<ol><li> Öncelikle ekstraksiyonu için 2 ml vidalı kapaklı şişeleri hazırlamak gerekir: 400ul ekstraksiyon tamponu ekle (1-propanol: H2O: konsantre HCl 2:1:0.002 hacim / hacim / hacim) ve ilgili iç standart 10 ul (10ng/μl ) bir flakon. Sıvı nitrojen içinde dondurun ve sonra seramik boncuklar ekleyin. </li><li> (50 ve 200 mg arasında) bitki materyali şişeleri hala sıvı nitrojen içinde konumlandırılmış ekleyin. Katma bitki materyali ağırlığı daha fazla işlem taze ağırlığı dayalı tam sayısallaştırma, daha sonra izin önce tahmin gerektiğini unutmayın. </li><li> Tüm sıvı azot flakon bir kapak ile sıkıca kapatmadan önce buharlaşmış olduğundan emin olun. Kap homojenizasyon sırasında solvent dökülmeleri ve böylece, ayıklanan bileşikler önlemek için kauçuk conta olması gerektiğini unutmayın. </li><li> 30 sn için 6000 bir hızda (Precellys için) bir FastPrep veya Precellys boncuk değirmeni Homojenizatör doku homojenize. </li><li> Şişeleri ayrı ayrı homojenizatör çıkarın, kapağı dikkatli bir şekilde açın ve her bir örnek için 1 ml diklorometan eklemek. Kapat şişeleri tekrar sıkıca ve 6000 (Precellys) 10-15sn için tekrar homojenize. </li><li> Bir masaüstü santrifüj şişeleri transferi ve organik faz 60sec için 10.000xg sulu faz ayrı. </li><li> Faz ayrıldıktan sonra temiz bir 4ml cam flakon şişeleri tek tek yeşil alt katman (organik faz, hormonlar ve iç standartları içerir) aktarın. Su transferi (üst faz) kaçının. Alternatif olarak, etil asetat, diklorometan yerine kullanılabilir. Bu durumda ethyacetate faz üst katmanı (yeşil) ve şimdi kaldırmak ve yeni bir 4ml cam flakon transfer daha kolay olacaktır. Daha önce olduğu gibi, su transferi kaçının. </li><li> Yaklaşık 10-25 dakika (örnek bağlıdır) manifoldu ile hava ile solvent kapalı darbe; örnekler kuru (dikkatle) tek bir hava akımı ucu ile kontrol edin. Overdry örnekleri etmeyin, bu bileşiklerin kaybına neden olabilir. </li><li> Örnekleri kurutulmuş sonra karboksilik metilasyon asit içeren örneklerde bileşikler başlayabilirsiniz. Birincisi, bir diethyether / metanol karışımı (09:01 hacim / hacim) 100 ul 2M trimethylsilyldiazomethane solüsyonu (hekzan, Sigma Aldrich) 4μl tarafından her flakon eklenir. Şişeler hemen Teflon kaplı silikon septum ile donatılmış bir üstü açık vidalı kapak ile kapatılır. Yavaşça çalkalayın ve 25 ila 30 dakika oda sıcaklığında inkübe edin. </li><li> 9. adımı tamamladıktan sonra, istenilen toplama sıcaklıkta ısıtma bloğu ve set açmak. Metillenmiş bileşiklerin volatilite, onların büyüklüğüne değil, aynı zamanda = O,-OH ve-NH gibi diğer, daha polar gruplar bağlıdır. Jasmonik asit ve salisilik asit metil ester bir koleksiyon sıcaklığı yaklaşık 80 ° C yeterli, oysa örneğin C18 yağ asitleri gibi diğer bileşikleri, Jasmonik asit amino asit konjugatları coronatine, ve 12-oxo-phytodienoic asit metil ester arasındaki sıcaklık gerektiren 180-200 ° C etkin bir şekilde gaz haline. </li><li> 30 dakika inkübasyondan sonra metilasyon reaksiyonu istenmeyen ikincil reaksiyon önlemek için durdurulması gerekir. Bu her flakon 2M bir asetik asit-çözümü (hekzan) 4 ul ekleyerek yapılır. Kapaklar ve kısa bir vorteks şişeleri kapattıktan sonra, numuneler, başka bir 30 dakika boyunca inkübe izin verilir. </li><li> Bu buhar faz ekstraksiyon için kullanılan filtreler, el yapımı ve ticari olarak sunulan formu mevcut değildir. Onlar bir taraftan 4cm hakkında uzunluğu iyi oturan bir cam tüpü içine bir dış teflon tüp (7-8cm uzunluğunda), oluşur. Diğer taraftan teflon tüp iç cam tüp ulaşıncaya dek bir paslanmaz çelik hasır disk tüp içine itilir üzerine yaklaşık 20-30mg adsorban (SuperQ 80/100 (üretilmiyor) veya HayeSep ® Q80/100 ya) eklenir. Başka bir paslanmaz çelik hasır disk takılı ve nihayet bir bardak ucu böylece dikkatle emici paslanmaz çelik hasır disk basarak, teflon tüp içine iter. Ayrıntılı bir resmi bir filtre, Şekil 1&#39;de gösterilmiştir. <img src="/files/ftp_upload/1127/1127fig1.jpg" alt="Şekil 1" /> Şekil 1. </li><li> Adım 11 inkübasyon süresi boyunca, filtreler, metil ve böylece, uçucu bitki hormonları toplanması için 200ul diklorometan her 2 yıkar sonra ilk 200ul metanol kullanarak yıkanacak, ve. Filtreler kalan Solvent sonra hava üflenir. Filtreler, buhar faz ekstraksiyon yoluyla uçucu hormonların yakalama için kullanılabilir. </li><li> Inkübasyon 30 dakika sonra 4ml flakon kapaklar kauçuk kapaklı bir neşter ile kesilir. Temizlenmiş filtreler 800ml/min hakkında bir akış hızında bireysel vakum hatları bağlı buhar faz ekstraksiyon bitki hormonları toplamak için. Filtreleri sonra kesme septa ile şişenin içine yerleştirilir. Küçük bir pipetlemeyin ucu da bir hava girişi olarak hareket eklenir. Prosedürün bu ilk bölümü sırasında eklenen filtreler ile şişeleri yapılacakflakon içinde herhangi bir sıvı filtre ucu, filtre ve daha sonra da GC / MS kirletici ile temas almak önlemek için dik bir pozisyonda. Filtre ile tüm sıvı buharlaşıncaya kadar şişeleri sonra elle tutulur. Sonra ekli filtreleri ile şişeleri ısıtma bloğu ve 3dk için toplanan ısı buharlaşmış bileşikler üzerine yerleştirilir. Sisteminin yanı sıra kurulum temel bileşenler Şekil 1&#39;de tasvir edilmektedir. Bu son buhar faz ekstraksiyon adımdan sonra hala bağlı filtreleri ile şişeleri rafa yerleştirilir ve soğuması için izin verilir. </li><li> Filtreleri tekrar oda sıcaklığına ulaşmış, bir ekleme ile donatılmış bir 1,5 ml GC-flakon diklorometan 150ml ile yıkandı. Şişeleri kapatılmış ve GC / MS ile analiz filtre kalan solvent üfleme Alter. </li></ol> Bölünmüş bir splitless enjektör / analizi için kullanılan kimyasal iyonizasyon modu (CI) işletilen bir kütle spektrometresi ile arayüzü (° C, enjeksiyon hacmi 1μ splitless modu 250)) ile donatılmış bir gaz kromatografisi. Bileşikler HP 1MS sütun ayrılmış (30m x 0.25mm x 025μm) 40 ° C enjeksiyondan sonra 1dk için düzenlenen ve daha sonra sıcaklık 15 programlanmış ° C / dak helyum ile 250 ° C (10dk), taşıyıcı olarak gaz (sabit akışı 0.7ml/min). MS-tabanlı analiz için iki Quadropole (örneğin Agilent HP5973) ve bir iyon tuzak tabanlı (örneğin Varian Satürn 2200) kütle spektrometresi olabilir. CI gaz ya izobütan (gaz tankları geliyor) veya metanol (bir sıvı rezervuar buharlar kullanılır) kullanımı gibi, ancak, her durumda iyonizasyon reaksiyon parçaları seçilen iyon sayısı (SIC) programları önce kontrol edilmelidir oluşturulur. Biz CI reaktif olarak Varian GC 3900/Saturn MS 2200 sistemi ve metanol için aşağıdaki programı kullanılır: 5.00-11.30min iyonları (M 1) + 153-158 (metil salisilat 153, metil 2 H 5-salisilat); 11.30-13.30min iyonları (M 1) + 207-228 (metil jasmonate 207 ve 225, metil dihydrojasmonate 209 ve 227); 13.30-16.30min iyonları (M 1) + 237-300 (metil absisik asit 261, metil 2 H 6-absisik asit 267 (absisik asit ağırlıklı olarak [MH 2 O 1] + iyonları). bileşikler otantik piyasada mevcut standartlar ile karşılaştırılması ile tespit edilmelidir. JA, SA, ve ABA Niceleme pik alanı ilişkilendirilerek yapılır ilgili internal standart pik alanı (çıkartılan iyonları) (iyonlar alınır) ve aynı zamanda kullanılan bitki materyali taze ağırlığı dayanmaktadır.

<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>

Burada sunulan yöntem, büyüklüğü ve kimyasal yapısı bileşik çıkarılan, yakacak ve buharlaşmış olan engellemez bitki sinyalizasyon bileşiklerin çok geniş bir dizi için güvenilir olduğu kanıtlanmıştır. Ayrıca, burada açıklandığı gibi metilasyon prosedürü türevlendirme tek yolu değildir. Silyllation ve asetilasyon alternatif yöntemler ve protokoller kolayca online olarak bulunabilir. Kütle spektrometresi tek iyon sürecinden çok fazla sayıda istenmeyen iyo...

monitoring plant hormones during stress responses

Bitki hormonları ve ilgili sinyalizasyon bileşikler, çeşitli çevresel uyaranlara ve gerilmeler bitki yanıtların düzenlenmesinde önemli bir rol oynamaktadır. En şiddetli gerilmeler arasında, böcek herbivory, patojen enfeksiyon, ve kuraklık stresi. Bunların her biri, böylece bitki hayatta kalmasını bunların ince ayarını yanıtları bilinen belirli bir hormon seti ve / veya kombinasyonları, vurgular. Bu yanıtların düzenlenmesinde yer alan önemli hormonların Jasmonik asit (JA), salisilik asit (SA) ve absisik asit (ABA). , Bolluk içinde bir zamansal hem de mekansal bir moda değişiklikleri daha iyi izlemek için bireysel hormonların yanı sıra gerekli bu tepkiler sırasında potansiyel etkileşim rolünü anlamak için. Bu ve diğer sinyalizasyon bileşikler, kolay, hassas ve tekrarlanabilir ölçümü için buhar faz ekstraksiyonu ve gaz kromatografisi / kütle spektrometresi (GC / MS) analizi (1, 2, 3, 4) dayanan bir yöntem geliştirdi. 1-propanol, diklorometan ile karıştırıldığında asidik sulu bitki doku ayıkladıktan sonra bu bileşiklerin karboksilik asit içeren bileşikler, mavi, ısı altında volatilized ve polimerik emici üzerinde toplanır. Analitler bir örnek şişesine elüsyon sonra gaz kromatografisi ile ayrılır ve kimyasal iyonizasyon kütle spektrometresi tarafından algılanır. Analit ve internal standart pik alanları ile ilgili basit ölçümü için uygun iç standartlarının kullanımı sağlar.

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

Monitoring Plant Hormones During Stress Responses

Küçük doku örneklerinde birden fazla bitki hormonların hızlı çıkarma ve analiz için izin veren basit bir yöntem sağlanır. Bu prosedür, buhar faz ekstraksiyon ağırbaşlı arıtma adımı olarak kullanır. Örnekleri, GC / MS ile ağırlıklı olarak üretir kimyasal iyonizasyon (M 1) + iyonları tarafından analiz edilir.

Stres Cevaplarının sırasında Bitki Hormonları İzlenmesi

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 Görüntüleme.  University of Texas. Küçük doku örneklerinde birden fazla bitki hormonların hızlı çıkarma ve analiz için izin veren basit bir yöntem sağlanır. Bu prosedür, buhar faz ekstraksiyon ağırbaşlı arıtma adımı olarak kullanır. Örnekleri, GC / MS ile ağırlıklı olarak üretir kimyasal iyonizasyon (M 1) + iyonları tarafından analiz edilir.

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.

Hücre tipi Özgül gösterilmeden FACS yardımlı kullanarak Doku ve Organ spesifik Fitokrom Yanıtları incelenmesi 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 ...

Biyoloji

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.

İnsektisit Direnci İzleme için biyoanalizlere

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.

Predasyon Risk, Herbivore Fizyolojik Stres ve Bitki Çöp mikrobiyal bozunma Bağlama

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.

Polizom profilleme Stres Durumlardaki Çeviri başlatılması Analizi

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.

Topraksız: A yönlü Sistemi Toksik Elements Besin Uygunluk ve Pozlama Besin Tahsis ve Bitki Cevapları Eğitim için

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.

Split yeşil flüoresan Protein sistemi effectors görselleştirmek için bakteri enfeksiyonu sırasında teslim.

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.

C. Eleganlarda Fizyolojik Stres Tepkilerinin Ölçüleri

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 E3 Ubiquitin Ligase fonksiyonunun analizi

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.

CRISPR/Cas9 Teknolojisi Kullanılarak Göçmen Locust'un Homozigot Mutantlarının İnşası

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.

Gen İfadesindeki Değişiklikleri ve Plasmodiophora brassicae'ye Fizyolojik Yanıtları Görselleştirmek için Temizleme ve Floresan Mikroskopisinin Birleştirilmesi

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