这项工作得到了江苏省自然科学基金 (BK20140919)、中国国家自然科学基金 (31470375)、江苏高等教育机构重点学术项目开发和青兰项目的支持。

1. 植物的准备 (8 周)
<ol><li>将 50烟草烟种子放在一个1.5 毫升的离心管中, 其中含有1毫升5% 次氯酸和0.1% 海卫 X-100 (v/v) 溶液, 并留出5分钟来消毒种子。</li>
	<li>用无菌水冲洗种子5次, 在200µL 的无菌水中暂停种子。</li>
	<li>小心地将单个种子放到 MS 琼脂培养基上 (1 L: 1x 村 & Skoog 盐, 10 克蔗糖, pH 5.8 (KOH), 1% 琼脂), 并在4° c 中储存介质板, 3 天以同步发芽。 注意: 为了避免微生物污染, 在干净的长凳上执行这一步。在灭菌过程中不需要摇动管子。</li>
	<li>将培养基板置于正常生长条件下 (22 ° c, 80-90 µmolm-2s-1白光强度) 为10天, 然后将发芽的幼苗转移到土壤中。 注意: 一定要将幼苗根部插入土壤中, 不要破坏根部。用透明的塑料盖盖在托盘上过夜, 以保持湿度。</li>
	<li>在一个受控的生长室中生长的植物在一个 16 h/8 的 h 光/暗循环和70% 湿度。7周前的N. 烟植物已准备好进行Agrobacteriumtumefaciens浸润 (图 1A)。 注: 水厂和控制害虫的需要, 保持植物的健康。</li>
</ol>2. 在n 烟叶片中的瞬态表达式 (7-10 天)
<ol><li>提供 150 ng 质粒 (pEGAD-哈卢-卢克控制粒, 空向量, 和构建质粒的检测, 在这种情况下, 我们使用 pCAMBIA1300-Nluc 和 pCAMBIA1300-Cluc 的质粒建设) 到.标准电穿孔法。</li>
	<li>在 Luria Bertani (LB) 琼脂培养基 (1 升:10 克氯化钠, 5 克酵母提取物, 10 g 胰, 1.5% 琼脂) 中含有50µg/毫升卡那霉素和50µg/毫升利福平在28° c 4 天的培养. 注: 抗生素的选择取决于所使用的载体和.</li>
	<li>将单个A. 农杆菌菌落从每个板块接种到3毫升的 LB 液体培养基中, 其中含有50µg/毫升的卡那霉素和50µg/毫升的利福平, 在28° c 时摇动 220 rpm, 以传播细胞培养。</li>
	<li>将75µL 的细菌培养物转化为15毫升的新鲜 LB 液体培养基, 其中含有50µg/毫升的卡那霉素和50µg/毫升的利福平, 其稀释系数为200。培养细菌在220转每分钟在30° c 为大约 8 h, 直到 OD600在0.5 到1.0 之间。</li>
	<li>将A. 农杆菌液体培养在15毫升离心管和离心机在 4000 x g 和4° c 为 15 min. 丢弃上清, 并在15毫升的转化溶液中悬浮颗粒以洗涤颗粒。</li>
	<li>旋转管在 4000 x g 和4° c 为15分钟和放弃上清液。再次重复洗涤步骤。</li>
	<li>在5毫升的转化液中重颗粒, 室温下保持2小时。使用转换解调整A. 农杆菌颗粒细胞的密度, 直到最终浓度的 OD600是 0.5, 或混合两个样本的大小相等, 以测试配对蛋白质的相互作用。</li>
	<li>将悬浮细胞渗透到 4th至 7th , 带1毫升针注射器 (图 1B-C)。入渗后, 用黑色塑料盖立即盖住植物, 在黑暗中放置托盘12小时。之后, 在发现卢克的活动之前, 像往常一样种植2到4天的植物。 注意: 选择大小相近的健康叶子。在一片叶子中, 四不同区域的渗透是好的。</li>
</ol>3. 检测荧光活动 (一天)
注意: 有两种方法可以监视荧光活动。一个是基于成像, 另一个是定量测量荧光活动。
<ol><li>
成像方法
	<ol>
<li>分离渗透的叶子, 把整个传单放在 MS 琼脂培养基上。</li>
		<li>用50毫升喷雾瓶将素工作缓冲液喷洒到叶片正面表面。保持材料在黑暗中5分钟, 以淬火叶绿素荧光。</li>
		<li>使用低光冷却的 CCD 成像设备 (冷却到-30 ° c) 捕获图像 (图 2)。light-filed 曝光时间为50毫秒, 发光检测时间为1分钟。 注: 根据机器说明书调整参数。较低的温度会提高信噪比, 提高图像质量。</li>
	</ol>
</li>
	<li>或者, 直接用商业计测量发光。<ol>
<li>小心地将叶子碎片 (大约0.25 厘米2) 从N. 烟的渗透区中取出, 然后将叶盘浸入100µL 的去离子水中, 在96井白板中 (图 3)。</li>
		<li>添加10µL 的素工作缓冲器, 离开5分钟。然后进行具体的处理, 研究蛋白质相互作用的动力学。 注意: 在不同的时间点检测发光与计, 以获得荧光活性动力学, 这代表了蛋白质-蛋白质相互作用的动力学在一定的治疗。该方法具有同时检测大量蛋白质对的优点。对于那些没有商业计, 检查荧光蛋白质丰度由西方印迹, 因此引用的卢克活动定量。如果没有必要的光, 只是保持板在计和测量发光在不同的时间点。否则, 在检测间隙中将板材移入生长腔。为了获得统计学意义上的结果, 使用至少三的生物复制。虽然卢克的活动从不同的渗透区将变化, 他们的变化模式是一致的正常化后。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Thines, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAZ+repressor+proteins+are+targets+of+the+SCF(COI1)+complex+during+jasmonate+signalling.">JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling.</a> Nature. 448, 661-665 (2007).</li><li>Chini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAZ+family+of+repressors+is+the+missing+link+in+jasmonate+signalling.">The JAZ family of repressors is the missing link in jasmonate signalling.</a> Nature. 448, 666-671 (2007).</li><li>Jones, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Border+control--a+membrane-linked+interactome+of+Arabidopsis.">Border control--a membrane-linked interactome of Arabidopsis.</a> Science. 344, 711-716 (2014).</li><li>Arabidopsis Interactome Mapping Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+network+evolution+in+an+Arabidopsis+interactome+map.">Evidence for network evolution in an Arabidopsis interactome map.</a> Science. 333, 601-607 (2011).</li><li>Yazaki, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+transcription+factor+interactome+networks+using+HaloTag+protein+arrays.">Mapping transcription factor interactome networks using HaloTag protein arrays.</a> Proc Natl Acad Sci U S A. 113, 4238-4247 (2016).</li><li>Kenworthy, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein-protein+interactions+using+fluorescence+resonance+energy+transfer+microscopy.">Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.</a> Methods. 24, 289-296 (2001).</li><li>Walter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> Plant J. 40, 428-438 (2004).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+luciferase+complementation+imaging+assay+for+protein-protein+interactions+in+plants.">Firefly luciferase complementation imaging assay for protein-protein interactions in plants.</a> Plant Physiol. 146, 368-376 (2008).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein-protein+interactions+by+thermal+stability+shift+analysis.">Quantitation of protein-protein interactions by thermal stability shift analysis.</a> Protein Sci. 20, 1439-1450 (2011).</li><li>Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+determination+of+binding+affinity+and+kinetics+using+surface+plasmon+resonance+biosensors.">Reliable determination of binding affinity and kinetics using surface plasmon resonance biosensors.</a> Curr Opin Biotechnol. 8, 498-502 (1997).</li><li>Huang, X., Ouyang, X., Deng, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+repression+of+photomorphogenesis:+role+switching+of+COP/DET/FUS+in+light+signaling.">Beyond repression of photomorphogenesis: role switching of COP/DET/FUS in light signaling.</a> Current opinion in plant biology. 21, 96-103 (2014).</li><li>Liu, B., Zuo, Z., Liu, H., Liu, X., Lin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+cryptochrome+1+interacts+with+SPA1+to+suppress+COP1+activity+in+response+to+blue+light.">Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light.</a> Genes Dev. 25, 1029-1034 (2011).</li><li>Lian, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue-light-dependent+interaction+of+cryptochrome+1+with+SPA1+defines+a+dynamic+signaling+mechanism.">Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism.</a> Genes Dev. 25, 1023-1028 (2011).</li><li>Zuo, Z., Liu, H., Liu, B., Liu, X., Lin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light-dependent+interaction+of+CRY2+with+SPA1+regulates+COP1+activity+and+floral+initiation+in+Arabidopsis.">Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis.</a> Curr Biol. 21, 841-847 (2011).</li></ol>

作者没有什么可透露的。

这里描述的协议是简单和可再生的研究在体内蛋白质-蛋白质相互作用, 特别适合检测蛋白质相互作用动力学在外在治疗之下。本试验的关键步骤是烟浸润。为了确保渗透成功, 植物必须非常健康。另一个关键因素是何时检查渗透后的荧光活动。这个问题没有正确的答案。研究人员被鼓励监测荧光活动在不同的日子后, 渗透判断时, 荧光是表达的最高水平, 因为我们观察到的蛋白质表达?...

为了协调生长与环境, 植物已经进化了优雅的信号通路, 感知, 传感器, 并响应信号提示。就像接力赛中的赛跑者一样, 蛋白质是植物信号传导的必要参与者。人们普遍认识到蛋白质-蛋白质相互作用 (PPI) 在细胞通讯中起着重要作用。蛋白质的磷酸化、再乙酰化和降解都依赖于靶蛋白与其修饰酶之间的物理相互作用。例如, 茉莉以域蛋白 (JAZ) 家族蛋白与转录因子 MYC2 相互作用并抑制其转录活性1,2。鉴于 PPI 的重要性, 植物中的 large-scale 蛋白 interactomes 最近已被探索3,4,5。这些 interactome 的结果进一步揭示了复杂的调控网络, 协调不同的细胞功能。
有一些既定的方法来监测 PPI。酵母两种杂交 (Y2H) 法是 PPI 检测中应用最广泛的方法。Y2H 很容易执行, 适合快速检测蛋白质相互作用。Y2H 也广泛用于筛选未知的交互合作伙伴的特定 protein-of-interest。然而, 由于 Y2H 完全是在酵母中进行的, 它不能反映植物细胞的真实情况, 并带来高假阳性率。另一个类似的策略是下拉法。一般来说, 两个蛋白质的表达和纯化从大肠杆菌细胞, 然后混合和固定的检测蛋白相互作用。虽然此方法不费时, 但它也无法获取在体内交互结果。对于体内交互目的, co-immunoprecipitation (co) 是最常用的检测方法, 它要求高质量的抗体 immunoprecipitate protein-of-interest, 不能排除间接质子的可能性。此外, 由于联合 IP 中的复杂实验程序, 结果通常与个人专长不同。
基于记者的重组分析大大推进了对体内PPI 的检测。这些方法包括荧光共振能量传递 (烦恼)6, 双荧光互补 (BiFC)7, 和萤火虫荧光互补成像 (的) 试验8。虽然这三种方法比 co 更好地反映直接的在体内PPI, 烦恼, 和 BiFC 需要特定的显微镜来检测荧光信号, 不能轻易量化的互动强度。相比之下, 该公司利用萤火虫荧光, 这将发光后与它的基底素反应。更重要的是, PPI 强度可以从荧光活动的价值来确定。因此, 它不仅表明两种蛋白质是否相互作用, 而且还提供了治疗时相互作用动力学的信息。虽然有一些建立的方法来监测交互动力学 (基于表面等离子体共振或热稳定转移)9,10, 这些方法需要微妙的仪器或特定的标签。相比之下, 它更易于执行和检测。
该方法的原理是将氨基末端和羧基末端的荧光 (分别命名为 n-卢克或 C-卢克) 与蛋白 A 和 B 融合, 并在植物细胞中同时表达这两种融合蛋白。如果蛋白质 a 与蛋白质 B 相互作用, 那么荧光的两个半部分将被重组为活性荧光酶。荧光活动可以通过计或 CCD 摄像头检测到。没有必要获得稳定的转化。瞬态表达式足以获得高质量的交互结果。
环型 E3 泛素连接本构 photomorphogenic 1 (COP1) 与无数转录因子相互作用, 通过26S 蛋白酶体11促进它们的降解。其中一些 COP1-targeted 转录因子是光的阳性调节剂。在黑暗中, COP1 与光敏 A-105 1 (SPA1) 的抑制器交互, 这增强了 COP1 E3 活动8。在对光的知觉之后, 光将废除 COP1-SPA1 相互作用抑制 COP1 活动, 然后稳定 COP1 基板12,13,14。这个例子说明了研究 ppi 和确定 ppi 动力学的生物学意义。

<table style="border-collapse:collapse;width:686pt" width="915">
	<colgroup>
		<col style="width:293pt" width="390" />
		<col style="width:140pt" width="186" />
		<col style="width:140pt" width="187" />
		<col style="width:114pt" width="152" />
	</colgroup>
	<tbody>
		<tr height="28" style="height:21.0pt">
			<td class="xl16" height="28" style="width:293pt;height:21.0pt" width="390">Transformation solution</td>
			<td class="xl16" style="width:140pt" width="186"></td>
			<td class="xl16" style="width:140pt" width="187"></td>
			<td style="width:114pt" width="152"></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">10 mM Morpholineethanesulfonic acid</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900336&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">27.8 mM Glucose</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900392</td>
			<td></td>
		</tr>
		<tr height="32" style="height:24.0pt">
			<td class="xl18" height="32" style="width:293pt;height:24.0pt" width="390">10 mM MgCl2&#215;6H2O</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900020&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">150 &#956;M Acetosyringone</td>
			<td class="xl17" style="width:140pt" width="186">ALDRICH</td>
			<td class="xl17" style="width:140pt" width="187">D134406&#160;</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">pH 5.7</td>
			<td class="xl17" style="width:140pt" width="186"></td>
			<td class="xl17" style="width:140pt" width="187"></td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl16" height="28" style="width:293pt;height:21.0pt" width="390">Luciferin working buffer</td>
			<td class="xl17" style="width:140pt" width="186"></td>
			<td class="xl17" style="width:140pt" width="187"></td>
			<td></td>
		</tr>
		<tr height="56" style="height:42.0pt">
			<td class="xl17" height="56" style="width:293pt;height:42.0pt" width="390">5 mM Luciferin potassium salt</td>
			<td class="xl17" style="width:140pt" width="186">GOLD BIOTECHNOLOGY</td>
			<td class="xl17" style="width:140pt" width="187">LUCK-100</td>
			<td></td>
		</tr>
		<tr height="28" style="height:21.0pt">
			<td class="xl17" height="28" style="width:293pt;height:21.0pt" width="390">0.025% Triton X-100</td>
			<td class="xl17" style="width:140pt" width="186">VETEC</td>
			<td class="xl17" style="width:140pt" width="187">V900502&#160;</td>
			<td></td>
		</tr>
		<tr height="32" style="height:24.0pt">
			<td class="xl17" height="32" style="width:293pt;height:24.0pt" width="390">H2O to 10 ml</td>
			<td class="xl21"></td>
			<td class="xl21"></td>
			<td></td>
		</tr>
	</tbody>
</table>

luciferase complementation imaging assay in nicotiana benthamiana leaves for transiently determining protein-protein interaction dynamics

蛋白质-蛋白质相互作用是在大多数细胞过程中中继信号传导的基本机制;因此, 鉴定新的蛋白质-蛋白质相互作用对和监测蛋白质相互作用动力学是特别感兴趣的揭示植物如何响应环境因素和/或发展信号。已经开发出过多的方法来检测蛋白质-蛋白质相互作用, 无论是体外还是体内。其中, 最近建立的荧光互补成像技术是最简单和最快的方法来演示体内蛋白-蛋白质相互作用。在这种检测中, 蛋白质 a 或蛋白 B 与氨基末端或羧基末端荧光的一半分别融合。当蛋白质 a 与蛋白质 B 相互作用时, 荧光的两个半部分将被重组, 形成功能性和活性的荧光酶。荧光活动可以用计或 CCD 摄像机记录。与其他方法相比, 基因组检测显示蛋白质-蛋白质相互作用的定性和定量。农杆菌在烟草烟叶片中的浸润是一种广泛应用的瞬态蛋白表达系统。通过 COP1 和瞬态表达式的结合, 表明茉莉处理后, SPA1 的物理相互作用逐渐降低。

在这个荧光的互补协议中, 有三主要的步骤可用于研究蛋白质-蛋白质相互作用的体内, 包括植物生长、烟草浸润和荧光测定。该协议中最关键的步骤是将液体A. 农杆菌渗透到N. 烟叶 (图 1)。
下面是这个技术的一个例子, 它证实了茉莉减少 COP1-SPA1 的相互作用。荧光的氨基末端和羧基端部...

Watch this Scientific Journal Video about Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics at JoVE.com

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

荧光互补成像检测在烟草烟叶中瞬时测定蛋白质-蛋白质相互作用动力学

这篇手稿描述了一个简单和快速的实验程序, 以确定蛋白质-蛋白质相互作用的基础上荧光活动的测量。

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

luciferase-complementation-imaging-assay-nicotiana-benthamiana-leaves

Research

JoVE Journal

Biology

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

13.8K 次观看.  Nanjing Normal University. 这篇手稿描述了一个简单和快速的实验程序, 以确定蛋白质-蛋白质相互作用的基础上荧光活动的测量。

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

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

科研

生物学

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

液相亲和捕获试验用磁珠研究蛋白质相互作用:脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

双分子荧光互补流式细胞仪分析:高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

马铃薯和尼科蒂亚娜本塔米亚纳的农业渗透和PVX农业感染

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

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

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

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

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

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

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...