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 border="0" cellpadding="0" cellspacing="0" width="1514">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:565px;">Cetyltrimethylammonium bromide (CTAB)</td>
			<td style="width:211px;">Applichem</td>
			<td style="width:324px;">A6284</td>
			<td style="width:415px;">for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trishydroxymethylaminomethane (Tris)</td>
			<td>Applichem</td>
			<td>A2264</td>
			<td>for media and buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodiumchloride (NaCl)</td>
			<td>Applichem</td>
			<td>A2942</td>
			<td>for media and buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethylenediaminetetraacetic acid Disodium salt (NaEDTA)</td>
			<td>Applichem</td>
			<td>A2937</td>
			<td>for media and buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">N-Lauroylsarcosin sodium salt</td>
			<td>Applichem</td>
			<td>A7402</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ammonium acetate&#160;</td>
			<td>Sigma-Aldrich (Fluka)</td>
			<td>9688</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2-mercaptoethanol</td>
			<td>Applichem</td>
			<td>A1108</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isopropanol (2-Propanol)</td>
			<td>Applichem</td>
			<td>A3928</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol</td>
			<td>Sigma-Aldrich (Fluka)</td>
			<td>51976</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RNAse</td>
			<td>Applichem</td>
			<td>A2760</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chloroform:Isoamyl 24:1</td>
			<td>Applichem</td>
			<td>A1935</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Proofreading Polymerase (e.g. iProof)</td>
			<td>Bio-Rad</td>
			<td>203433</td>
			<td>for PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EcoRI</td>
			<td>Thermo Scientific</td>
			<td>ER0271</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SalI</td>
			<td>Thermo Scientific</td>
			<td>ER0641</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Column-based DNA Purification Kit (e.g. QIAquick)</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kanamycin Sulfate</td>
			<td>Applichem</td>
			<td>A1493</td>
			<td>for microbiological selection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">3-Amino-1,2,4-Triazole (3-AT)</td>
			<td>Sigma-Aldrich</td>
			<td>A8056</td>
			<td>for self-activation assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Arginine Monohydrochloride&#160;</td>
			<td>Applichem</td>
			<td>A3680</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Isoleucine&#160;</td>
			<td>Applichem</td>
			<td>A3642</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-lysine Monohydrate&#160;</td>
			<td>Applichem</td>
			<td>A3448</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Methionine&#160;</td>
			<td>Applichem</td>
			<td>A3897</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Phenylalanine&#160;</td>
			<td>Applichem</td>
			<td>A3464</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Threonine&#160;</td>
			<td>Applichem</td>
			<td>A3946</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Tyrosine&#160;</td>
			<td>Applichem</td>
			<td>A3401</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Uracile</td>
			<td>Applichem</td>
			<td>A0667</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Valine&#160;</td>
			<td>Applichem</td>
			<td>A3406</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Adenine Hemisulfate Salt&#160;</td>
			<td>Applichem</td>
			<td>A1596</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.22 &#181;m Pore Filter</td>
			<td>Sartorius</td>
			<td>16541</td>
			<td>for sterile filtration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Histidine Monohydrochloride</td>
			<td>Applichem</td>
			<td>A3719</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Leucine&#160;</td>
			<td>Applichem</td>
			<td>A3496</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-Tryptophane&#160;</td>
			<td>Applichem</td>
			<td>A3410</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Yeast Nitrogen Base&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>51483</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-Glucose Monohydrate&#160;</td>
			<td>Applichem</td>
			<td>A1349</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Agar</td>
			<td>Fisher Scientific</td>
			<td>BP2641-1</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Peptone</td>
			<td>Applichem</td>
			<td>A2208</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polyethylene Glycol 4000 (PEG)</td>
			<td>Applichem</td>
			<td>A1249</td>
			<td>for yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lithium Acetate Dihydrate (LiOAc)</td>
			<td>Applichem</td>
			<td>A3478</td>
			<td>for yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Salmon Sperm DNA&#160;</td>
			<td>Applichem</td>
			<td>A2159</td>
			<td>for yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Antibody against Lex-A tag (e.g. Anti-LexA DNA binding region)</td>
			<td>Millipore</td>
			<td>06-719</td>
			<td>for Western blot</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Plasmid Miniprep kit (e.g. GenElute Plasmid Miniprep Kit)</td>
			<td>Sigma-Aldrich</td>
			<td>PLN350</td>
			<td>for plasmid purification from yeast and bacteria</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acid Washed Glass Beads</td>
			<td>Sigma-Aldrich</td>
			<td>G8772</td>
			<td>for plasmid purification from yeast</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ampicillin Sodium Salt</td>
			<td>Applichem</td>
			<td>A0839</td>
			<td>for microbiological selection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Yeast Extract</td>
			<td>Applichem</td>
			<td>A1552</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum concentrator centrifuge (e.g. Concentrator 5301)</td>
			<td>Eppendorf</td>
			<td>Z368245 (Sigma)</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bait vector (e.g. pLexA-N)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Prey vector (e.g. pGAD-HA)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Control prey vector (e.g. pLexA-p53)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Control bait vector (e.g. pACT-largeT)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrocompetent E. coli (e.g. MegaX DH10B T1R Electrocomp Cells)</td>
			<td>Invitrogen</td>
			<td>C640003</td>
			<td>for cloning</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Yeast reporter strain (NMY51:MATahis3&#916;200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ ade2::(lexAop)8-ADE2 GAL4, e.g. NMY51)</td>
			<td>Dualsystems</td>
			<td>P01004</td>
			<td>for Y2H</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Plastic paraffin film (e.g. Parafilm M)</td>
			<td>Sigma-Aldrich</td>
			<td>P7793-1EA</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gas permeable sealer (e.g. BREATHseal)</td>
			<td>Greiner</td>
			<td>676 051</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PCR Cycler (e.g. T100 Thermal Cycler)</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td>for PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">quantitative PCR Cycler (e.g. CFX96 Touch Real-Time PCR Detection System)</td>
			<td>Bio-Rad</td>
			<td>1855195</td>
			<td>for quantitative PCR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge (e.g. Centrifuge 5417 R)</td>
			<td>Eppendorf</td>
			<td>5417 R</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Centrifuge (e.g. Centrifuge 5804 R)</td>
			<td>Eppendorf</td>
			<td>5804 R</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">Scalpell</td>
			<td>Swann-Morton</td>
			<td>0301</td>
			<td>for DNA preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile filter (0.22 &#181;m; Polyethersulfone))</td>
			<td>VWR-International</td>
			<td>514-0073 (European Catalogue number)</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri dishes &#216; 90 mm&#160;</td>
			<td>Greiner Bio-One</td>
			<td>633180</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Petri dishes &#216; 150 mm&#160;</td>
			<td>Greiner Bio-One</td>
			<td>639161</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Photometer (e.g. BioPhotometer)</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Photometer Cuvettes</td>
			<td>Brand</td>
			<td>759015</td>
			<td>for yeast culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water Bath (e.g. Water Bath Memmert WB7)</td>
			<td>Memmert</td>
			<td>WB7</td>
			<td>for yeast transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Western Blot Equipment</td>
			<td>Bio-Rad</td>
			<td>diverse</td>
			<td>for protein detection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">dNTPs</td>
			<td>5Prime</td>
			<td>2201210</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Shaker (Erlenmeyer) Flasks (100 ml; 1000 ml; 2000 ml) and breathable cotton plugs (e.g. Duran Erlenmeyer narrow-neck flasks)</td>
			<td>Sigma Aldrich</td>
			<td>Z232793, Z232858, Z232866</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Shaking Incubator (e.g. GFL 3031)</td>
			<td>GFL</td>
			<td>3031</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Incubator</td>
			<td>Binder</td>
			<td>KB 53 (E3.1)</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ice Maker (e.g. Ice Maker IF 825)</td>
			<td>Omniwash</td>
			<td>IF 825</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">Pipettes and disposable pipette Tips (different volumina: 10-1000 &#181;l)</td>
			<td>Eppendorf</td>
			<td>diverse</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spreader</td>
			<td>Sigma-Aldrich</td>
			<td>SPR-L-S01</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vortex shaker (e.g. MS 3 Minishaker)</td>
			<td>IKA</td>
			<td>3617000</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">T4-Ligase</td>
			<td>Thermo Fisher</td>
			<td>EL0011</td>
			<td>for cloning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fridge (4&#176;C) (e.g. Liebherr Medline FKEX 5000)</td>
			<td>Liebherr</td>
			<td>81.767.580.4</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Freezer (-20&#176;C) (e.g. Liebherr Comfort Professional G5216)</td>
			<td>Liebherr</td>
			<td>G5216</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Graduated Cylinder (e.g. Brand)</td>
			<td>Sigma Aldrich</td>
			<td>Z327352; Z327417; Z327441&#160;&#160;</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Serological Pipettes (e.g. Fisherbrand)</td>
			<td>Thermo Fisher</td>
			<td>S55701</td>
			<td>for yeast and bacteria culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mechanical Pipettor (e.g. Pipetboy acu 2)</td>
			<td>Integra-Biosciences</td>
			<td>155000</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cuvettes for Electroporation (1 mm)</td>
			<td>Molecular Bio Products</td>
			<td>5510-11</td>
			<td>for bacteria transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electroporator (e.g. Eporator)</td>
			<td>Eppendorf</td>
			<td>4309000019</td>
			<td>for bacteria transformation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gel and Blot imaging system (e.g. ChemiDoc MP System)</td>
			<td>Bio-Rad</td>
			<td>1708280</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tubes (Polypropylene) (50 ml)</td>
			<td>VWR-International&#160;</td>
			<td>525-0403 (European Catalogue number)</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tubes (Polypropylene) (13 ml)</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td>general lab equipment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dithiothreitol (DTT, e.g. DTT, 1M)</td>
			<td>Thermo Scientific</td>
			<td>P2325</td>
			<td>for ligation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">adenosine triphosphate (ATP, e.g. ATP Solution (10 mM))</td>
			<td>Ambion&#160;</td>
			<td>AM8110G (distributed by Thermo Scientific)</td>
			<td>for ligation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dropout mix without histidine, leucine, tryptophan, and adenine (DO, e.g. Yeast Synthetic Drop-out Medium Supplements)</td>
			<td>Sigma-Aldrich</td>
			<td>Y2021 Sigma&#160;</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 ...

unravelling-function-bacterial-effector-from-non-cultivable-plant

Universidad de Cartagena UDEC

Research

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

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

"<em&gt;枸橼酸rodentium</em&gt;鼠标产品型号:研究感染性结肠炎的病原体和宿主的贡献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

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.

检测皮质片段在细菌孢子萌发

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

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

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

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此视频中的章节

用荧光素酶在小鼠细菌性感染的形象

由细菌病原体入侵人体细胞

在细菌粘附的研究剪应力

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

移植物抗宿主病的诱导和

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

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自动分离

检测皮质片段在细菌孢子萌发

评估视网膜小胶质细胞吞噬功能