<ol><li>まず、抽出のために2ミリリットルのスクリューキャップ付きバイアルを準備する必要があります：400ul抽出バッファーを加え（1 - プロパノール：H2O：濃塩酸2:1:0.002体積/体積/体積）とそれぞれの内部標準の10μlのは（10ng/μl ）バイアルに。液体窒素で凍結し、セラミックビーズを追加。 </li><li>バイアルがまだ液体窒素内に位置している間（50〜200mgの間）植物材料を追加します。追加された植物材料の重量は、生体重に基づいて正確な定量化、のために後で可能にするためにさらに処理する前に推定する必要があることに注意してください。 </li><li>すべての液体窒素がしっかりとキャップでバイアルを閉じる前に蒸発されていることを確認します。溶剤の流出を防ぐためにキャップがゴム製のシールを持っている必要があることに注意して、したがって、均質化中に化合物を抽出した。 </li><li> 30秒のための6000の速度（Precellys用）でFastPrepまたはPrecellysビードのグラインダーのホモジナイザーで組織をホモジナイズ。 </li><li>個別にホモジナイザーからバイアルを取り外し、慎重にキャップを開き、各試料にジクロロメタンの1mlを加える。しっかりと再びバイアルを閉じて、6000（Precellys）で10 - 15秒のために再度ホモジナイズする。 </li><li>卓上型遠心機にバイアルに移し、60秒のための10.000xgで水相から有機相を分離。 </li><li>相分離の後にクリーンな4ミリリットルガラスバイアルに、個々のバイアルから下の緑色の層（有機相、ホルモンおよび内部標準を含む）を転送する。水（上相）の転送を避ける。また、酢酸エチル、ジクロロメタンの代わりに使用できます。その場合、ethyacetateフェーズでは、上位層（緑色）になると、今削除して、新鮮な4ミリリットルガラスバイアルに移し方が簡単ですでしょう。前と同様に、水の移動を避ける。 </li><li> 10月25日分程度（サンプルに依存）のためのマニホールドを通して空気と溶剤を吹き飛ばす、サンプルが乾燥している場合（慎重に）単一の気流の先端に確認してください。これは化合物の損失を引き起こす可能性のための過乾燥するサンプルは、しないでください。 </li><li>サンプルは乾燥されると我々は、カルボン酸含有我々のサンプル中の化合物のメチル化を開始できます。最初に、diethyether /メタノール混合物（9:1 vol / vol）の100μlの2Mトリメチルシリルジアゾメタン溶液（ヘキサン、シグマアルドリッチで）の4μlに続く各バイアルに添加されています。バイアルは直ちにテフロンライニングシリコンセプタムを装備したオープントップスクリューキャップで閉じられます。穏やかに振盪し、25〜30分間室温でインキュベートする。 </li><li>手順9を終えた後、目的のコレクションの温度で加熱ブロックとセットの電源を入れます。メチル化化合物の揮発性は、その大きさではなく、= O、- OH、及び- NHのような他の、より多くの極性基に依存する。ジャスモン酸とサリチル酸のメチルエステルのコレクションの温度に対して約80 ° Cで十分である、一方、例えば、C18脂肪酸のような他の化合物、ジャスモン酸 - アミノ酸複合体、コロナチン、および12 ​​- オキソ- phytodienoic酸メチルエステルの間の温度を必要とする180から200 ° Cを効果的に揮発する。 </li><li> 30分インキュベーションした後にメチル化反応は、不要な副反応を避けるために停止する必要があります。これは、各バイアルに2M酢酸-溶液（ヘキサン中）4μlを加えることにより行われます。キャップと短いボルテックスでバイアルを閉じた後、サンプルは別の30分間インキュベートされている。 </li><li>この気相抽出に使用されるフ​​ィルタは、ハンドメイドと表示されるフォームで市販されていない。彼らは、片側から約4cm長さのよくフィットガラス管が挿入されている外側のテフロンチューブ（7〜8センチメートル長い約）、で構成されています。それは内側のガラス管に到達するまでテフロンチューブの反対側にステンレスメッシュディスクは、管の内部に押されていることへの約20 - 30mgの吸着剤（SuperQ 100分の80（販売終了）またはHayeSep ® Q80/100のどちらか）追加されます。別のステンレス鋼のメッシュディスクが挿入され、最終的にガラスチップは、それによって慎重に吸収剤でステンレスメッシュディスクを押して、テフロンチューブにプッシュされています。詳細な画像フィルタを図1に示されています。 <img src="/files/ftp_upload/1127/1127fig1.jpg" alt="図1" /> 図1。 </li><li>ステップ11のインキュベーション時間の間に、このようにメチル化し、揮発性の植物ホルモンのコレクションのためのフィルタは、200ulのジクロロメタン各2回洗浄後、メタノールの最初の200ulを使用して洗浄す​​る必要があります、と。フィルタ内の残りの溶剤を空気で吹き飛ばします。フィルタは、今気相抽出を通じて揮発性ホルモンの捕捉に使用することができます。 </li><li>インキュベーションの30分後に4ミリリットルバイアルキャップのラバーセプタムをメスでカットされます。気相抽出によって植物ホルモンを収集するにはクリーニングフィルターは約800ml/minの流量で、個々の真空ラインに接続されています。次に、フィルターをカットセプタムバイアルに挿入されます。小さなピペットチップは、空気入口として機能するように挿入されます。この手順の最初の部分の間に挿入されたフィルタを使用したバイアルは、保持する必要がその後、フィルタともGC / MSを汚染するフィルターチップ、と接触して得るためにバイアル内の液体のいずれかを避けるために、直立位置。すべての液体がフィルターを介して蒸発するまで、バイアルを手で保持されています。その後、付属のフィルター付きバイアルを加熱ブロック上に配置し、熱蒸発化合物は、3分のために収集されます。システムの基本コンポーネントと同様にセットアップを図1に示されている。この最後の蒸気相抽出工程の後にまだ接続されているフィルタを使用してバイアルをラック上に配置し、クールダウンに許可されています。 </li><li>フィルタは、再び室温に達したときは、インサートを装着した1.5​​ mlのGC -バイアルにジクロロメタンの150ミリリットルで溶出されています。バイアルに蓋をし、GC / MSによって分析されているフィルタから、残りの溶剤を吹き変える。 </li></ol>スプリット/化学イオン化モード（CI）で動作質量分析計とのインターフェーススプリットレスインジェクター（スプリットレスモード250℃、注入量1μ）の分析に使用する必要がある装備、ガスクロマトグラフ）。化合物は、40で開催されたHP - 1MS列（30M X 0.25ミリメートルX025μm）·キャリアとしてヘリウム注入後1分間、C、およびその後15でプログラムされた温度° C / minで250℃まで（10分間）、上で分離されていますガス（定流量0.7ml/min）。 MSベースの分析の両方の場合は、quadropole（例えば、アジレントHP5973）とイオントラップベース（例：バリアンサターン2200）質量分析計を使用することができます。 CIガスイソブタン（ガスのタンクに来る）やメタノール（液体タンクからの蒸気が使用されている）のどちらかを使用することができますよう、しかし、それぞれのケースでイオン化反応のフラグメントは、選択されたイオン数（SIC）プログラムの前にチェックしてください作成されます。我々は、CIの試薬 ​​としてバリアンGC 3900/Saturn MS 2200のシステムおよびメタノールについては、次のプログラムに使用されている：5.00 - 11.30minイオン（M +1）+ 153〜158（サリチル酸メチル153、メチル2 H 5 -サリチル酸）; 11.30 - 13.30minイオン（M +1）+ 207〜228（ジャスモン酸メチル207と225、メチルジヒドロジャス209および227）、13.30 - 16.30minイオン（M +1）+ 237〜300（メチルアブシジン酸261、メチル2 H 6 -アブシジン酸267（アブシジン酸は、主に[MH 2 O +1] +イオンを生成する）。すべての化合物は、本物の市販の標準との比較によって識別されるべきである。JA、SA、およびABAの定量はピーク面積を相関させることによって行われます。それぞれの内部標準のピーク面積（抽出されたイオン）と（イオンを抽出）し、また使用される植物材料の生体重に基づいている。

<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>

ここで紹介する方法は、化合物のメチル化、サイズおよび化合物の化学的性質が抽出されることを防ぐしないことを提供し、気化をシグナリング植物の広い配列のために確実に動作することが実証されています。また、それは、本明細書に記載されているメチル化法は、誘導体化の唯一の方法ではありません。 Silyllationとアセチル化は、別の方式とプロトコルであり、簡単にオンラインで見?...

monitoring plant hormones during stress responses

植物ホルモンと関連するシグナリング化合物は、種々の環境刺激やストレスに対する植物の応答の調節に重要な役割を果たす。最も深刻なストレスの中で昆虫草食、病原体感染、および乾燥ストレスがあります。これらのそれぞれについて、その特定のホルモンのセットおよび/またはそれらの組み合わせを強調することにより、植物の生存を確保する、微調整応答することが知られています。これらの応答の調節に関与する主要なホルモンは、ジャスモン酸（JA）、サリチル酸（SA）、およびアブシジン酸（ABA）です。より個々のホルモンの役割だけでなく、これらの応答の間に彼らの潜在的な相互作用を理解するには、空間的な形でだけでなく、時間的にそれらの存在量の変化を監視する必要があります。これらおよび他のシグナリング化合物の、簡単で、感度が高く、再現性の定量化のために我々は気相抽出とガスクロマトグラフィー/質量分析（GC / MS）分析（1、2、3、4）に基づく方法を開発した。酸性水溶液、1 - プロパノールによる植物組織からこれらの化合物を抽出した後、ジクロロメタンと混合カルボン酸含有化合物は、メチル化熱で揮発し、高分子吸収剤で収集されます。サンプルバイアルに溶出後の検体は、ガスクロマトグラフィーによって分離され、化学イオン化質量分析法で検出。適切な内部標準の使用は、分析対象物と内部標準のピーク面積を関連付けることによって、単純な定量が可能になります。

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

Monitoring Plant Hormones During Stress Responses

簡単な方法は、小さな組織サンプルから複数の植物ホルモンの迅速抽出と分析ができるようにその提供されています。手順は、厳粛な精製工程として気相の抽出を使用しています。サンプルは主に生成する化学イオン化（M +1）とGC / MS +イオンによって分析されます。

ストレス応答時には植物ホルモンのモニタリング

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 ビュー.  University of Texas. 簡単な方法は、小さな組織サンプルから複数の植物ホルモンの迅速抽出と分析ができるようにその提供されています。手順は、厳粛な精製工程として気相の抽出を使用しています。サンプルは主に生成する化学イオン化（M +1）とGC / MS +イオンによって分析されます。

ビデオ: 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.

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ</em

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

生物学

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.

モニタリングの殺虫剤抵抗性のためのバイオアッセイ

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.

捕食リスク、草食動物生理的ストレスと植物リターの微生物分解を結ぶ

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.

ポリソームプロファイリングによってストレス状態の間に翻訳開始の分析

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.

水耕：栄養アベイラビリティと毒性元素への曝露に栄養の割り当てと植物の応答を研究するための汎用性の高いシステム

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.

緑の蛍光蛋白質感染時に細菌から配信されるエフェクターを視覚化するシステムを分割します。

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.

エレガンスにおける生理的ストレス応答の測定

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.

イン・ヴィトロ E3ユビキチンリガーゼ関数の解析

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技術を用いた渡りバッタのホモ接合変異体の構築

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.

透明化顕微鏡と蛍光顕微鏡を組み合わせて、アブラナ原虫に対する遺伝子発現の変化と生理学的応答を視覚化

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