彼の microrespirometer の使用のための博士ポール霜とニコール ワーグナーとアレックス ベルのテクニカル サポートに感謝したいと思います。この作品資金が供給された B.J.S. するレベルの補助金

1. トウモロコシ穂軸感染
<ol><li>トウモロコシ(品種黄金のバンタム) の成長まで、穂軸が形成され、シルク (約 60 日) を開始しています。</li>
	<li>互換性のある半数米アルテリシジンの文化系統の前述の標準プロトコルを使用して説明7。</li>
	<li>前述7として標準プロトコルを使ってトウモロコシの穂軸に感染します。</li>
</ol>2. 冬の収穫
<ol><li>オートクレーブ装置 (ファンネルを Büchner、フラスコを Büchner、ミキサー、遠心瓶 250 mL、フラットのへら、水) 121 ° C (標準的な水の液体のサイクル) で少なくとも 30 分滅菌のサイクル乾燥標準を使用して。</li>
	<li>かみそりを使用して植物 (約 28-35 日ポスト感染) から感染したコブを削除し、ベンチ プロテクターで覆われているトレイに穂軸を設定します。</li>
	<li>かみそりの刃で穂軸から腫瘍を削除し、ビーカーに収集します。</li>
	<li>腫の約 1/3 フルまで 250 mL 研究所ブレンダー カップを埋めるし、ブレンダー カップが約 3/4 までオートクレーブ dH2O を追加検討します。腫瘍が均質になるまで低速、ミキサーをパルスで中断されます。</li>
	<li>水トラップを放置して 1 L 吸引ビンは、真空ポンプを接続します。</li>
	<li>フラスコを Büchner の大きい漏斗を挿入し、寒冷紗の 4 つの層を Büchner じょうごの底を並べ。</li>
	<li>真空ポンプをオンにします。</li>
	<li>ヘラで寒冷紗とこすりを通じて均質化腫瘍の部分を注ぐ。</li>
	<li>寄主をフラッシュするために、寒冷紗にいくつか dH2O を注ぐ。</li>
	<li>DH2まで繰り返し、寒冷紗を通って来る O は、明らかです。</li>
	<li>最大冬復旧できるようにフィルターに均質化腫瘍物質を含む寒冷紗を絞る。</li>
	<li>1 L 吸引ビンが近くなっている場合完全な大きなエルレンマイヤー フラスコに空、脇に置いておきます。</li>
	<li>寒冷紗の新しい作品を入れて、フィルターと腫瘍のすべてが破壊されているし、フィルターまで (2.7-2.12) の手順を繰り返します。</li>
	<li>フィルター処理された寄主を注ぎオートクレーブ 250 ml 遠心分離機、1,000 x g で 5 分間遠心し、上清をデカントします。</li>
	<li>遠心分離によってペレットすべてフィルター処理された寄主のされるまで、手順 2.14.</li>
	<li>少量の水と 50 mL 遠沈管への転送でペレットを中断します。</li>
	<li>5 分、1,000 x g でチューブを遠心し、上清をデカントします。</li>
	<li>DH2O の約 50 mL にペレットを中断、5 分、1,000 x g でチューブを遠心し、上清をデカントします。優しくへらと処分灰色のトップ層をこすり。上はグレーのレイヤーがされなくなるまでを繰り返します。</li>
	<li>乾燥試料は真空デシケータで一夜します。</li>
	<li>使用するまで 4 ° C で乾燥した寄主を格納します。</li>
	<li>
必要な場合は、発芽に誘導する前に硫酸銅8寄主を扱います。扱われていない場合は、細菌や他の汚染を欠いている、純粋な寄主を表すサンプルを確認する寄主の徹底的な顕微解析を実行します。
	<ol>
<li>50 mg を約 1.5 mL 遠心チューブに寄主を重量を量る。</li>
		<li>寄主を含む 1.5 mL 遠心チューブに 0.75% CuSO4の約 1.0 mL を追加します。ピペットの上下の攪拌 3 h のサンプルに続いて CuSO4ソリューション、寄主を中断します。</li>
		<li>5 分間 2,500 x g でサンプルを遠心し、上清を除去します。滅菌水で冬のペレットを再懸濁します、遠心分離を繰り返し、上澄みを除去します。</li>
		<li>2.21.3 のステップを 2 回繰り返します。</li>
	</ol>
</li>
</ol>3. 冬の生存率及び発芽試験
<ol><li>彼らの生存率を評価するために 1.5 mL 遠心チューブに米国アルテリシジン寄主の約 10 mg と発芽のタイミングを重量を量る。</li>
	<li>バイオ セーフティ キャビネット、ストレプトマイシン硫酸塩 (160 μ g/mL) を添加したジャガイモ デキスト ロース スープ (PDB、24 g/L) を準備します。</li>
	<li>PDB の 500 μ L で、寄主を中断します。優しくのピペットをミックスし、寄主のすべての塊を破る。</li>
	<li>冬のペンションをオートクレーブ 250 mL PDB の 10 ml 三角フラスコに転送します。</li>
	<li>28 ° c 12-16 h 90 rpm で振盪フラスコを孵化させなさい。</li>
	<li>バイオ セーフティ キャビネット、発芽し、顕微鏡スライドを準備する誘導寄主の 20 μ L のサンプルを削除します。</li>
	<li>
顕微鏡を使用して、存在している発芽の段階と細菌汚染の有無を視覚的に評価します。
	<ol>
<li>寄主の数を数える V 診断の使用を行うし、発芽率を決定します。</li>
		<li>場合にのみステージ I 寄主が存在、冬発芽を評価する前に 24 時間の合計はフラスコをインキュベートし続けます。非実行可能なサンプルを認める前に最大 48 時間の培養を続けます。</li>
		<li>細菌汚染が存在する場合ストレプトマイシン硫酸塩 (160 μ g/mL)、硫酸カナマイシン (50 μ g/mL) PDB を補完し、手順 3.1 から 3.7。細菌汚染が解決しない場合、硫酸銅と寄主を扱う、手順 3.1 から 3.7.</li>
	</ol>
</li>
</ol>4. 呼吸を監視するため発芽の誘導
<ol><li>同量の重量を量る (e.g。、50 mg) 各呼吸実験の寄主の。</li>
	<li>バイオ セーフティ キャビネット、寄主をオートクレーブ呼吸室に追加します。</li>
	<li>ストレプトマイシン硫酸塩 (160 μ g/mL) とカナマイシン硫酸塩 (50 μ g/mL) 塗りつぶし PDB (24 g/L) とチャンバーが補われます。</li>
	<li>上下のピペット冬の懸濁液を作成します。</li>
	<li>気密のシールを作成する商工会議所の商工会議所のふたを配置します。</li>
</ol>5. 酸素消費率 (OCR) 測定値を得る
<ol><li>商工会議所ラック (28 ° C に予熱) 水浴中でチャンバーを配置します。</li>
	<li>オープニングの中商工会議所の O2プローブを配置します。</li>
	<li>「SensorTrace レート」プログラム上でリアルタイムに表示されるデータ ポイントを監視し、、プローブが (~ 3 分商工会議所にプローブを配置後) を安定化します。</li>
	<li>2 秒間隔で記録した測定値と 6 時間の継続的に O2レベルを測定する「測定」をクリックします。</li>
	<li>測定を停止し、各サンプルを分析するための手順 4.1-5.4 を繰り返します。</li>
	<li>クリックして、データを Microsoft Excel にエクスポート"ファイル |エクスポート |.Xls として保存"。</li>
</ol>6. データの分析
<ol><li>
OCR を計算します。
	<ol>
<li>エクスポートされた Excel ファイル"Within_Rates"タブの下でそれぞれの商工会議所の測定 (nmol/h)「率」を記録します。</li>
		<li>各実験のサンプルでは、空白のチャンバー修正された OCR 値を取得するに実験試料室の「率」からの「率」を減算し、この数値の絶対値を取る。</li>
		<li>質量使用携帯修正絶対 OCR 値で除算して寄主の mg 当たり合計 OCR を計算します。</li>
		<li>平均は、各系統の"寄主の mg 当たり OCR"値をレプリケートします。</li>
	</ol>
</li>
	<li>適切な統計的手法を用いたデータ分析 (e.g。、スチューデントのt-テスト、分散分析) 他の統計ソフトウェアの Microsoft Excel を使用して。</li>
	<li>生データをグラフ化、グラフ化したい場合は、各時点の残存酸素の割合を計算します。単独で最初の読書を分割 (100% 酸素残存) 100 倍、各後続の読み取りの最初の読書、除算し、100 倍チャンバ内酸素の割合を取得します。</li>
</ol>7. 冬発芽発芽の段階で寄主を分離するための誘導
<ol><li>バイオ セーフティ キャビネットのストレプトマイシン硫酸塩 (160 μ g/mL) を添加した PDB (24 g/L) を準備します。</li>
	<li>1.5 mL 遠心チューブに米国アルテリシジン寄主の約 10 mg を配置します。</li>
	<li>PDB の 500 μ L で、寄主を中断します。優しくピペット培地で寄主の塊がなくなるまで混ぜます。</li>
	<li>冬サスペンションを含む PDB ストレプトマイシン硫酸塩を添加したオートクレーブ 250 mL 三角フラスコに転送します。</li>
	<li>一晩で 28 ° C 90 rpm で振動フラスコを孵化させなさい。</li>
</ol>8. シャーレおよびマイクロマニピュレーターの準備
<ol><li>
マイクロキャピ ラリーの準備、およびサンプル コレクションの液滴行数のピペッティングしてシャーレ (57 cm2) を準備します。
	<ol>
<li>ペトリ皿の上部に 5 μ L (x 4) dH2O 液滴をピペットします。</li>
		<li>2 μ L (x3) サンプル コレクションに使用するシャーレに RNA 安定化溶液をピペットします。</li>
		<li>ペトリ皿に寄主を発芽 5 (30) x μ L 水滴をピペットします。</li>
	</ol>
</li>
	<li>鉱物油の 15 mL をシャーレに追加します。すべての液滴が進む前に油で覆われていることを確認します。</li>
	<li>マイクロキャピ ラリー ホルダーにそれを置き、鉱物油に水没して毛細管は、マイクロキャピ ラリーを入力する鉱物油になります 15 μ m の内径、1 mm フランジ、55 ミリメートルの長さ、20 ° の先端角とマイクロキャピ ラリーを準備します。水液滴に持ち込む前に、マイクロキャピ ラリー内の圧力のリリースです。吸引水でマイクロキャピ ラリーを準備します。</li>
</ol>9. ステージ固有の発芽の寄主の分離
<ol><li>発芽粒のいずれかに準備マイクロキャピ ラリーを移動マニピュレーターのコントロールを使用する。液滴を浸透、マイクロキャピ ラリーを下げ、関心の発芽の段階で発芽冬までマイクロキャピ ラリーの口をもたらします。</li>
	<li>ゆっくりと発芽の冬をキャプチャする吸引します。一度、冬を迎えて、マイクロキャピ ラリーを吸引を停止します。繰り返して、マイクロキャピ ラリーを約 5 寄主があります。</li>
	<li>マニピュレーターにマイクロキャピ ラリーを上げるし、RNA 安定化ソリューションのコレクション液滴にそれをもたらします。液滴を浸透し、寄主の液滴を注入します。</li>
	<li>約 1,000 の寄主がキャプチャされるまで 8.1 に 8.3 の手順を繰り返します。</li>
</ol>10 コレクション液滴の回収
<ol><li>コレクション液滴をピペットし、RNase 空き DNase の 2.0 mL 遠心チューブの蓋に転送。コレクション液滴を乱すことがなく、ピペットと鉱物油を慎重に取り外します。</li>
	<li>RNA の隔離など下流用、寄主を使用します。</li>
</ol>

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<li>Christensen, J. J. <a target="_blank" href="https://trove.nla.gov.au/work/14819898">Monograph Number 2</a>. , The American Phytopathological Society. Worcester, MA. (1963).</li>
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<li>Warburg, O. Metabolism of tumours. Biochemische Zeitschrift. 142, 317-333 (1923).</li>
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</ol>

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担子菌赤植物病原菌は、毎年作物の損失の数十億ドルを引き起こします。これらの病原体の大半は、真菌の散布と有性生殖に不可欠な寄主を生成します。寄主の発芽と開発の知識を得ることは、これらの菌によって引き起こされる破壊的な病気の広がりを理解する重要です。主な制御点で分子の変更を識別するために我々 は、生理学的変化と発芽の段階で寄主を分離する別のタイミングを識...

黒穂病菌 (Ustilaginales) から成っている牧草、トウモロコシ、大麦、小麦の重要な穀物を含む作物の損失のドルの十億を引き起こす感染以上 1,600 種年間1。これらの菌は、寄主細胞壁を発色が暗く、散布剤の生産によって特徴付けられます。寄主はホスト植物の間分散のストレスの中に遺伝物質を保護するために機能し、2年間休止状態で保持できます。そのため、寄主は病気の広がりの重要なコンポーネントです。
冬の生物学を研究するためには、当研究室は、モデル黒穂病菌経路(米国アルテリシジン) 'トウモロコシの共通黒穂病' 病気の原因物質であるを利用しています。成熟した米国アルテリシジン寄主は増殖停止、減らされた細胞の代謝と細胞呼吸3の低レベルによって特徴付けられます。有利な環境条件 (e.g。、特定の糖の存在)、米国アルテリシジン寄主が発芽し、減数分裂、生産担子胞子による感染症の新ラウンドを開始することができますこれを完了。発芽は、高められた呼吸、代謝に戻り活動、発芽4の観察可能な形態学的段階を経て進行が特徴です。
発芽の初期段階に高められた呼吸と代謝機能が含まれています、ただし、変更の形態学的兆候がないです。米国アルテリシジンの呼吸の変化の元の測定は Warburg フラスコ装置5マノメーターで高い酸素消費を測定 50 年以上前に行われました。クラーク型 microrespirometer を使用して発芽の時間経過とともに酸素消費量を測定することによって冬発芽時における呼吸の正確な変化を研究する新しい、シンプルな方法を行った。野生型の呼吸速度の変化を研究するこのメソッドが以前米菌細胞の倍加と不良ミトコンドリア6、変異体中に冬の呼吸速度の変化を勉強するここのプロトコルを適応していると発芽。これは早い分子でき事を調査するため発芽開始後の適切な時期に寄主を対象することができますようにする呼吸の変更のタイミングを正確に識別できる手段を提供します。一度、冬から、promycelia が出てくるが、非同期の性質は調査の特定の段階で十分な寄主の分離を抑制、発芽の進行は顕微鏡的続くことができます。我々 は、物理的に発芽の形態学的段階で寄主を収集する体外受精に使用するものと同様レーザーマイクロダイ セクション技術を開発しました。

<table><tbody><tr><td>Streptomycin Sulfate</td><td>BioShop</td><td>STP101</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>BioShop</td><td>KAN201</td><td></td></tr><tr><td>Potato Dextrose Broth</td><td>BD Difco</td><td>254920</td><td></td></tr><tr><td>1 L Waring Laboratory blender</td><td>Waring</td><td>7011S</td><td></td></tr><tr><td>Cheesecloth</td><td>VWR</td><td>470150-438</td><td></td></tr><tr><td>Nalgene Polypropylene Desiccator with Stopcock</td><td>ThermoFisher Scientific</td><td>5310-0250</td><td></td></tr><tr><td>&lt;em&gt;Unisense MicroRespiration system&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>MicroRespiration Sensor (O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Unisense</td><td>OX10</td><td></td></tr><tr><td>MicroOptode Meter Amplifier</td><td>Unisense</td><td>N/A</td><td></td></tr><tr><td>MR-Ch Small</td><td>Unisense</td><td>MR-Ch</td><td></td></tr><tr><td>SensorTrace Rate Software</td><td>Unisense</td><td>N/A</td><td></td></tr><tr><td>MicroRespiration Rack</td><td>Unisense</td><td>MR2-Rack</td><td></td></tr><tr><td>MicroRespiration Stirrer</td><td>Unisense</td><td>MR2-Co</td><td></td></tr><tr><td>&lt;em&gt;Microdissection system&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Axio Vert.A1 Inverted Light Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Coarse Manipulator</td><td>Narishige</td><td>MMN-1</td><td></td></tr><tr><td>Three-axis Hanging Joystick Oil Hydraulic Micromanipulator</td><td>Narishige</td><td>MMO-202ND</td><td></td></tr><tr><td>Pneumatic Microinjector</td><td>Narishige</td><td>IM-11-2</td><td></td></tr><tr><td>TransferTip (ES)</td><td>Eppendorf</td><td>5175107004</td><td></td></tr></tbody></table>

investigating teliospore germination using microrespiration analysis and microdissection

クロボ菌類は、いくつかの壊滅的な農業病気の病因のエージェントです。彼らは肉厚分散エージェントである寄主の生産によって特徴付けられます。寄主は、何十年も休眠することができます。休眠は、低代謝率によって特徴付けられる、高分子生合成を一時停止し、呼吸のレベルを大幅に削減します。必要な環境シグナルを受け取ったら、寄主は感染症の新ラウンドを開始することができます半数体の細胞を生産する発芽します。冬の発芽は、高分子合成、高められた呼吸および劇的な形態学的変化の再開によって特徴付けられます。発芽の初期段階中に細胞呼吸の変化を正確に測定、するためにクラーク型呼吸度計を用いた単純なプロトコルを開発しました。発芽の後の段階は特定の形態学的変化によって区別されますが、発芽は非同期です。我々 は異なる発芽段階で寄主を収集するために私たちができる顕微解剖技術を開発しました。

休眠寄主が呼吸 (1,075 ~ µmol/h/mg) 発芽と比較しての低レベルを示すことを確認した冬の休眠および発芽における呼吸速度の変化を測定するクラーク型 microrespirometer ベース メソッドを使用して、寄主 (2,614 ~ µmol/h/mg;図 1 a)。これは、休眠寄主と発芽を誘導されて寄主の呼吸率は平均で ~2.4-fold 変更を表します。さらに、発芽に誘導されている寄主酸...

Watch this Scientific Journal Video about Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection at JoVE.com

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

クロボ菌類は、壊滅的な農業の多くの病気を引き起こします。彼らは環境のキューに応答で発芽休眠の寄主として分散しています。発芽過程における分子の変化を調査する 2 つの方法の概要を説明: 呼吸増加代謝活性化を検出するための測定と形態学的段階に寄主を分離することによって変化する分子イベントを評価します。

Microrespiration 分析と顕微解剖を使用して冬の発芽を調査

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

investigating-teliospore-germination-using-microrespiration-analysis

Research

JoVE Journal

Biology

7.2K ビュー.  Trent University. クロボ菌類は、壊滅的な農業の多くの病気を引き起こします。彼らは環境のキューに応答で発芽休眠の寄主として分散しています。発芽過程における分子の変化を調査する 2 つの方法の概要を説明: 呼吸増加代謝活性化を検出するための測定と形態学的段階に寄主を分離することによって変化する分子イベントを評価します。

ビデオ: Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

の解剖サッカロマイセスセレビシエ ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

生物学

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

交配との四分子の分離クラミドモナス遺伝子解析用

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

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 ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

細菌胞子の発芽中に皮質断片を検出

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

免疫・感染学

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

非常に小さいスペースで先端成長植物細胞の伸長機能を勉強するマイクロ流体デバイスの開発

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

生物工学

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Germinosomes と枯草菌胞子の内部の膜の可視化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

の長期、高分解能共焦点タイムラプスイメージング<em&gt;シロイヌナズナ子葉</em発芽過程&gt;表皮

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

トロピック応答におけるカロース媒介Plasmodesmalゲーティングの役割を検証するための戦略

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

有害な効果の低圧プラズマ滅菌を使用して住んでいる携帯顕微鏡枯草菌胞子の生存に調査

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

菌従属栄養植物組織における真菌のコロニー形成と種子の共生発芽を解釈するための顕微鏡技術

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...