Name: unravelling the function of a bacterial effector from a non-cultivable plant pathogen using a yeast two-hybrid screen
Uploaded: 2017-01-20T13:00:00.000+00:00
Duration: 11 min 30 s
Description: Watch this Scientific Journal Video about Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen at JoVE.com

我们感谢恭Kerschbamer，托马斯Letschka，萨宾Oettl，玛戈Raffeiner和弗洛里安Senoner从Dualsystems生物技术公司的技术支持和Julia施特罗布尔的Laimburg研究中心和Mirelle博尔赫斯迪亚斯Schnetzer校对稿件。这项工作是为APPL2.0项目的一部分进行和波森/意大利博尔扎诺自治省和南蒂罗尔苹果联盟被部分资助。

作者宣称，他们没有竞争的经济利益。

在Y2H屏幕，潜在效应蛋白与不同假想相互作用蛋白（步骤7）共表达。每个生长酵母克隆含有诱饵但一个（可能）不同交互件。的相互作用的蛋白质被编码在被克隆到捕食的质粒cDNA文库。诱饵和猎物质粒对酵母转录因子的一部分，每个合作cistronically代码。在诱饵（效应）和猎物质粒编码交互件之间的物理相互作用的情况下，两个转录因子份（DNA结合结构域和活化结构域）团结，并且报告基因的表达被诱导。酵母是能够在组氨酸和腺嘌呤的贫的SD选择平板上生长。那种猎物cDNA文库的是依赖于筛选效应，必须相应地选择。猎物和诱饵载体，以及酵母菌株，必须兼容。在此屏幕上，一个LexA的DNA结合结构域和GAL4激活domaiÑ被用作相容酵母转录因子单元。 cDNA文库可以单独构成，定制，或商购获得。该cDNA文库猎物的准备是不是该协议的一部分。在这个协议中，使用的是来自苹果点¯x家蝇叶RNA的自行建造，归一化的cDNA文库，并克隆到pGAD-HA 41。 -expressing酵母菌株的效应（诱饵）与捕食文库转化，并选择对组氨酸和腺嘌呤的贫的SD选择平板的克隆。提取的酵母菌落的质粒DNA，细菌的基于列的DNA质粒mini试剂盒中的组合，建议使用玻璃珠，以提高酵母裂解（步骤8）的机械破碎步骤。在此质粒纯化，诱饵和猎物载体将同时在相对低浓度纯化。然而，质粒DNA的量足以通过测序机智识别相互作用配偶豪特之前质粒传播（步骤8.4）。作为替代，在步骤8.4纯化DNA可以转化到感受态大肠杆菌和猎物载体介导的抗生素抗性选择（ 例如，在pGAD-HA的情况下氨苄青霉素的）。选定的大肠杆菌菌落仅携带pGAD-HA文库质粒，和高产质粒纯化可以用这些克隆进行。 pLexA-N和pGAD-HA结构的成功转型补充NMY51的色氨酸和亮氨酸营养缺陷。效应和蛋白质（编码在pGAD-HA）之间的相互作用导致了报告系统的在NMY51菌株的活化。在自激活的情况下，NMY51转化有表达pLexA-N结合的空文库载体pGAD-HA生长在缺乏色氨酸-LEU-他-ADE，选择性平板效应由于NMY51报告系统的不希望的活化40。自激活可以为wEAK和可发生在没有trp启动-LEU-他的，或激活能强和其特征在于生长上的trp-LEU-他-阿德板41。自激活可引起假阳性克隆中的实际Y2H屏幕大规模背景。为了避免这种情况，与诱饵的自激活测试必须进行，其中，所述效应子表达载体是共转化用空文库载体。在这个实验设置，报告基因的表达不能被诱导。如果自激活（ 即，在选择性平板上生长）是在测试可见，培养基可以补充有不同浓度的3-氨基-1,2,4-三唑45（3-AT）的。 3-AT是imidazoleglycerol磷酸脱水酶（HIS3），酶组氨酸生物合成46中重要的抑制剂。补充3-AT可以减少在Y2H屏幕47自激活的作用较弱，48。不同浓度的3-AT的进行测试。在这个协议中，1 - 40毫米3-AT中使用。这导致自我激活抑制的最低浓度应随后在Y2H屏幕使用。自活化测定的选择性平板可以，如果共转化的SD-TRP-Leu平板含有足够数量的克隆只进行评估。作为指示≥每90毫米（直径）的培养皿500个菌落是足够的。确定准确的转染效率，建议以制备共转化的酵母的连续稀释液并把它们散布在SD-TRP-Leu平板。 以减少自激活，效应可以克隆到pLexA-C，即融合了LexA的标签的蛋白质的C末端的诱饵表达载体。莱克斯-A标签的方向可以减轻自激活24。然而，这是不可能在所有情况下，以减少或消除自激活。的形成红色或淡红色菌落表示弱或假阳性的互动。 NMY51的ADE2报告基因，当涉及到一个蛋白质-蛋白质相互作用，仅激活这反过来块红色染料的在此酵母菌株40的积累。在缺乏互动，NMY51都偏红，同时搭载强大的干扰作用的菌落是白色的。在选择平板的菌落颜色的观察因此提供进一步的重要的暗示以判断如果各个菌落道道或假阳性相互作用物。 它是最终要适应或相对于该诱饵的性质和作用特性的更改某些测定法设置。现在，一些改进和共同Y2H的衍生物技术已经建立，以允许在不同的主机系统相当困难蛋白质相互作用的分析。通过Stynen 等进行审查。 49地址的第二描述Y2H，其改进和适应的各个方面，从而提供了有关如何选择适当的互动分析有用的信息。 Y2H屏幕和衍生技术已被广泛应用于不同的研究领域，任何需要二进制蛋白质相互作用的鉴定。即使关键控制被执行，如诱饵的自激活试验和鉴定相互作用的蛋白质的一到一个重新变换，在Y2H是容易产生错误的结果26，50，51。酵母作为模型并不适用于所有的相互作用研究。酵母并不一定构成支持适当的翻译后修饰和折叠为每个蛋白24的蜂窝环境。此外，在Y2H设置，蛋白质过表达，并且它们的表达是不控制其天然启动子领导。在Y2H迫使蛋白质相互作用配偶到细胞核，这不一定是它们的天然亚细胞目标。相互作用的天然细胞环境因此可能不会通过酵母中反映出来，并可能导致假阴性或假阳性结果。最Y2H基于酵母营养缺陷型互补作为选择原则。此营养选择的特征是高灵敏度，但在相对于其他的选择性降低的成本（ 例如，发色记者）测定49。如果不可还原自激活（见上文）或发生了一定的效应的其他限制，Y2H不是合适的测定25。在表1和图1中 ，自激活试验的预期结果中给出。缺乏真正的相互作用（ 即假阴性），可通过蛋白的毒性，不正确的翻译蛋白质引起的必要的互动24处理，互动的空间位阻，在猎物库未被充分代表的互动合作伙伴，诱饵膜定位，或缺少的组件。 建议从头扩增来自cDNA的所识别的相互作用蛋白的全长基因，亚克隆入pGAD-HA，并且通过转化产生的pGAD-HA构建成执行一对一的相互作用测定诱饵表达NMY51应变。这是必要的，因为存在于猎物文库观察到的交互器可能仅是一个更大的蛋白质的片段。但是，对于相应的全长基因的信息是只有当相当的基因组和转录组序列数据可用访问。变换协议为这里所描述的自活化试验可以应用于这样一对一的测定中，以及在诱饵和交互件之间的相互作用必须是可重复的。 海棠点¯x家蝇的TCP转录因子的相互作用在植物证实在本塞姆氏烟草双分子荧光互补（附设）原生质体28（未此协议的一部分）。的效应蛋白ATP_00189从植物病原体P.马里而得。 在植物附设就这样选择了验证与海棠点¯x家蝇转录因子相互作用ATP_00189 先前在Y2H 28标识。附设是不需要的相互作用蛋白到细胞核的亚细胞易位以激活报告系统&lt;蛋白质 - 蛋白质相互作用测定法SUP类="外部参照"&gt; 52。此外， 在植物中表达和修改机器模仿在其植物宿主植物细菌效应之间的自然交互的环境中。然而，使用附设一个全局画面是不可行的。 有在，必须谨慎处理的协议几个步骤。当扩增的利息（步骤4）的基因，它排除引物结合到植物DNA是非常重要的。因此，来自非感染植物的DNA必须包含作为阴性对照。感兴趣的基因的序列不能包含用于插入件的定向克隆的EcoRI和SalI限制性位点。如果序列确实包含这些网站上，用于克隆不同的限制酶必须选择。酵母转化是该协议中的核心方法。转染效率依赖于酵母细胞的能力，它们的生存能力，生长状态，并转染REAG的质量耳鼻喉科53,54。对于所提出的协议，这是强烈建议在执行变换时，使用从新鲜平板酵母不超过两周（保持在4℃）以上。通常情况下，当选择液体培养物与来自旧琼脂平板的菌落接种转化的酵母的生长被延迟。这也有利于用液态起子培养作为实际实验的接种物，而不是直接从板使用酵母。转染效率低，当猎物进入诱饵表达酵母可导致某些猎物蛋白（潜在相互作用者）的人数不足，因此可以扭曲整个屏幕。在这个协议中，在Y2H〜15万CFU /微克DNA转染的酵母转染效率运作良好。 知道了Y2H技术的缺陷和弊端，并在关键的和适当的方式解释结果是必不可少的绘制科尔等结论。 Y2H测定和衍生物已被使用多年，并已进行了许多改进和调整相对于所述不同的诱饵特性，相互作用的亚细胞定位，这可能是必需的相互作用的预期的结合伙伴，以及其他因素（见Y3H 55，56，57）。最近，基于阵列的Y2H屏幕已经开发了允许对数以百万计的猎物58的众多诱饵自动化，高通量分析。将来很可能在于自动化和高通量的方法来该测定，以允许在不同的研究领域复杂信号传导途径和interactomes的澄清。

<html>酵母双杂交屏幕（Y2H）的大约27年前1开发，至今已被广泛地应用于各个领域，以确定特定的蛋白质-蛋白质相互作用2，3，4，5，6，7，8，9，10，11 。与主机靶蛋白的细菌效应的物理相互作用常常是用于本目标蛋白质的功能操作的前提条件。分析这些相互作用已经转移到感染生物学12，13，14，15多个不同行业的焦点，"&gt; 16，17，18。在Y2H屏幕的原则是，两种蛋白质的相互作用导致驱动的报告基因的表达的功能性转录因子的重构。已知效应（的"诱饵"）被翻译融合至DNA结合结构域的转录因子（DBD），和潜在的相互作用伙伴（在"猎物"）被融合到相应的转录因子的活化结构域（AD），由于相互作用伴侣是未知的，潜在的库交互件是由克隆的cDNA插入合适的猎物载体的表达质粒编码的AD是与编码DBD诱饵载体兼容克隆到所谓的"猎物库"。通常，该库是由在相互作用的情况下，该重组转录因子诱导报道基因，其通常使酵母的生长选择的表达。该交互件识别是通过测序与特定的质粒引物的猎物质粒实现。 细菌已经制定各种策略来操纵和利用其宿主的代谢或逃避防御机制，并通过不同的细菌分泌系统19，20，21分泌效应分子。确定这些细菌效应的结合配偶从而在识别在宿主中操纵途径的第一个步骤。这导致特定致病机制的改善的理解。 Y2H屏幕进行phytoplasmoses期间识别细菌效应相互作用，常常，Y2H为分子致病机制植研究22，23中的进一步表征了至关重要的初始实验。然而，遇到了在大多数出版物HOD描述是相当稀少，并且这些技术通常外包给生物技术公司。要提醒大家注意这种方法的可行性，该协议提供了一步一步的信息来确定它的自然宿主细菌效应分子相互作用的合作伙伴。 尽管广泛使用和Y2H屏幕的快进的方式，它不应该忘记，某些相互作用可能不会在酵母系统发生。这是由于酵母细胞被用作一种" 体内反应容器"具有一定的生物限制的事实。几位作者已经解决的优点和Y2H及其衍生物11，24，25，26，27的缺点。一般考虑为，例如，酵母细胞可能无法提供适当的基因的表达，（后）的平移，或者针对各个蛋白质translocational条件正在研究中。这可能导致在屏幕假阴性结果。阳性相互作用可以反过来是人工制品和（在适当的宿主，即，在效应的情况下）可能不会发生在自然情况。它因此不可缺少确认以更密切相关的生物系统从与独立相互作用测试异源酵母表达系统中的相互作用。 在这项研究中，效应ATP_00189的从非可培养植物病原体暂定植原马里（P.马里）的结合伴侣进行鉴定。研究结果提供了重要的见解苹果增殖28，导致受影响的苹果种植区高昂的经济损失在29欧洲疾病的症状发展的分子机制。

<table><tbody><tr><td>Cetyltrimethylammonium bromide (CTAB)</td><td>Applichem</td><td>A6284</td><td>for DNA preparation</td></tr><tr><td>Trishydroxymethylaminomethane (Tris)</td><td>Applichem</td><td>A2264</td><td>for media and buffer</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Applichem</td><td>A2942</td><td>for media and buffer</td></tr><tr><td>Ethylenediaminetetraacetic acid Disodium salt (NaEDTA)</td><td>Applichem</td><td>A2937</td><td>for media and buffer</td></tr><tr><td>N-Lauroylsarcosin sodium salt</td><td>Applichem</td><td>A7402</td><td>for DNA preparation</td></tr><tr><td>ammonium acetate&amp;nbsp;</td><td>Sigma-Aldrich (Fluka)</td><td>9688</td><td>for DNA preparation</td></tr><tr><td>2-mercaptoethanol</td><td>Applichem</td><td>A1108</td><td>for DNA preparation</td></tr><tr><td>Isopropanol (2-Propanol)</td><td>Applichem</td><td>A3928</td><td>for DNA preparation</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich (Fluka)</td><td>51976</td><td>for DNA preparation</td></tr><tr><td>RNAse</td><td>Applichem</td><td>A2760</td><td>for DNA preparation</td></tr><tr><td>Chloroform:Isoamyl 24:1</td><td>Applichem</td><td>A1935</td><td>for DNA preparation</td></tr><tr><td>Proofreading Polymerase (&lt;em&gt;e.g.&lt;/em&gt; iProof)</td><td>Bio-Rad</td><td>203433</td><td>for PCR</td></tr><tr><td>EcoRI</td><td>Thermo Scientific</td><td>ER0271</td><td>for cloning</td></tr><tr><td>SalI</td><td>Thermo Scientific</td><td>ER0641</td><td>for cloning</td></tr><tr><td>Column-based DNA Purification Kit (&lt;em&gt;e.g.&lt;/em&gt; QIAquick)</td><td>Qiagen</td><td>28104</td><td>for cloning</td></tr><tr><td>Kanamycin Sulfate</td><td>Applichem</td><td>A1493</td><td>for microbiological selection</td></tr><tr><td>3-Amino-1,2,4-Triazole (3-AT)</td><td>Sigma-Aldrich</td><td>A8056</td><td>for self-activation assay</td></tr><tr><td>L-Arginine Monohydrochloride&amp;nbsp;</td><td>Applichem</td><td>A3680</td><td>for yeast culture</td></tr><tr><td>L-Isoleucine&amp;nbsp;</td><td>Applichem</td><td>A3642</td><td>for yeast culture</td></tr><tr><td>L-lysine Monohydrate&amp;nbsp;</td><td>Applichem</td><td>A3448</td><td>for yeast culture</td></tr><tr><td>L-Methionine&amp;nbsp;</td><td>Applichem</td><td>A3897</td><td>for yeast culture</td></tr><tr><td>L-Phenylalanine&amp;nbsp;</td><td>Applichem</td><td>A3464</td><td>for yeast culture</td></tr><tr><td>L-Threonine&amp;nbsp;</td><td>Applichem</td><td>A3946</td><td>for yeast culture</td></tr><tr><td>L-Tyrosine&amp;nbsp;</td><td>Applichem</td><td>A3401</td><td>for yeast culture</td></tr><tr><td>L-Uracile</td><td>Applichem</td><td>A0667</td><td>for yeast culture</td></tr><tr><td>L-Valine&amp;nbsp;</td><td>Applichem</td><td>A3406</td><td>for yeast culture</td></tr><tr><td>L-Adenine Hemisulfate Salt&amp;nbsp;</td><td>Applichem</td><td>A1596</td><td>for yeast culture</td></tr><tr><td>0.22 &amp;micro;m Pore Filter</td><td>Sartorius</td><td>16541</td><td>for sterile filtration</td></tr><tr><td>L-Histidine Monohydrochloride</td><td>Applichem</td><td>A3719</td><td>for yeast culture</td></tr><tr><td>L-Leucine&amp;nbsp;</td><td>Applichem</td><td>A3496</td><td>for yeast culture</td></tr><tr><td>L-Tryptophane&amp;nbsp;</td><td>Applichem</td><td>A3410</td><td>for yeast culture</td></tr><tr><td>Yeast Nitrogen Base&amp;nbsp;</td><td>Sigma-Aldrich</td><td>51483</td><td>for yeast culture</td></tr><tr><td>D-Glucose Monohydrate&amp;nbsp;</td><td>Applichem</td><td>A1349</td><td>for yeast culture</td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>BP2641-1</td><td>for yeast and bacteria culture</td></tr><tr><td>Peptone</td><td>Applichem</td><td>A2208</td><td>for yeast culture</td></tr><tr><td>Polyethylene Glycol 4000 (PEG)</td><td>Applichem</td><td>A1249</td><td>for yeast transformation</td></tr><tr><td>Lithium Acetate Dihydrate (LiOAc)</td><td>Applichem</td><td>A3478</td><td>for yeast transformation</td></tr><tr><td>Salmon Sperm DNA&amp;nbsp;</td><td>Applichem</td><td>A2159</td><td>for yeast transformation</td></tr><tr><td>Antibody against Lex-A tag (&lt;em&gt;e.g.&lt;/em&gt; Anti-LexA DNA binding region)</td><td>Millipore</td><td>06-719</td><td>for Western blot</td></tr><tr><td>Plasmid Miniprep kit (&lt;em&gt;e.g.&lt;/em&gt; GenElute Plasmid Miniprep Kit)</td><td>Sigma-Aldrich</td><td>PLN350</td><td>for plasmid purification from yeast and bacteria</td></tr><tr><td>Acid Washed Glass Beads</td><td>Sigma-Aldrich</td><td>G8772</td><td>for plasmid purification from yeast</td></tr><tr><td>Ampicillin Sodium Salt</td><td>Applichem</td><td>A0839</td><td>for microbiological selection</td></tr><tr><td>Yeast Extract</td><td>Applichem</td><td>A1552</td><td>for yeast culture</td></tr><tr><td>Vacuum concentrator centrifuge (&lt;em&gt;e.g.&lt;/em&gt; Concentrator 5301)</td><td>Eppendorf</td><td>Z368245 (Sigma)</td><td>for DNA preparation</td></tr><tr><td>Bait vector (&lt;em&gt;e.g.&lt;/em&gt; pLexA-N)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Prey vector (&lt;em&gt;e.g.&lt;/em&gt; pGAD-HA)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Control prey vector (&lt;em&gt;e.g.&lt;/em&gt; pLexA-p53)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Control bait vector (&lt;em&gt;e.g.&lt;/em&gt; pACT-largeT)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Electrocompetent &lt;em&gt;E. coli&lt;/em&gt; (&lt;em&gt;e.g.&lt;/em&gt; MegaX DH10B T1R Electrocomp Cells)</td><td>Invitrogen</td><td>C640003</td><td>for cloning</td></tr><tr><td>Yeast reporter strain (NMY51:MATahis3&amp;Delta;200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ ade2::(lexAop)8-ADE2 GAL4, &lt;em&gt;e.g.&lt;/em&gt; NMY51)</td><td>Dualsystems</td><td>P01004</td><td>for Y2H</td></tr><tr><td>Plastic paraffin film (&lt;em&gt;e.g.&lt;/em&gt; Parafilm M)</td><td>Sigma-Aldrich</td><td>P7793-1EA</td><td>for yeast and bacteria culture</td></tr><tr><td>Gas permeable sealer (&lt;em&gt;e.g.&lt;/em&gt; BREATHseal)</td><td>Greiner</td><td>676 051</td><td>for yeast culture</td></tr><tr><td>PCR Cycler (&lt;em&gt;e.g.&lt;/em&gt; T100 Thermal Cycler)</td><td>Bio-Rad</td><td>1861096</td><td>for PCR</td></tr><tr><td>quantitative PCR Cycler (&lt;em&gt;e.g.&lt;/em&gt; CFX96 Touch Real-Time PCR Detection System)</td><td>Bio-Rad</td><td>1855195</td><td>for quantitative PCR</td></tr><tr><td>Microcentrifuge (&lt;em&gt;e.g.&lt;/em&gt; Centrifuge 5417 R)</td><td>Eppendorf</td><td>5417 R</td><td>general lab equipment</td></tr><tr><td>Centrifuge (&lt;em&gt;e.g.&lt;/em&gt; Centrifuge 5804 R)</td><td>Eppendorf</td><td>5804 R</td><td>general lab equipment</td></tr><tr><td>Nuclease-free water (DNAse-free, RNAse-free, Protease-free)</td><td>5Prime</td><td>2500010</td><td>for PCR and DNA works</td></tr><tr><td>Scalpell</td><td>Swann-Morton</td><td>0301</td><td>for DNA preparation</td></tr><tr><td>Sterile filter (0.22 &amp;micro;m; Polyethersulfone)</td><td>VWR-International</td><td>514-0073 (European Catalogue number)</td><td>for yeast and bacteria culture</td></tr><tr><td>Petri dishes &amp;Oslash; 90 mm&amp;nbsp;</td><td>Greiner Bio-One</td><td>633180</td><td>for yeast and bacteria culture</td></tr><tr><td>Petri dishes &amp;Oslash; 150 mm&amp;nbsp;</td><td>Greiner Bio-One</td><td>639161</td><td>for yeast culture</td></tr><tr><td>Photometer (&lt;em&gt;e.g.&lt;/em&gt; BioPhotometer)</td><td>Eppendorf</td><td>550507804</td><td>for yeast culture</td></tr><tr><td>Photometer Cuvettes</td><td>Brand</td><td>759015</td><td>for yeast culture</td></tr><tr><td>Water Bath (&lt;em&gt;e.g.&lt;/em&gt; Water Bath Memmert WB7)</td><td>Memmert</td><td>WB7</td><td>for yeast transformation</td></tr><tr><td>Western Blot Equipment</td><td>Bio-Rad</td><td>diverse</td><td>for protein detection</td></tr><tr><td>dNTPs</td><td>5Prime</td><td>2201210</td><td>for cloning</td></tr><tr><td>Shaker (Erlenmeyer) Flasks (100 mL; 1,000 mL; 2,000 mL) and breathable cotton plugs (&lt;em&gt;e.g.&lt;/em&gt; Duran Erlenmeyer narrow-neck flasks)</td><td>Sigma Aldrich</td><td>Z232793, Z232858, Z232866</td><td>for yeast and bacteria culture</td></tr><tr><td>Shaking Incubator (&lt;em&gt;e.g.&lt;/em&gt; GFL 3031)</td><td>GFL</td><td>3031</td><td>for yeast and bacteria culture</td></tr><tr><td>Incubator</td><td>Binder</td><td>KB 53 (E3.1)</td><td>for yeast and bacteria culture</td></tr><tr><td>Ice Maker (&lt;em&gt;e.g.&lt;/em&gt; Ice Maker IF 825)</td><td>Omniwash</td><td>IF 825</td><td>general lab equipment</td></tr><tr><td>0.2 mL, 1.5 mL and 2.0 mL Reaction Vials (Polypropylene)</td><td>Eppendorf</td><td>0030.124.332 (0.2 ml); 0030.123.328 (1.5 ml); 0030.123.344 (2.0 ml)</td><td>general lab equipment</td></tr><tr><td>Pipettes and disposable pipette Tips (different volumina: 10 - 1,000 &amp;micro;L)</td><td>Eppendorf</td><td>diverse</td><td>general lab equipment</td></tr><tr><td>Spreader</td><td>Sigma-Aldrich</td><td>SPR-L-S01</td><td>for yeast and bacteria culture</td></tr><tr><td>Vortex shaker (&lt;em&gt;e.g.&lt;/em&gt; MS 3 Minishaker)</td><td>IKA</td><td>3617000</td><td>general lab equipment</td></tr><tr><td>T4-Ligase</td><td>Thermo Fisher</td><td>EL0011</td><td>for cloning</td></tr><tr><td>Fridge (4 &amp;deg;C) (&lt;em&gt;e.g.&lt;/em&gt; Liebherr Medline FKEX 5000)</td><td>Liebherr</td><td>81.767.580.4</td><td>general lab equipment</td></tr><tr><td>Freezer (-20 &amp;deg;C) (&lt;em&gt;e.g.&lt;/em&gt; Liebherr Comfort Professional G5216)</td><td>Liebherr</td><td>G5216</td><td>general lab equipment</td></tr><tr><td>Graduated Cylinder (&lt;em&gt;e.g.&lt;/em&gt; Brand)</td><td>Sigma Aldrich</td><td>Z327352; Z327417; Z327441&amp;nbsp;&amp;nbsp;</td><td>general lab equipment</td></tr><tr><td>Serological Pipettes (&lt;em&gt;e.g.&lt;/em&gt; Fisherbrand)</td><td>Thermo Fisher</td><td>S55701</td><td>for yeast and bacteria culture</td></tr><tr><td>Mechanical Pipettor (&lt;em&gt;e.g.&lt;/em&gt; Pipetboy acu 2)</td><td>Integra-Biosciences</td><td>155000</td><td>general lab equipment</td></tr><tr><td>Cuvettes for Electroporation (1 mm)</td><td>Molecular Bio Products</td><td>5510-11</td><td>for bacteria transformation</td></tr><tr><td>Electroporator (&lt;em&gt;e.g.&lt;/em&gt; Eporator)</td><td>Eppendorf</td><td>4309000019</td><td>for bacteria transformation</td></tr><tr><td>Gel and Blot imaging system (&lt;em&gt;e.g.&lt;/em&gt; ChemiDoc MP System)</td><td>Bio-Rad</td><td>1708280</td><td>general lab equipment</td></tr><tr><td>Conical centrifuge tubes (Polypropylene) (50 mL)</td><td>VWR-International&amp;nbsp;</td><td>525-0403 (European Catalogue number)</td><td>general lab equipment</td></tr><tr><td>Conical centrifuge tubes (Polypropylene) (13 mL)</td><td>BD Falcon</td><td>352096</td><td>general lab equipment</td></tr><tr><td>Dithiothreitol (DTT; 1 M)</td><td>Thermo Scientific</td><td>P2325</td><td>for ligation</td></tr><tr><td>adenosine triphosphate (ATP, &lt;em&gt;e.g.&lt;/em&gt; ATP Solution (10 mM))</td><td>Ambion&amp;nbsp;</td><td>AM8110G (distributed by Thermo Scientific)</td><td>for ligation</td></tr><tr><td>Dropout mix without histidine, leucine, tryptophan, and adenine (DO, &lt;em&gt;e.g.&lt;/em&gt; Yeast Synthetic Drop-out Medium Supplements)</td><td>Sigma-Aldrich</td><td>Y2021 Sigma&amp;nbsp;</td><td>for yeast culture (ready-made mix)</td></tr></tbody></table>

unravelling the function of a bacterial effector from a non-cultivable plant pathogen using a yeast two-hybrid screen

Unravelling疾病表现的分子机制重要的是要了解植物科学病理和症状的发展。细菌已经进化不同的战略来操纵其主机的新陈代谢为自己的利益。此细菌操作常常与严重症状的发展，或受影响植物的死亡。确定负责主机操纵特异性细菌分子已经成为微生物研究的一个重要领域。这些细菌分子，称为鉴定后"效应，"这阐明它们的功能是非常重要的。一个简单的方法来确定的效应的功能是通过酵母双杂交（Y2H）屏幕，以确定在其自然宿主中的蛋白质结合伴侣。通常主机怀有不能由任何在硅片算法充分预测许多潜在的结合配偶体。因此，这是对PERFO最好的选择RM屏幕与对表达宿主蛋白的全库中的假想效应。如果病原体是uncultivable植一样，是特别具有挑战性。该协议提供了从植原体感染的木本寄主植物的潜在效应的放大，工厂的分子相互作用合作伙伴的后续识别与屏幕Y2H DNA纯化一步一步的指示。即使Y2H屏幕常用，有这种技术外包给生物技术公司，在一个成本提供Y2H服务的趋势。该协议提供了有关如何使用标准实验室技术的任何体面的装备分子生物学实验室进行Y2H指令。

可以执行实际Y2H屏幕之前诱饵必须为自激活进行测试。这是通过用空猎物文库载体一起转化诱饵表达载体，并检查在选择性平板上生长实现的。 分析phytoplasmal蛋白ATP_00189是自激活，如第5诱饵质粒补充色氨酸和猎物质粒酿酒酵母 NMY51 40列伊营养缺陷的描述进行自我激活测试。因此，一个成功的合作转型的特点是生长在缺乏色氨酸和亮氨酸选择性平板。诱饵和猎物的蛋白质相互作用导致NMY51的他和阿德营养缺陷型的互补。如果出现自激活由诱饵在不存在相互作用，酵母生长在缺乏他和ADE选择性平板。强与弱的自我可以进行激活。诱饵的强自活化的特征是在trp启动-LEU-他-阿德耗尽选择平板的共转化的酵母的生长。弱自激活导致生长在TRP-LEU-他，但不是在TRP-列伊 - 他 - 埃德耗尽选择平板。合适的阳性对照是用于解释自激活测定法的结果不可缺少的。自活化测定的预期结果的总结和它们的解释在表1中提供了与图1中显示。 <img alt="图1" src="/files/ftp_upload/55150/55150fig1.jpg" /> 图1: 一个诱饵的诱饵自我激活试验的实施例执行Y2H屏幕前 。 酿酒酵母 NMY51被共转化与相互作用诱饵（PLEX化p53）和猎物（PACT-largeT）作为阳性对照（ 左图:交流电 ），弱自激活加上一个空的猎物天秤RY向量（ 中图:DF）和不能自我激活诱饵加上一个空的猎物库矢量（ 右图:GI）。 ，trp启动，亮氨酸和他的（ 中图:B，E，H）:该共转化的酵母被上缺乏trp和亮氨酸（A，D，G上面板 ）的SD平板上培养和trp，亮氨酸，他和ADE（ 低级面板:C，F，I）。上介质缺乏色氨酸和亮氨酸的选择是成功共转化的阳性对照，作为诱饵载体补充了色氨酸营养缺陷和猎物载体NMY51（在SD-TRP-亮氨酸生长）的亮氨酸营养缺陷型。在诱饵和猎物或诱饵的自活化之间的相互作用的情况下，NMY51的报告基因的表达被接通并补充了他和ADE营养缺陷型（在SD-TRP-LEU-他-ADE生长）。弱自激活的特点是生长在SD-TRP-LEU-他的板（ 中间面板，DF）。诱饵弱自激活必须分析诱饵前的被削弱在Y2H屏幕， 例如通过添加3-AT到选择性培养基。氨基酸枯竭都标有" - "在各自的媒体名称。 <a href="http://ecsource.jove.com/files/ftp_upload/55150/55150fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> pLexA-N-atp00189 </td><td>没有</td><td>群落</td><td>没有增长</td><td>没有增长</td><td>没有增长</td><td>没有增长</td></tr><tr><td>没有</td><td> pGAD-HA </td><td>没有增长</td><td>群落</td><td>没有增长</td><td>没有增长</td><td>没有增长</td></tr><tr><td> pLexA-N </td><td>没有</td><td>群落</td><td>没有增长</td><td>没有增长</td><td>没有增长</td><td>没有增长</td></tr><tr><td> pLexA-N </td><td> pGAD-HA </td><td>群落</td><td>群落</td><td&gt;菌落</td><td>没有增长</td><td>没有增长</td></tr><tr><td> pLexA-P53 </td><td>的pACT-largeT </td><td>群落</td><td>群落</td><td>群落</td><td>群落</td><td>群落</td></tr><tr><td> pLexA-N-atp00189 </td><td> pGAD-HA </td><td>群落</td><td>群落</td><td>群落</td><td>没有增长</td><td>没有增长</td></tr></tbody></table> 表1:诱饵自活化实验的预期结果。 酿酒酵母 NMY51的转型，基于共同转化诱饵和猎物的特点选择性培养基不同的生长产生。在选择性平板上生长，在30℃下经72小时后培养不同载体的组合（AF）的转化进行评价。弱自激活的特点是酵母生长在SD-TRP-LEU-他并通过在SD-TRP-列伊 - 他 - 埃德选择开发平台的强劲增长自激活ES在不存在相互作用配偶的。所述pLexA-N编码phytoplasmal效应ATP_00189不表现自激活，其特点是不能转化NMY51在没有他和ADE的生长诱饵载体。 根据不同的饵，一Y2H屏幕能获得大量的酵母克隆选择性平板上生长。所有克隆必须分析和检查可能的冗余。即使归一化的cDNA文库已被使用，它很可能是一个交互件在许多不同克隆表示。根据库克隆技术，它也有可能是仅完整基因的片段被插入一些猎物载体。它因此可取的从头 （从cDNA）扩增和亚克隆交互件的全长基因，并测试在一对一的Y2H分析（ 图2）的相互作用。 <img aLT ="图2"SRC ="/文件/ ftp_upload / 55150 / 55150fig2.jpg"/&gt; 图2: 在Y2H实验诱饵和猎物之间的相互作用的例子。进行酵母双杂交（Y2H）屏幕，并纯化从正相互作用分子克隆质粒。猎物载体测序， 海棠点¯x家蝇宿主相互作用的合作伙伴MdTCP24和MdTCP25进行鉴定。作为阴性对照MdTCP34（对于没有交互件是在Y2H库筛选中鉴定）并联亚克隆。全长基因从苹果的cDNA扩增，克隆到捕食载体（共cistronically表达的激活域AD）和从头共转化用表达耦合至DNA结合结构域（BD）的细菌效应ATP_00189诱饵载体。这个数字是从28所。 <a href="http://ecsource.jove.com/files/ftp_upload/55150/55150fig2large.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。

Watch this Scientific Journal Video about Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen at JoVE.com

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

细菌效应蛋白是用于建立成功的感染很重要的。这个协议描述在其天然植物宿主细菌效应蛋白的蛋白质结合配偶的实验鉴定。识别通过酵母双杂交屏幕这些效应的相互作用已成为解开分子致病性策略的一个重要工具。

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

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JoVE Journal

Immunology and Infection

11.4K 次观看.  Laimburg Research Centre. 细菌效应蛋白是用于建立成功的感染很重要的。这个协议描述在其天然植物宿主细菌效应蛋白的蛋白质结合配偶的实验鉴定。识别通过酵母双杂交屏幕这些效应的相互作用已成为解开分子致病性策略的一个重要工具。 

视频: Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

科研

生物化学

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

酵母 2-混合筛在批次中比较蛋白质相互作用

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

遗传学

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

免疫与感染

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

植物病原体分泌效应器中RNA沉默抑制器的筛选和鉴定

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

生物学

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

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.

分离绿色荧光蛋白系统可视化细菌在感染过程中传递的效应

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 ...

使用酵母双杂交技术的生物分子相互作用研究 

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

作者聚焦:增强酵母中相互作用零突变体的分离

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

使用酵母双杂交试验分离相互作用无效/受损突变体

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...