我们承认Joris Winderickx教授的实验室，该实验室提供配有常规紫外灯的紫外线交联设备。 KG由KU Leuven研究基金支持，并承认FWO授予G065713N的支持。

作者没有什么可以披露的。

我们成功应用开发用于酵母和人类细胞的mRNA相互作用捕获，植物叶肉叶原生质体。叶肉叶细胞是植物叶片中主要类型的地面组织。该方法的主要优点是其使用体内交联从生理环境中发现蛋白质。 在这个协议中，我们主要存在许多的优化实验条件50（ 例如，原生质体的数量以作为起始原料和UV辐射的持续时间使用）。当使用至少10 7个原?...

真核生物使用多个RNA生物发生调控途径来维持细胞生物学过程。在已知类型的RNA，mRNA的是非常多样的并携带的蛋白质的编码能力和它们的同种型1个 .The PTGR通路引导预的mRNA 2,3的命运。来自不同基因家族的RBPs控制RNA的调节，并且在PTGR中，特异性mRBP通过直接物理相互作用指导mRNAs，形成功能性mRNP。因此，识别和表征mRBPs及其mRNP对于了解细胞mRNA代谢的调节至关重要2。在过去三十年中，各种体外方法 - 包括RNA电泳迁移率转移（REMSA）测定法，通过指数富集测定系统进化配体（SELEX），基于文库衍生构建体，RNA Bind-n-Seq（RBNS），放射性标记或定量荧光RNA结合测定法，X-射线晶体学和NMR光谱法4，5，6，7，8，9 -已被广泛应用到限制性商业惯例的研究，主要是从哺乳动物细胞。这些哺乳动物RBP研究的结果可以通过RNA结合蛋白DataBase（RBPDB）进行搜索，收集了已发表的观察10 。 尽管这些体外方法是强大的工具，但它们确定了来自给定的RNA序列库的结合RNA基序，因此在发现新的靶RNA的能力方面受到限制。计算策略预测基因组范围的RBP也是如此，它们基于蛋白质序列和结构15的保守性。为了克服这个，一个新的实验方法哈已被确定，其允许识别感兴趣的RBP相互作用的RNA图案，以及用于确定结合的精确位置。这种称为"交联和免疫沉淀"（CLIP）的方法由体内紫外线交联，然后进行免疫沉淀11 。早期研究表明，DNA和RNA核苷酸的光激活可以发生在大于245nm的激发UV波长处。通过胸苷的反应似乎是有利的（按光反应性降低的顺序排列:dT≥dC&gt; rU&gt; rC，dA，dG） 12 。使用波长为254nm（UV-C）的紫外光，观察到当RNA核苷酸和蛋白质残基之间的共价键仅在几埃（Å）的范围内时产生。因此，这种现象称为RNA和RBP的"零长度"交联。之后可以进行严格的净化程序<html>很少背景13,14杜热。 与CLIP相辅相成的策略是将体内紫外线交联与蛋白质鉴定相结合，以描述RBP的景观。许多这样的全基因组基因结合的蛋白质组已经从酵母细胞，胚胎干细胞（ESC），并使用该新的实验方法的人细胞系（ 即，HEK293和HeLa）中分离，被称为"mRNA的相互作用组捕获" 18 19，20，21。该方法由体内紫外线交联，然后是mRNP纯化和基于MS的蛋白质组学。通过应用这种策略，已经发现了许多含有非典型RBD的新型"月光"RBP，并且已经清楚的是，更多的蛋白质具有比以前所想象的RNA结合能力"&gt; 15，16，17，使用这种方法允许新的应用程序和调查限制性商业惯例时，回答新的生物学问题的能力。例如，最近的一项研究调查了mRNA的结合蛋白质（核心RBP的保护蛋白质组）酵母和人类细胞之间22 。 已经发现植物限制性商业惯例参与生长和发育（ 例如 ，在开花时间的转录后调节，昼夜节律钟，并在线粒体和叶绿体基因表达）的24，25，26，27，28，29 。此外，他们被认为在响应非生物胁迫的细胞过程中进行功能（ 例如，感冒，干旱，salinity，和脱落酸（ABA））31，32，33，34。基于RNA识别基序（RRM）和K同源性（KH）结构域序列基因， 拟南芥基因组中有超过200种预测的RBP基因;在水稻，大约250已注意到35,36。值得注意的是，许多预测限制性商业惯例似乎是独特的植物（ 例如，没有后生动物直向同源物，以含有RRM结构域预测拟南芥限制性商业惯例的约50％）35，这表明许多可以用作新的功能。大多数预测RBP的功能仍然没有表征23 。 通过使用来自拟南芥的幼苗，叶组织，培养的根细胞和叶肉叶原生质体的mRNA结合的蛋白质组分离基因相互作用组捕获近日有报道称38，39。这些研究表明在不久的将来系统地对植物功能性RBP进行编目的潜力很大。在这里，我们提出了从植物原生质体（ 即没有细胞壁的细胞）捕获mRNA相互作用的方案。 拟南芥叶片叶肉原生质体是叶细胞的主要类型。分离的原生质体允许UV光最佳地接近细胞。这种细胞类型可以在瞬时表达功能表征40,41蛋白质测定中。此外，原生质体已被应用到其它几种植物细胞类型和物种42，43，44（ 例如，彼得森等人 ，2009;和巴格曼伯恩鲍姆，2010;和Hong 等人 ，2012）。 &lt;p class ="jove_content"&gt;该方法总共包含11个步骤（ 图1A ）。首先分离拟南芥叶肉叶肉原生质体（步骤1），随后UV照射形成交联的mRNP（步骤2）。当原生质体在变性条件下裂解（步骤3）时，将交联的mRNP释放在裂解/结合缓冲液中，并通过oligo-d（T） 25珠（步骤4）下拉。经过几轮严格洗涤后，将mRNPs纯化并进一步分析。在纯化交联的mRNA之前，通过蛋白酶K消化mRBPs的变性肽，并通过qRT-PCR验证RNA质量（步骤5和6）。经RNA酶处理和蛋白质浓缩（步骤7）后，蛋白质质量由SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）和银染法（步骤8）进行控制。在交联样品（CL）和非交联样品（非CL;交联样品）之间，蛋白质带图案的差异可以容易地可视化。来自不经受紫外线照射的原生质体的阴性对照样品）。蛋白质的鉴定通过基于MS的蛋白质组学获得。通过一维聚丙烯酰胺凝胶电泳（1D-PAGE）分离来自CL样品的蛋白质以除去可能的背景污染，并使用胰蛋白酶将其"凝胶内消化"成短肽，并进行纯化（步骤9）。偶联到质谱（纳米LC-MS）的纳米反相液相色谱法允许测定mRNA结合蛋白质组中的确定蛋白质的量（步骤10）。最后，使用生物信息学分析对所鉴定的mRBP进行表征和编目（步骤11）。

<table><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.8 M Mannitol</td><td>Sigma</td><td>M1902-500G</td><td>Primary isotonic Enzyme solution &amp;amp;&lt;br/&gt; MMg solution</td></tr><tr><td>2 M KCl</td><td>MERCK</td><td>Art. 4935</td><td>Primary isotonic Enzyme solution &amp;amp;&lt;br/&gt; W5 buffer</td></tr><tr><td>0.2 M MES (pH 5.7)&lt;br/&gt; (4-morpholineethanesulfonic acid)</td><td>Sigma-aldrich</td><td>M2933</td><td>Primary isotonic Enzyme solution,&lt;br/&gt; W5 buffer &amp;amp;&lt;br/&gt; MMg solution,&lt;br/&gt; Filtration sterilization</td></tr><tr><td>Cellulase R10</td><td>Yakult Pharmaceutical Industry Co., Ltd.</td><td>CELLULASE&lt;br/&gt; &amp;ldquo;ONOZUKA&amp;rdquo;&lt;br/&gt; R-10, 10 g</td><td>Final isotonic enzyme solution</td></tr><tr><td>Macerozyme R10</td><td>Yakult Pharmaceutical Industry Co., Ltd.</td><td>MACEROZYME R-10, 10g</td><td>Final isotonic enzyme solution</td></tr><tr><td>10% (&lt;em&gt;w/v&lt;/em&gt;) BSA&lt;br/&gt; (Bovine Serum Albumin)</td><td>Sigma-aldrich</td><td>A7906-100G</td><td>Final isotonic enzyme solution &amp;amp;&lt;br/&gt; Filtration sterilization</td></tr><tr><td>1 M CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Chem-Lab NV</td><td>CL00.0317.1000</td><td>Final isotonic enzyme solution,&lt;br/&gt; W5 buffer &amp;amp;&lt;br/&gt; Digestion buffer</td></tr><tr><td>1 M NaCl</td><td>Fisher Chemical</td><td>S/3160/60</td><td>W5 buffer</td></tr><tr><td>2 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266-100G</td><td>MMg solution</td></tr><tr><td>1 M LiCl&lt;br/&gt; (Lithium Chloride)</td><td>Acros</td><td>199885000</td><td>Lysis/binding buffer,&lt;br/&gt; Wash buffer 1,&lt;br/&gt; Wash buffer 2 &amp;amp;&lt;br/&gt; Low salt buffer</td></tr><tr><td>5% (&lt;em&gt;w/v&lt;/em&gt;) LiDS&lt;br/&gt; (Lithium Dodecyl Sulphate)</td><td>Sigma-aldrich</td><td>L4632-25G</td><td>Lysis/binding buffer,&lt;br/&gt; Wash buffer 1 &amp;amp;&lt;br/&gt; Filtration sterilization</td></tr><tr><td>1 M DTT&lt;br/&gt; (Dithiothreitol)</td><td>Thermo Fisher Scientific&lt;br/&gt; Wash buffer 1 &amp;amp;&lt;br/&gt; Wash buffer 2</td><td>307866</td><td>Lysis/binding buffer,</td></tr><tr><td>1 M Tris-HCl (pH 7.5) (Tris(hydroxymethyl)aminomethane, Hydrochloric acid S.G. (HCl))</td><td>Acros &amp;amp;&lt;br/&gt; Fisher Chemical</td><td>167620010 &amp;amp;&lt;br/&gt; H/1200/PB15</td><td>Lysis/binding buffer,&lt;br/&gt; Wash buffer 1,&lt;br/&gt; Wash buffer 2,&lt;br/&gt; Low salt buffer &amp;amp;&lt;br/&gt; Elution buffer</td></tr><tr><td>0.5 M EDTA (pH 8.0) (Ethylenediaminetetraacetic acid)</td><td>Sigma-aldrich</td><td>ED-500G</td><td>Lysis/binding buffer,&lt;br/&gt; Wash buffer 1,&lt;br/&gt; Wash buffer 2,&lt;br/&gt; Low salt buffer &amp;amp;&lt;br/&gt; Elution buffer</td></tr><tr><td>Tween 20</td><td>MERCK</td><td>8.22184.0500</td><td>Regeneration of oligo-d(T)&lt;sub&gt;25&lt;/sub&gt; beads</td></tr><tr><td>0.1 M NaOH</td><td>VWR PROLABO CHEMICALS</td><td>28244.295</td><td>Regeneration of oligo-d(T)&lt;sub&gt;25&lt;/sub&gt; beads</td></tr><tr><td>1x PBS (pH 7.4)&lt;br/&gt; (Phosphate Buffered Saline)&lt;br/&gt; containing&lt;br/&gt; (NaCl; KCl; Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;; KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Chemical, MERCK,&lt;br/&gt; Sigma-aldrich &amp;amp; SAFC</td><td>S/3160/60,&lt;br/&gt; Art. 4935,&lt;br/&gt; 71640-250G &amp;amp;&lt;br/&gt; 60230</td><td>Regeneration of oligo-d(T)&lt;sub&gt;25&lt;/sub&gt; beads</td></tr><tr><td>Proteinase K solution (2 &amp;mu;g/&amp;mu;L)</td><td>Thermo Fisher Scientific</td><td>11789020</td><td>Protein digestion</td></tr><tr><td>Loading dye</td><td>Invitrogen</td><td>LC5925</td><td>SDS-PAGE</td></tr><tr><td>qPCR master mix</td><td>Promega</td><td>A6001</td><td>qRT-PCR assay</td></tr><tr><td>RNase Cocktail</td><td>Thermo Fisher Scientific</td><td>AM2286</td><td>RNA digestion</td></tr><tr><td>Methanol</td><td>Sigma-aldrich</td><td>322415</td><td>Gel fixation and gel destaining</td></tr><tr><td>Acetic acid</td><td>Sigma-aldrich</td><td>537020</td><td>Gel fixation and gel destaining</td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Thermo Fisher Scientific</td><td>20278</td><td>Gel staining</td></tr><tr><td>1 M NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;&lt;br/&gt; (Ammonium bicarbonate)&lt;br/&gt; &amp;nbsp;</td><td>Sigma-aldrich</td><td>09830-500G</td><td>Gel hydration &amp;amp;&lt;br/&gt; Digestion buffer</td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;CN&lt;br/&gt; (Acetonitrile)</td><td>Sigma-aldrich</td><td>34851-100ML</td><td>Gel dehydration &amp;amp;&lt;br/&gt; Peptide dissolving solution</td></tr><tr><td>IAA&lt;br/&gt; (Iodoacetic acid)</td><td>Sigma-aldrich</td><td>I4386-10G</td><td>Alkylating agent</td></tr><tr><td>TFA&lt;br/&gt; (Trifluoroacetic acid)</td><td>Sigma-aldrich</td><td>302031-10X1ML</td><td>Peptide dissolving solution</td></tr><tr><td>FA&lt;br/&gt; (Formic acid)</td><td>Sigma-aldrich</td><td>06554-5G</td><td>Peptide extraction</td></tr><tr><td>Trypsin solution (6 ng/&amp;mu;L)</td><td>Promega</td><td>V5280</td><td>Digestion buffer</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Soil</td><td>Peltracom</td><td>LP2D</td><td>Plant growth</td></tr><tr><td>Vermiculite 3</td><td>Sibli AS</td><td>05VERMICULIET</td><td>Plant growth</td></tr><tr><td>Petri dish (150 x 20 mm)</td><td>Sarstedt</td><td>82.1184.500</td><td>Carrier for protoplast suspension</td></tr><tr><td>0.22 &amp;mu;m filter</td><td>Millipore</td><td>SE2M229104</td><td>Homogenization of final isotonic enzyme solution</td></tr><tr><td>Razorblade</td><td>Agar Scientific</td><td>T585</td><td>Rosette leaf strips</td></tr><tr><td>35-75 &amp;mu;m nylon mesh</td><td>SEFAR NITEX</td><td>74010</td><td>Protoplast suspension filtration</td></tr><tr><td>50 mL round bottom tubes</td><td>Sigma-aldrich</td><td>T1918-10EA</td><td>Carrier for protoplast suspension</td></tr><tr><td>Hemocytometer&lt;br/&gt; (B&amp;uuml;rker hemocytometer)</td><td>MARIENFELD</td><td>650030</td><td>Protoplast cell counting</td></tr><tr><td>UV crosslinking apparatus&lt;br/&gt; (HL-2000 HybriLinker)</td><td>UVP, LLC</td><td>UVP95003101</td><td>&lt;em&gt;in vivo&lt;/em&gt; UV crosslinking</td></tr><tr><td>UV lamp&lt;br/&gt; (Sankyo-Denki G8T5)</td><td>SANKYO&lt;br/&gt; DENKI</td><td>SD G8T5</td><td>&lt;em&gt;in vivo&lt;/em&gt; UV crosslinking</td></tr><tr><td>50 mL glass syringe</td><td>FORTUNA Optima</td><td>Z314560</td><td>Homogenization of protoplast lysate</td></tr><tr><td>Narrow needle (0.9 x 25 mm)</td><td>Becton Dickinson microlance 3</td><td>2021-04</td><td>Homogenization of protoplast lysate</td></tr><tr><td>Rotator Model L26</td><td>Labinco BV</td><td>26110912</td><td>Sample incubation by rotating</td></tr><tr><td>Oligo-d(T)&lt;sub&gt;25&lt;/sub&gt; magnetic beads&lt;br/&gt; (5 mg/mL)</td><td>New England BioLabs</td><td>S1419S</td><td>mRNPs and mRNAs binding and pull-down</td></tr><tr><td>Magnetic rack</td><td>Invitrogen</td><td>CS15000</td><td>mRNPs and mRNAs binding and pull-down</td></tr><tr><td>Centrifugal filter units&lt;br/&gt; (Amicon Ultra-4 centrifugal filter units)</td><td>EMD Millipore</td><td>UFC800308</td><td>mRBP concentration</td></tr><tr><td>Pierce Silver Stain Kit</td><td>Thermo Fisher Scientific</td><td>24612</td><td>Silver-staining assay</td></tr><tr><td>RNA purification kit&lt;br/&gt; (InviTrap Spin Plant RNA Mini Kit)</td><td>STRATEC Molecular</td><td>1064100300</td><td>RNA purification</td></tr><tr><td>Spectrophotometer device (NanoDrop 1000 Spectrophotometer)</td><td>Thermo Fisher Scientific</td><td>ND-1000</td><td>RNA quality and quantity</td></tr><tr><td>Real-Time PCR cycler&lt;br/&gt; (StepOne Real-Time PCR cycler)</td><td>Thermo Fisher Scientific</td><td>4376600</td><td>cDNA quantification</td></tr><tr><td>&amp;micro;-C18 columns&lt;br/&gt; (Millipore Zip Tip &amp;micro;-C18 columns)</td><td>Sigma-aldrich</td><td>720046-960EA</td><td>Peptide purification</td></tr><tr><td>Mass spectrometer&lt;br/&gt; (Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer)</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMAZR</td><td>Mass spectrometry-based proteomics</td></tr><tr><td>Liquid chromatography instrument (Ultimate 3000 ultra-high performance liquid chromatography (UHPLC) instrument)</td><td>Thermo Fisher Scientific</td><td>ULTIM3000RSLCNANO</td><td>Mass spectrometry-based proteomics</td></tr><tr><td>C18 column&lt;br/&gt; (Easy Spray Pepmap RSLC C18 column)</td><td>Thermo Fisher Scientific</td><td>ES800</td><td>Mass spectrometry-based proteomics</td></tr><tr><td>C18 precolumn&lt;br/&gt; (Acclaim Pepmap 100 C18 precolumn)</td><td>Thermo Fisher Scientific</td><td>160321</td><td>Mass spectrometry-based proteomics</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primers for qRT-PCR assay&lt;/strong&gt;</td><td>&lt;strong&gt;Sequences&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>&lt;em&gt;UBQ10&lt;/em&gt; mRNA&lt;br/&gt; (Li &lt;em&gt;et al.&lt;/em&gt;, 2014)</td><td>Fw: AACTTTGGTGGTTTGTGTTTTGG&lt;br/&gt; Rv: TCGACTTGTCATTAGAAAGAAAGAGATAA</td><td></td><td></td></tr><tr><td>18S rRNA&lt;br/&gt; (Durut &lt;em&gt;et al.&lt;/em&gt;, 2014)</td><td>Fw: CGTAGTTGAACCTTGGGATG&lt;br/&gt; Rv: CACGACCCGGCCAATTA</td><td></td><td></td></tr></tbody></table>

mrna interactome capture from plant protoplasts

RNA结合蛋白（RBP）决定RNA的命运。他们参与所有的RNA生物发生途径，特别有助于信使RNA（mRNA）的转录后基因调控（PTGR）。在过去几年中，已经通过使用称为"mRNA相互作用捕获"的新方法成功地分离了许多来自酵母和哺乳动物细胞系的mRNA结合蛋白质组，其允许鉴定mRNA结合蛋白（mRBP）直接从生理环境。该方法由体内紫外（UV）交联，寡核苷酸（dT）珠的信使核糖核蛋白复合物（mRNP）的下拉和纯化以及随后通过质谱（MS）鉴定交联蛋白质组成。最近，通过应用相同的方法，已经从不同的拟南芥组织来源同时报道了几种植物mRNA结合的蛋白质组:病毒幼苗，叶组织，叶肉叶原生质体和培养根细胞。在这里，我们提出了拟南芥叶肉叶片原生质体的优化的mRNA相互作用捕获方法，这是一种细胞类型，其用作包括各种细胞测定的实验的通用工具。最佳蛋白质产量的条件包括起始组织的量和UV照射的持续时间。在从中等规模实验（10 7个细胞）获得的mRNA结合蛋白质组中，发现具有RNA结合能力的RBP被超表达，并且鉴定出许多新的RBP。实验可以放大（10 9个细胞），优化的方法可以应用于其他植物细胞类型和物种，以广泛分离，编目和比较植物中mRNA结合的蛋白质组。

在洗涤步骤4.3中用洗涤缓冲液2（ 图1B ）观察到围绕CL样品中的珠粒的特征性晕圈。尽管尚未进行研究，但是这种现象可能通过在磁捕获过程中交联的mRNP复合物与珠聚集的干扰来解释，从而导致形成更扩散的聚集体。这表明oligo-d（T） 25珠捕获是有效的14 。 在非CL对照和CL样品中?...

Watch this Scientific Journal Video about mRNA Interactome Capture from Plant Protoplasts at JoVE.com

mRNA Interactome Capture from Plant Protoplasts

在这里，我们提出了应用于拟南芥叶肉叶肉原生质体的相互作用捕获方案。该方法严格依赖于体内紫外线交联，并允许从生理环境中分离和鉴定植物mRNA结合蛋白。

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

mrna-interactome-capture-from-plant-protoplasts

Research

JoVE Journal

Biochemistry

9.1K 次观看.  KU Leuven. 在这里，我们提出了应用于拟南芥叶肉叶肉原生质体的相互作用捕获方案。该方法严格依赖于体内紫外线交联，并允许从生理环境中分离和鉴定植物mRNA结合蛋白。 

视频: mRNA Interactome Capture from Plant Protoplasts

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

生物化学

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

生物学

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

发育生物学

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

基于 TurboID 的接近标签，用于蛋白质-蛋白质相互作用网络的植物中识别

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

免疫与感染

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...