatilisés par la chaleur, et recueilli sur un absorbant polymères. Après élution dans un flacon d&#39;échantillon des analytes sont séparés par chromatographie gazeuse et détectés par spectrométrie de masse à ionisation chimique. L&#39;utilisation de normes internes appropriées permet alors de la simple quantification en reliant les zones pic de l&#39;étalon analyte et internes.

Watch this Scientific Journal Video about Monitoring Plant Hormones During Stress Responses at JoVE.com

Monitoring Plant Hormones During Stress Responses

Une méthode simple est fourni qui permet l&#39;extraction rapide et l&#39;analyse des hormones végétales multiples à partir de petits échantillons de tissu. La procédure utilise l&#39;extraction en phase vapeur comme l&#39;étape de purification solennelle. Les échantillons sont analysés par des ions + GC / MS avec ionisation chimique qui produit principalement des (M +1).

Surveillance des hormones végétales Durant les réponses au stress

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

monitoring-plant-hormones-during-stress-responses

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JoVE Journal

Biology

16.5K vues.  University of Texas. Une méthode simple est fourni qui permet l'extraction rapide et l'analyse des hormones végétales multiples à partir de petits échantillons de tissu. La procédure utilise l'extraction en phase vapeur comme l'étape de purification solennelle. Les échantillons sont analysés par des ions + GC / MS avec ionisation chimique qui produit principalement des (M +1).

Vidéo: Monitoring Plant Hormones During Stress Responses

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Étude des tissus et des organes spécifiques Réponses phytochrome utilisant FACS assistée type cellulaire profilage expression spécifique dans Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Recherche

Biologie

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Bioessais pour la résistance aux insecticides de surveillance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

Lier le risque de prédation, stress physiologique et Herbivore décomposition microbienne de la litière végétale

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Analyse de la traduction Initiation Pendant conditions de stress par polysome profilage

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponique: un système polyvalent pour étudier et répartition des plantes Réponses des éléments nutritifs à la disponibilité des éléments nutritifs et l&#39;exposition aux éléments toxiques

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Protéine fluorescente verte bibloc pour visualiser les effecteurs livré de bactéries lors de l’Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Mesures des réponses physiologiques au stress à C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Analyse de la fonction E3 Ubiquitin Ligase

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

Construction de mutants homozygotes du criquet migrateur à l’aide de la technologie CRISPR/Cas9

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

Combinaison de la microscopie de compensation et de fluorescence pour visualiser les changements dans l’expression génique et les réponses physiologiques à Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...