<ol><li> Per prima cosa dobbiamo preparare il 2 ml con tappo a vite fiale per l&#39;estrazione: Aggiungi 400ul tampone di estrazione (1-propanolo: H2O: HCl concentrato 2:1:0.002 vol / vol / vol) e 10 ml di standard rispettivi interno (10ng/μl ) in una sacca. Congelare in azoto liquido e quindi aggiungere le perline in ceramica. </li><li> Aggiungere il materiale vegetale (tra 50 e 200 mg), mentre le fiale sono ancora posizionati nel azoto liquido. Si noti che il peso del materiale vegetale aggiunto deve essere valutato prima di proseguire per consentire in seguito per la quantificazione esatta, che si basa sul peso fresco. </li><li> Assicurarsi che tutti l&#39;azoto liquido è evaporato prima a chiusura ermetica la fiala con un tappo. Si noti che il tappo deve avere una guarnizione di gomma per evitare fuoriuscite di solventi e quindi, composti estratti durante l&#39;omogeneizzazione. </li><li> Omogeneizzare il tessuto in una FastPrep o Precellys omogeneizzatore smerigliatrice tallone ad una velocità di 6000 (per Precellys) per 30 sec. </li><li> Rimuovere fiale da omogeneizzatore singolarmente, aprire con cautela il tappo e aggiungere 1 ml di diclorometano per ogni campione. Fiale richiudere saldamente e omogeneizzare di nuovo per 10-15sec a 6000 (Precellys). </li><li> Trasferimento fiale per una centrifuga da tavolo e separare la fase organica dalla fase acquosa 10.000xg per 60sec. </li><li> Dopo separazione delle fasi trasferire lo strato inferiore verde (fase organica, contiene gli ormoni e gli standard interni) fiale da individuo a una fiala di vetro pulito 4ml. Evitare il trasferimento di acqua (fase superiore). In alternativa, acetato di etile può essere usato al posto di diclorometano. In questo caso la fase ethyacetate sarà lo strato superiore (verde) e ora è più facile da rimuovere e trasferito in una fiala di vetro fresco 4ml. Come prima, evitare il trasferimento di acqua. </li><li> Soffiare via il solvente con l&#39;aria attraverso un collettore per circa 10-25 minuti (dipende dal campione); controllare con una punta del flusso d&#39;aria solo se i campioni sono a secco (con attenzione). Non asciugare eccessivamente campioni, per questo potrebbe causare perdite di composti. </li><li> Una volta che i campioni sono asciugati possiamo iniziare la metilazione del carbossilico composti contenenti nei nostri campioni. In primo luogo, 100 l di una diethyether / miscela di metanolo (9:1 vol / vol) vengono aggiunti a ogni flacone seguita da 4μl di una soluzione trimethylsilyldiazomethane 2M (in esano, Sigma Aldrich). I flaconi sono immediatamente chiuso con un open-top tappo a vite dotato di un setto in Teflon rivestito di silicone. Agitare delicatamente ed incubare a RT per 25 a 30 min. </li><li> Dopo aver terminato il punto 9, girare su un blocco di riscaldamento e alla temperatura impostata collezione desiderata. La volatilità dei composti metilati dipende dalle loro dimensioni, ma anche su altri gruppi, come più polare = O,-OH, e-NH. Per l&#39;acido salicilico e jasmonic esteri metilici degli acidi una temperatura collezione di circa 80 ° C sono sufficienti, mentre per esempio altri composti come acidi grassi C18, jasmonic acido-amino acido coniugati, coronatine, e 12-oxo-phytodienoic estere metilico dell&#39;acido richiedono di temperatura tra 180-200 ° C per volatilizzare in modo efficace. </li><li> Dopo una incubazione di 30 minuti la reazione di metilazione deve essere fermata per evitare indesiderate reazione secondaria. Questo viene fatto aggiungendo 4 ml di acido acetico 2M uno-soluzione (in esano) per ogni flacone. Dopo aver chiuso le fiale con tappi e un vortex breve, i campioni sono autorizzati a incubare per altri 30 min. </li><li> I filtri utilizzati per questa estrazione in fase vapore sono fatti a mano e nella forma presentata non disponibili in commercio. Sono composti da un tubo esterno in teflon (circa 7-8 centimetri di lunghezza), in cui da un lato si inserisce un ben raccordo tubo di vetro di circa 4 centimetri di lunghezza. Dall&#39;altra parte del tubo in Teflon uno maglia di acciaio inossidabile disco viene spinto all&#39;interno del tubo fino a raggiungere la camera d&#39;aria di vetro e su che circa 20-30mg di adsorbenti (sia SuperQ 80/100 (fuori produzione) o HayeSep ® Q80/100) sono aggiunti. Un altro disco in acciaio inox viene inserito in rete e infine una punta di vetro si spinge nel tubo in Teflon, così accuratamente premendo il disco di rete d&#39;acciaio inox sul assorbente. Un quadro dettagliato di un filtro è mostrato in figura 1. <img src="/files/ftp_upload/1127/1127fig1.jpg" alt="Figura 1" /> Figura 1. </li><li> Durante il periodo di incubazione del punto 11, i filtri per la raccolta del metilato e quindi, ormoni vegetali volatili, devono essere lavati con 200ul prima di metanolo, e poi 2 lavaggi con diclorometano 200ul ciascuno. Solvente rimanente nei filtri è poi saltato fuori per via aerea. I filtri possono ora essere utilizzati per la cattura di ormoni volatili attraverso l&#39;estrazione in fase vapore. </li><li> Dopo 30 min di incubazione i setti di gomma della fiala 4ml tappi vengono tagliati con un bisturi. Per raccogliere gli ormoni vegetali per estrazione fase vapore sono collegati i filtri puliti per linee di vuoto individuo ad un flusso di circa 800ml/min. I filtri vengono poi inseriti nel flaconcino attraverso i setti taglio. Un consiglio pipetta piccolo è anche inserito di agire come una presa d&#39;aria. Durante questa parte iniziale della procedura le fiale con i filtri inseriti devono essere tenutein posizione verticale per evitare di liquido all&#39;interno della fiala di entrare in contatto con la punta filtro, che potrebbe contaminare il filtro e successivamente anche il GC / MS. Le fiale sono poi tenuti a mano fino a quando tutto il liquido è evaporato attraverso il filtro. Poi le fiale con i filtri attaccati sono immessi sul blocco di riscaldamento e calore evaporato composti raccolti per 3min. I componenti principali del sistema e la configurazione sono rappresentati in figura 1. Dopo questo ultimo passaggio di estrazione in fase vapore le fiale con i filtri ancora attaccato sono posti su una griglia e lasciate raffreddare. </li><li> Quando i filtri hanno raggiunto la temperatura ambiente nuovo sono eluiti con 150 ml di diclorometano in un 1,5 ml GC-flacone dotato di un inserto. Alter soffia il solvente residuo dal filtro sono ricoperti i flaconi e analizzati mediante GC / MS. </li></ol> Un gascromatografo dotato di un iniettore split / splitless (splitless modalità 250 ° C, volume di iniezione 1μ) interfacciato ad uno spettrometro di massa operante a ionizzazione chimica (CI) deve essere utilizzato per l&#39;analisi). I composti sono separati nei sistemi HP-1MS colonna (30m x 0,25 millimetri x 025μm) ha tenuto a 40 ° C per 1 minuto dopo l&#39;iniezione, e poi la temperatura programmata a 15 ° C / min a 250 ° C (per 10 minuti), con elio come il vettore gas (0.7ml/min flusso costante). Per gli Stati membri a base di analisi di entrambi, un quadropole (ad es Agilent HP5973) e uno ione trappola-based (ad esempio, Varian Saturn 2200) spettrometro di massa può essere utilizzato. Come gas CI sia isobutano (viene in serbatoi di gas) o metanolo (vapori da un serbatoio di liquido vengono utilizzate) si possono utilizzare, comunque, in ogni caso i frammenti della reazione di ionizzazione deve essere verificata prima che il numero di ioni selezionati (SIC) programmi sono state create. Abbiamo usato il seguente programma per un sistema GC Varian 3900/Saturn MS 2200 e il metanolo come reagente CI: 5.00-11.30min ioni (M +1) + 153-158 (metil salicilato 153, metil 2 H 5-salicilato); 11.30-13.30min ioni (M +1) + 207-228 (metil jasmonate 207 e 225, metil dihydrojasmonate 209 e 227); 13.30-16.30min ioni (M +1) + 237-300 (metil acido abscissico 261, metil 2 H 6-acido abscissico 267 (acido abscissico produce prevalentemente [MH 2 O +1] + ioni). Tutti i composti devono essere individuate tramite il confronto con autentici standard disponibili in commercio. Quantificazione dei JA, SA, e ABA è fatto, correlando l&#39;area del picco (estratto ioni) con l&#39;area del picco (ioni estratta) dello standard rispettivi ordinamenti ed è anche in base al peso fresco del materiale vegetale utilizzato.

<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>

Il metodo presentato qui ha dimostrato di funzionare in modo affidabile per una vasta gamma di composti vegetali segnalazione a condizione che la dimensione e la natura chimica del composto non gli impediscono di essere estratta, metilato, e vaporizzato. Inoltre, la procedura di metilazione in quanto è qui descritto non è l&#39;unico modo di derivatizzazione. Silyllation e acetilazione sono metodi alternativi e protocolli possono essere trovati facilmente on-line. Se lo spettrometro di mas...

monitoring plant hormones during stress responses

Ormoni vegetali e composti correlati segnalazione svolgono un ruolo importante nella regolazione delle risposte delle piante ai vari stimoli ambientali e sottolinea. Tra le sollecitazioni più severe sono insetti erbivori, infezioni degli agenti patogeni e stress da siccità. Per ognuna di queste sollecitazioni uno specifico insieme di ormoni e / o loro combinazioni sono noti per mettere a punto le risposte, garantendo così la sopravvivenza della pianta. I principali ormoni coinvolti nella regolazione di queste risposte sono l&#39;acido jasmonic (JA), acido salicilico (SA), e l&#39;acido abscissico (ABA). Per meglio comprendere il ruolo degli ormoni individuali e la loro potenziale interazione durante queste risposte è necessario monitorare i cambiamenti nella loro abbondanza in un temporale e in un modo spaziale. Per la quantificazione semplice, sensibile e riproducibile di questi e di altri composti segnalazione abbiamo sviluppato un metodo basato sulla estrazione in fase vapore e gas cromatografia / spettrometria di massa (GC / MS) analisi (1, 2, 3, 4). Dopo l&#39;estrazione di questi composti dal tessuto vegetale da acquosi acidi 1-propanolo mescolato con il diclorometano carbossilico contenenti composti sono metilate, volatilizzato a caldo, e raccolto su un assorbente polimerici. Dopo l&#39;eluizione in una fiala campione gli analiti siano separati mediante gascromatografia e spettrometria di massa rilevati da ionizzazione chimica. L&#39;uso di appropriati standard interno consente poi, per la quantificazione semplice mettendo in relazione le aree del picco dello standard dell&#39;analita ed interna.

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

Monitoring Plant Hormones During Stress Responses

Un metodo semplice è previsto che consente l&#39;estrazione rapida e analisi di ormoni vegetali più da piccoli campioni di tessuto. La procedura di estrazione utilizza vapore fase come la fase di purificazione solenne. I campioni vengono analizzati mediante GC / MS con ionizzazione chimica che produce principalmente (M +1) + ioni.

Monitoraggio ormoni vegetali Durante risposte allo 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

Research

JoVE Journal

Biology

16.5K Views.  University of Texas. Un metodo semplice è previsto che consente l'estrazione rapida e analisi di ormoni vegetali più da piccoli campioni di tessuto. La procedura di estrazione utilizza vapore fase come la fase di purificazione solenne. I campioni vengono analizzati mediante GC / MS con ionizzazione chimica che produce principalmente (M +1) + ioni.

Video: 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.

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in 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 ...

Ricerca

Biologia

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.

Saggi biologici per la resistenza di monitoraggio Insetticida

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.

Collegamento rischio di predazione, stress Herbivore fisiologica e decomposizione microbica dei Rifiuti vegetali

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.

Analisi della traduzione innesco durante condizioni di stress da polisoma Profiling

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.

Hydroponics: un sistema versatile per studiare nutrienti allocazione e vegetali risposte a disponibilità di nutrienti e l&#39;esposizione a elementi tossici

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.

Proteina fluorescente verde sistema di visualizzare gli effettori consegnati dai batteri durante l'infezione Split

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.

Misurazioni delle risposte allo stress fisiologico in 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 Analisi della funzione E3 Ubiquitina ligasi

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.

Costruzione di mutanti omozigoti di locuste migratorie utilizzando la tecnologia 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.

Combinazione di microscopia a compensazione e fluorescenza per visualizzare i cambiamenti nell'espressione genica e le risposte fisiologiche a Plasmodiophora brassicae

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