<ol><li> En primer lugar tenemos que preparar a los 2 ml con tapa de rosca viales para la extracción: Agregar 400ul de tampón de extracción (1-propanol: H2O: HCl concentrado 2:1:0.002 vol / vol / vol) y 10 l de la norma interna respectiva (10ng/μl ) a un vial. Congelar en nitrógeno líquido y luego agregar las cuentas de cerámica. </li><li> Añadir el material vegetal (entre 50 y 200 mg), mientras que los viales siguen siendo colocado en el nitrógeno líquido. Tenga en cuenta que el peso del material vegetal añadido tiene que ser estimado antes de proseguir para permitir que más tarde para la cuantificación exacta, que se basa en el peso fresco. </li><li> Asegúrese de que todo el nitrógeno líquido se evapora antes de cerrar bien el frasco con una tapa. Tenga en cuenta que la tapa tiene que tener un sello de goma para evitar derrames de solventes y por lo tanto, los compuestos extraídos durante la homogeneización. </li><li> Homogeneizar el tejido en un FastPrep o Precellys homogeneizador molino de bolas a una velocidad de 6000 (para Precellys) durante 30 segundos. </li><li> Retire los viales de homogeneizador de forma individual, abra la tapa y añada 1 ml de diclorometano para cada muestra. Viales se cierran de nuevo con fuerza y ​​homogeneizar durante 10-15seg en 6000 (Precellys). </li><li> Transferencia de viales de una centrífuga de mesa y separar la fase orgánica de la fase acuosa a 10.000xg de 60 segundos. </li><li> Después de la separación de fases de transferencia de la capa inferior verde (fase orgánica, contiene las hormonas y las normas internas) de los viales individuales a un vial de 4 ml de vidrio limpio. Evitar la transferencia de agua (fase superior). Por otra parte, acetato de etilo se puede utilizar en lugar de diclorometano. En ese caso la fase de ethyacetate será la capa superior (verde) y ahora es más fácil de quitar y se transfiere a un vial de vidrio de 4 ml fresco. Al igual que antes, evitar la transferencia de agua. </li><li> Sople el disolvente con el aire a través de un colector de unos 10-25 minutos (depende de la muestra), consulte con una punta de caudal de aire solo si las muestras están secas (con cuidado). No muestras resecar, para que esto podría causar pérdidas de compuestos. </li><li> Una vez que las muestras se secan, podemos iniciar la metilación de los ácidos carboxílicos que contienen compuestos en las muestras. En primer lugar, 100 l de una mezcla de diethyether / metanol (9:1 vol / vol) se añaden a cada vial seguido por 4μl de una solución 2M trimethylsilyldiazomethane (en hexano, Sigma Aldrich). Los viales se cierran inmediatamente con un tapón de rosca descapotable equipado con un tabique de silicona recubierto de teflón. Agitar suavemente e incubar a temperatura ambiente durante 25 a 30 minutos. </li><li> Después de terminar el paso 9, a su vez en un bloque de calefacción y ajuste a la temperatura de la colección que desee. La volatilidad de los compuestos metilados depende de su tamaño, sino también en otros grupos más polares como = O,-OH, y NH. Para el ácido jasmónico y ésteres metílicos de ácidos salicílico una temperatura de recogida de alrededor de 80 ° C son suficientes, mientras que, por ejemplo, otros compuestos como ácidos grasos C18, jasmónico aminoácidos conjugados de ácido, coronatina y éster metílico de ácido 12-oxo-phytodienoic requieren temperaturas entre 180-200 ° C para volatilizar con eficacia. </li><li> Después de una incubación de 30 minutos la reacción de metilación tiene que ser detenido para evitar una reacción secundaria no deseada. Esto se hace mediante la adición de 4 l de un ácido acético 2M-solución (en hexano) a cada vial. Después del cierre de los viales con tapas y una agitación pocas palabras, las muestras se dejan incubar durante otros 30 minutos. </li><li> Los filtros utilizados para esta extracción en fase vapor son hechos a mano y en la forma presentada no está disponible comercialmente. Se componen de un tubo exterior de teflón (alrededor de 7 y 8 cm de largo), en la que de un lado se inserta un tubo de vidrio que ajuste bien de unos 4 cm de longitud. En el otro lado del tubo de teflón un disco de acero inoxidable de malla se inserta dentro del tubo hasta que llega al tubo de cristal interior y en que alrededor de 20-30mg de adsorbente (ya sea SuperQ 80/100 (suspendido) o HayeSep ® Q80/100) se agregan. Otro disco de acero inoxidable de malla se inserta y, finalmente, una punta de vidrio se lleva en el tubo de teflón, con lo que presionar con cuidado el disco de malla de acero inoxidable en el absorbente. Una descripción detallada de un filtro se muestra en la figura 1. <img src="/files/ftp_upload/1127/1127fig1.jpg" alt="Figura 1" /> Figura 1. </li><li> Durante el tiempo de incubación del paso 11, los filtros para la recogida de la metilación y por lo tanto, hormonas vegetales volátiles, tienen que ser lavados con 200ul primero de metanol, y luego de 2 lavados con diclorometano 200ul cada uno. Disolvente que queda en los filtros es entonces volado por el aire. Los filtros se pueden utilizar ahora para la captura de las hormonas volátiles a través de la extracción en fase vapor. </li><li> Después de 30 minutos de incubación de los septos de goma del vial de 4 ml tapas se cortan con un bisturí. Para recoger las hormonas de plantas por extracción en fase vapor de limpieza de los filtros se conectan a las líneas de vacío individual en un caudal de aproximadamente 800ml/min. Los filtros se insertan en el vial a través de los tabiques de corte. Una pequeña punta de la pipeta se inserta también para actuar como una entrada de aire. Durante esta parte inicial del procedimiento de los viales con los filtros han insertado, que se celebraráen posición vertical para evitar que cualquiera de los líquidos en el interior del vial para ponerse en contacto con la punta de filtro, que contaminaría el filtro y, posteriormente, también el GC / MS. Los viales están entonces en poder de la mano hasta que el líquido se evapora a través del filtro. A continuación, los viales con los filtros adjunta se colocan en el bloque de calentamiento y se evapora al calor compuestos recogidos por 3 minutos. Los principales componentes del sistema, así como la configuración se muestra en la figura 1. Después de esta etapa de extracción final de la fase de vapor de los viales con los filtros siguen unidos se colocan sobre una rejilla y dejar enfriar. </li><li> Cuando los filtros han llegado a la temperatura ambiente una vez más se eluyó con 150 ml de diclorometano en un 1,5 ml GC-vial equipado con una inserción. Alterar soplar el disolvente restante del filtro de los frascos se tapan y se analizaron por GC / MS. </li></ol> Un cromatógrafo de gases equipado con un split / splitless inyector (el modo splitless 250 ° C, el volumen de inyección 1μ) interfaz con un espectrómetro de masas que opere en modo de ionización química (CI) debe ser utilizado para el análisis). Compuestos se separan en HP-1MS columna (30m x 0.25mm x 025μm) se mantiene a 40 ° C durante 1 minuto después de la inyección, y luego la temperatura programada a 15 ° C / min a 250 ° C (de 10 minutos), con helio como el portador gas (0.7ml/min flujo constante). Para el análisis basado en MS tanto, un quadropole (por ejemplo, Agilent HP5973) y una trampa de iones basados ​​en (por ejemplo, Varian Saturn 2200) espectrómetro de masa se puede utilizar. Como un gas isobutano o CI (viene en los tanques de gas) o metanol (los vapores de un depósito de líquido se utilizan) se puede utilizar, sin embargo, en cada caso, los fragmentos de la reacción de ionización se debe comprobar antes de que el selectivo de iones contar (SIC) los programas de se crean. Hemos utilizado el siguiente programa para un sistema de GC Varian 3900/Saturn MS 2200 y el metanol como reactivo IC: 5.00-11.30min iones (M +1) + 153-158 (salicilato de metilo 153, metil 2 H 5-salicilato); 11.30-13.30min iones (M +1) + 207 a 228 (metil jasmonato 207 y 225 de metilo dihidrojasmonato 209 y 227); 13.30-16.30min iones (M +1) + 237-300 (metil ácido abscísico 261, metil 2 H-6 ácido abscísico 267 (ácido abscísico produce predominantemente [MH 2 O +1] + iones). Todos los compuestos deben ser identificados por comparación con estándares auténticos disponibles en el mercado. Cuantificación de JA, SA, y ABA se realiza mediante la correlación de la superficie del pico (extraído iones) con el área del pico (iones extraídos) de la norma interna respectiva y también se basa en el peso fresco del material vegetal utilizado.

<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>

El método que aquí se presenta se ha demostrado que funciona de forma fiable para una amplia gama de compuestos de señalización de las plantas, siempre que el tamaño y la naturaleza química del compuesto no impedir que se extrae, desnaturalizado, y se vaporiza. Además, el procedimiento de metilación como se describe en este documento no es la única forma de derivación. Silyllation y la acetilación de métodos alternativos y los protocolos se pueden encontrar fácilmente en línea. ...

monitoring plant hormones during stress responses

Hormonas de las plantas y los compuestos relacionados con la señalización desempeñan un papel importante en la regulación de la respuesta de las plantas a diversos estímulos ambientales y las tensiones. Entre las tensiones más graves son la herbivoría de insectos, la infección por patógenos, y la sequía. Para cada una de estas tensiones de un conjunto específico de las hormonas y / o combinaciones de ellos son conocidos para afinar las respuestas, asegurando así la supervivencia de la planta. Las principales hormonas implicadas en la regulación de estas respuestas son el ácido jasmónico (JA), el ácido salicílico (SA), y el ácido abscísico (ABA). Para comprender mejor el papel de las hormonas individuales, así como su potencial interacción durante estas respuestas es necesario monitorear los cambios en su abundancia en un temporal, así como de una manera espacial. Para la cuantificación sencilla, sensible y reproducible de estos y otros compuestos de señalización se desarrolló un método basado en la extracción en fase de vapor y la cromatografía de gases / espectrometría de masas (GC / MS) análisis (1, 2, 3, 4). Después de la extracción de estos compuestos en el tejido de la planta por acuosa ácida 1-propanol se mezcla con el diclorometano que contiene ácido carboxílico compuestos metilados, se volatiliza con el calor, y se recogen en un polímero absorbente. Tras la elución a un vial de los analitos se separan por cromatografía de gases y espectrometría de masas detectadas por ionización química. El uso apropiado de las normas internas se permite la cuantificación sencilla, relacionando las áreas de pico de analito estándar e internos.

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

Monitoring Plant Hormones During Stress Responses

Un método simple es a condición de que permite la rápida extracción y análisis de hormonas de plantas múltiples de pequeñas muestras de tejido. El procedimiento utiliza la extracción en fase vapor de la etapa de purificación solemne. Las muestras se analizaron por GC / MS con ionización química que produce principalmente (M +1) + iones.

Monitoreo hormonas vegetales en las respuestas de estrés

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 vistas.  University of Texas. Un método simple es a condición de que permite la rápida extracción y análisis de hormonas de plantas múltiples de pequeñas muestras de tejido. El procedimiento utiliza la extracción en fase vapor de la etapa de purificación solemne. Las muestras se analizaron por GC / MS con ionización química que produce principalmente (M +1) + iones.

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.

La investigación de los tejidos y órganos específicos de las respuestas fitocromo usando FACS asistida por células de tipo Perfiles de expresión específica en 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 ...

Investigación

Biología

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.

Bioensayos para vigilar la resistencia insecticida

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.

Vinculación de riesgo de depredación, Estrés Fisiológico herbívoro y descomposición microbiana de los desechos de las plantas

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.

Análisis de la Traducción Iniciación Durante condiciones de estrés por 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.

La hidroponía: un sistema versátil para el Estudio de nutrientes Las respuestas de las plantas a la asignación y disponibilidad de nutrientes y la exposición a elementos tóxicos

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.

Sistema proteína verde fluorescente para visualizar efectores entregado de las bacterias durante una infección

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.

Mediciones de las respuestas fisiológicas de estrés en 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 Análisis de la función E3 Ubiquitina Ligasa

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.

Construcción de mutantes homocigotos de langostas migratorias utilizando tecnología 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.

Combinación de microscopía de limpieza y fluorescencia para visualizar cambios en la expresión génica y respuestas fisiológicas 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 ...