<ol><li> Primeiro, precisamos preparar o 2ml tampa de rosca frascos para extração: tampão de extração Adicionar 400ul (1-propanol: H2O: HCl concentrado 2:1:0.002 vol / vol / vol) e 10 ml do respectivo padrão interno (10ng/μl ) para um frasco. Congelamento em nitrogênio líquido e em seguida, adicione as contas de cerâmica. </li><li> Adicione o material vegetal (entre 50 e 200mg), enquanto os frascos ainda estão posicionados no nitrogênio líquido. Note que o peso do material vegetal adicionado deve ser estimado antes do processamento, que permitisse mais tarde para a quantificação exata, que é com base no peso fresco. </li><li> Certifique-se que todo o nitrogênio líquido é evaporado antes de fechar hermeticamente o frasco com uma tampa. Note que a tampa deve ter um selo de borracha para evitar derramamentos de solventes e, portanto, compostos extraídos durante a homogeneização. </li><li> Homogeneizar o tecido em um moedor FastPrep ou homogeneizador bead Precellys a uma velocidade de 6000 (para Precellys) por 30 seg. </li><li> Remova frascos de homogeneizador individualmente, abra cuidadosamente a tampa e adicione 1 ml de diclorometano a cada amostra. Frascos de fechar novamente com força e homogeneizar novamente por 10-15s em 6000 (Precellys). </li><li> Transferência de frascos para uma centrífuga de mesa e separar a fase orgânica da fase aquosa, a 10.000xg para 60seg. </li><li> Depois da separação de fase de transferência da camada inferior verde (fase orgânica, contém os hormônios e as normas internas) de frascos individuais a um frasco de vidro limpo 4ml. Evitar a transferência de água (fase superior). Alternativamente, acetato de etila pode ser usado ao invés de diclorometano. Nesse caso, a fase ethyacetate será a camada superior (verde) e é agora mais fácil de remover e transferido para um frasco de vidro fresco 4ml. Como antes, evitar a transferência de água. </li><li> Sopre o solvente com ar através de uma variedade de cerca de 10-25 min (depende da amostra); verificar com uma ponta de fluxo de ar único se as amostras são secas (cuidadosamente). Não amostras overdry, pois isso pode causar perdas de compostos. </li><li> Depois que as amostras são secas, podemos começar a metilação do ácido carboxílico contendo compostos nas amostras. Primeiro, 100 ul de uma mistura diethyether / metanol (9:1 vol / vol) são adicionados a cada frasco seguido por 4μl de uma solução trimethylsilyldiazomethane 2M (em hexano, Sigma Aldrich). Frascos são imediatamente fechadas com uma tampa de rosca open-top equipado com um septo de silicone recoberta com teflon. Agitar suavemente e incubar em temperatura ambiente por 25 a 30min. </li><li> Depois de terminar a etapa 9, por sua vez em um bloco de aquecimento e ajuste na temperatura desejada coleção. A volatilidade dos compostos metilados depende de seu tamanho, mas também em outros grupos mais polares, como = O,-OH e-NH. Para o ácido jasmônico e ésteres de ácido salicílico metil uma coleção de temperaturas em torno de 80 ° C são suficientes, enquanto que, por exemplo, outros compostos como ácidos graxos C18, conjugados do ácido jasmônico-amino ácido, coronatina, e éster metílico de 12-oxo-ácido phytodienoic requerem temperatura entre 180-200 ° C para efetivamente volatilizam. </li><li> Após incubação de 30 minutos a reação de metilação precisa ser interrompido para evitar a reação secundária indesejados. Isto é feito pela adição de 4 mL de uma solução 2M de ácido acético (em hexano) para cada frasco. Depois de fechar os frascos com tampa e um vórtice curto, as amostras estão autorizados a incubar por mais 30min. </li><li> Os filtros usados ​​para essa extração em fase vapor são feitas à mão e na forma apresentada, não disponível comercialmente. Eles consistem em um tubo de Teflon exterior (cerca de 7-8cm de comprimento), em que de um lado um tubo de vidro bem-encaixe de cerca de 4 centímetros de comprimento é inserido. Do outro lado do tubo de Teflon um disco de malha de aço inoxidável é empurrado para dentro do tubo até chegar ao tubo de vidro interior e em que cerca de 20-30mg de adsorvente (ou SuperQ 80/100 (descontinuado) ou HayeSep ® Q80/100) são adicionados. Outro disco malha de aço inoxidável é inserido e, finalmente, uma dica de vidro é empurra para dentro do tubo de teflon, assim, cuidadosamente pressionando o disco de malha de aço inoxidável sobre o absorvente. A imagem detalhada de um filtro é mostrado na figura 1. <img src="/files/ftp_upload/1127/1127fig1.jpg" alt="Figura 1" /> Figura 1. </li><li> Durante o tempo de incubação da etapa 11, os filtros para a coleta do metilado e, portanto, hormônios vegetais voláteis, devem ser lavados com 200ul primeiro de metanol, e depois duas lavagens com diclorometano 200ul cada. Solvente remanescente nos filtros é, então, arrancada por via aérea. Os filtros podem ser usados ​​para a captura de hormonas voláteis através de extração em fase vapor. </li><li> Após 30min de incubação dos septos de borracha do frasco de 4ml tampas são cortados com um bisturi. Para recolher os hormônios vegetais por extração em fase vapor os filtros limpos são conectados a linhas de vácuo individuais em um fluxo de cerca de 800ml/min. Os filtros são então inseridas no frasco através dos septos de corte. A ponta da pipeta pequenas também é inserido para atuar como uma entrada de ar. Durante esta parte inicial do procedimento os frascos com os filtros têm inserido a ser realizadaem posição vertical para evitar qualquer do líquido dentro do frasco para entrar em contato com a ponta de filtro, que contaminaria o filtro e, posteriormente, também o GC / MS. Os frascos são então detidas pela mão até todo o líquido é evaporado através do filtro. Em seguida, os frascos com os filtros ligados são colocados no bloco de aquecimento e calor evaporou compostos coletados para 3min. Os componentes principais do sistema, bem como a configuração são descritas na figura 1. Após esta última etapa de extração de fase vapor os frascos com os filtros ainda ligado são colocados em um rack e deixar arrefecer. </li><li> Quando os filtros de ter atingido a temperatura ambiente novamente, eles são eluídos com 150ml de diclorometano em um frasco de 1,5 ml GC-equipado com um insert. Alter soprar o solvente restante do filtro os frascos são limitadas e analisadas por GC / MS. </li></ol> Um cromatógrafo a gás equipado com um injetor split / splitless (modo splitless 250 ° C, volume de injeção 1μ) interfaceado a um espectrômetro de massa operado em modo de ionização química (CI) devem ser utilizados para a análise). Compostos são separados em HP-1MS coluna (30m x 0,25 milímetros x 025μm), realizada a 40 ° C por 1min após a injeção, e depois a temperatura programada a 15 ° C / min a 250 ° C (por 10min), com hélio como o portador gás (0.7ml/min fluxo constante). Para a análise baseada em MS ambos, uma quadropole (por exemplo, Agilent HP5973) e um ion trap baseada em espectrômetro de massa (por exemplo, Varian Saturn 2200) pode ser usado. Como um gás CI ou isobutano (vem em tanques de gás) ou metanol (vapores de um reservatório de líquido são usados) pode ser usado, no entanto, em cada caso, os fragmentos da reação de ionização deve ser verificado antes da iões seleccionados count (SIC) programas são criados. Temos utilizado o seguinte programa para um 3900/Saturn GC Varian MS sistema de 2200 e metanol como reagente CI: 5.00-11.30min íons (M +1) + 153-158 (metil salicilato 153, metil salicilato 2 H 5); 11,30-13.30min íons (M +1) + 207-228 (metil jasmonato 207 e 225, metil-diidrosjaminato 209 e 227); 13,30 16.30min-íons (M +1) + 237-300 (metil ácido abscísico 261, metil 2 H do ácido abscísico 6-267 (ácido abscísico produz predominantemente [MH 2 O +1] + íons). Todos os compostos devem ser identificados por comparação com padrões autênticos disponíveis comercialmente. Quantificação do JA, SA, e ABA é feito correlacionando a área do pico (extraído íons) com a área de pico (íons extraídos) do respectivo padrão interno e também com base no peso fresco do material vegetal usado.

<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>

O método apresentado aqui tem sido comprovada para o trabalho de forma confiável para uma grande variedade de compostos de plantas de sinalização desde que o tamanho ea natureza química do composto não impedir que ele seja extraído, desnaturado, e vaporizado. Além disso, o processo de metilação como é descrito aqui não é a única maneira de derivatização. Silyllation e acetilação são métodos alternativos e protocolos pode ser facilmente encontrado on-line. Se o espectrôme...

monitoring plant hormones during stress responses

Hormônios vegetais e compostos relacionados sinalização desempenhar um papel importante na regulação da resposta das plantas a diversos estímulos ambientais e tensões. Entre as tensões mais graves são herbivoria de insetos, infecção do patógeno e estresse hídrico. Para cada um destes sublinha um conjunto específico de hormônios e / ou suas combinações, são conhecidos por fazer um ajuste fino das respostas, garantindo assim a sobrevivência da planta. Os principais hormônios envolvidos na regulação dessas respostas são o ácido jasmônico (JA), ácido salicílico (SA), e ácido abscísico (ABA). Para entender melhor o papel dos hormônios individuais, bem como sua interação em potencial durante essas respostas é necessário para monitorar as mudanças em sua abundância em um temporal, assim como de uma forma espacial. Para a quantificação fácil, sensível e reprodutível destes e de outros compostos de sinalização foi desenvolvido um método baseado na extração em fase vapor e cromatografia / espectrometria de massa de gás análise (GC / MS) (1, 2, 3, 4). Depois de extrair estes compostos a partir do tecido vegetal por aquosa ácida 1-propanol misturado com diclorometano contendo o ácido carboxílico compostos são metilados, volatilizado sob o calor, e recolhidos em um absorvente polimérico. Após eluição em um frasco de amostra dos analitos são separados por cromatografia gasosa e detectada por espectrometria de massa de ionização química. O uso de normas internas apropriadas, em seguida, permite a quantificação simples, relacionando as áreas dos picos de analito e padrão interno.

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

Monitoring Plant Hormones During Stress Responses

Um método simples é fornecida que permite a extração rápida e análise de hormônios vegetais múltiplos a partir de pequenas amostras de tecido. O procedimento utiliza extração em fase vapor como a etapa de purificação solene. As amostras são analisadas por íons GC / MS com ionização química que produz principalmente (M +1) +.

Monitoramento hormônios vegetais Durante Respostas ao Estresse

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 visualizações.  University of Texas. Um método simples é fornecida que permite a extração rápida e análise de hormônios vegetais múltiplos a partir de pequenas amostras de tecido. O procedimento utiliza extração em fase vapor como a etapa de purificação solene. As amostras são analisadas por íons GC / MS com ionização química que produz principalmente (M +1) +.

Vídeo: 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.

Investigando Tecido e órgãos específicos Responses Fitocromo usando FACS assistida Célula tipo Profiling expressão específica no 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 ...

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.

Bioensaios de Resistência a Inseticidas Monitoramento

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.

Ligando o risco de predação, Stress herbívoro fisiológica e decomposição da maca Usina

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.

Análise da Tradução Iniciação Durante condições de estresse por polysome 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.

Hidroponia: um sistema versátil para estudar nutrientes de alocação e de plantas As respostas a disponibilidade de nutrientes e exposição 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 de proteína verde fluorescente Split Visualizar efetores entregue de bactérias durante a infecção

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.

Medições de Respostas de Estresse Fisiológico em 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 Análise da Função Ligase E3 Ubiquitin

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.

Construção de mutantes homozigotos de gafanhotos migratórios utilizando 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.

Combinando Microscopia de Limpeza e Fluorescência para Visualização de Mudanças na Expressão Gênica e Respostas 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 ...