研究在加尼姆实验室是由研究经费不支持。 908-42.12/2006从德国和以色列基金会（GIF），不授予。 IS-4062-07从美国和以色列两个民族组成的农业研究和发展基金（BARD），以及研究经费没有。 884/07由以色列科学基金会（ISF），以MG

1。一般白粉虱，植物和病毒的准 ​​备FISH分析<ol><li>对棉花幼苗（ 陆地棉品种。阿卡拉）后B和Q型烟粉虱和里面的25±2℃，相对湿度60％标准条件下防虫笼和增长间保持，和一个14小时光照/ 10小时黑暗的光照。 </li><li>进行PCR与利用共生细菌特异性引物如前所述15-19内共生菌感染测试。 </li><li>从商业幼儿园或植物番茄种子购买番茄苗（ 期Zn2 +茄品种。牛排）。 </li><li>与上述相同的饲养条件下保持，直到植物达到4真叶阶段，患者的年龄为粉虱介导的接种TYLCV 15的最合适的（见第4.1段）。 </li></ol> 2。驱虫处理，固定，探针设计，杂交和可视化远藤鱼共生体<ol><li>收集10个成人粉虱从棉花植株吸出，或选择10 第 3或第 4龄若虫从棉花的叶子。 </li><li>立即浸入冷水中卡诺固定液（氯仿 - 乙醇 - 冰醋酸，6点03分01秒，体积/体积）的Eppendorf管内。定为2小时至过夜（最好过夜）。 </li><li>彻底去除固定剂和脱色的6％H 2 O 2在乙醇2小时。 </li><li>保留几天的无水乙醇的样品至数周，在室温下或继续到下一个步骤。 </li><li>设计的探头根据每个细菌的16S核糖体RNA基因的反向互补的DNA引物。对于设计的探头，考虑到该帐户的同样的考虑来设计PCR引物。使用寡核苷酸探针BTP1 5&#39;-Cy5的-TGTCAGTGTCAGCCCAGAAG-3&#39;，Rb1的5&#39;-Cy3标记TCCACGTCGCCGTCTTGC-3&#39;，BTH 5&#39;-Cy3标记CCAGATTCCCAGACTTTACTCA-3&#39;和ARS2 5&#39;-Cy3标记TCATGACCACAACCTCCAAA-3&#39;为Portiera，立克次体，Hamilonella和Arsenophonus，分别具体目标。 </li><li>每毫升过夜杂交的样品在黑暗中在杂交缓冲液（20mM的Tris-HCl，pH值8.0，0.9 M氯化钠，0.01％[重量/体积]十二烷基硫酸钠，30％[体积/体积]甲酰胺）中含有10皮摩尔荧光探针。如果有必要结合一个以上的探针用不同的荧光染料。使用未感染粉虱与靶细菌和无探针的样品作为阴性对照。 </li><li>装载样品整体，在显微镜载玻片上，在包含有液体阻滞剂杂交缓冲液，盖上盖玻片，用密封指甲油，并根据荧光或共聚焦显微镜视图。 </li></ol> 3。粉虱器官解剖，固定，杂交和可视化TYLCV鱼<ol><li>收集成人粉虱由愿望和ANE持续饲养TYLCV感染的番茄植株通过暴露于纸巾浸渍丙酮sthetize。揭露粉虱只有1-2分钟太长时间暴露在丙酮中可以修复粉虱组织。用饲养在被感染的番茄植株，无探头样品对照nonviruliferous粉虱，以确认具体的杂交。 </li><li>对抑郁症的显微镜山粉虱幻灯片，使用立体显微镜器官解剖。 </li><li>从前胸拉开昆虫头部和解剖唾液腺在1x PBS中添加1％甲苯胺蓝染色进行更好的可视化，由于其体积小。允许的色斑吸收2-3分钟。 </li><li>在胸部和腹部，观察并驱逐出肠之间的交界处拉虫分开。通过与上腹部尖镊子推驱逐腹部的内容。 </li><li>轻轻地从成功的解剖取出PBS和甲苯胺蓝，轻轻地加入300微升卡诺固定液固定器官5分钟。 </li><li>一从凹陷以及一个侧面而不是从顶部西元固定剂，以防止解剖器官的失控的移动。一旦触摸固定液，该机关将坚持以玻璃，并在整个过程中，他们不会移动。 </li><li>除去固定液，加入500微升杂交缓冲液补充有10皮摩尔的荧光DNA探针的Cy5-5&#39;-GGAACATCAGGGCTTCGATA-3&#39;，它互补于在TYLCV外壳蛋白基因的序列组成。 </li><li>通过孵育滑动的小湿润室用湿毛巾纸在下面组成的小塑料盒内的样品杂交过夜，在室温下在黑暗中。 </li><li>继杂交，拿起机关用细解剖工具，并迅速将其移至含有30微升的新的杂交缓冲液补充用DAPI（0.1 mg / ml的1×PBS中），并包含有液体阻滞剂新鲜显微镜幻灯片。 </li><li>盖上盖玻片的标本，密封机智ħ指甲油和视域下的荧光或共聚焦显微镜。 </li></ol> 4。厂处理，手工切片，固定和杂交的鱼TYLCV <ol><li>使用带毒粉虱介导的接种接种番茄幼苗TYLCV。可以用接种后一周无症状植物这种鱼方法检测病毒。典型的疾病的症状是明显的后三周接种。使用被感染的番茄植株也没有探测样品对照，以确认具体的杂交。 </li><li>使用锋利的组织学刀片编制番茄手切2-4厘米长的纵向或横截面的茎和叶。 </li><li>在Eppendorf管中，固定在卡诺固定液的章节2小时至室温过夜。 </li><li>除去固定液和保存在无水乙醇中的章节数天至数周，在室温下或继续到下一个步骤。 </li><li>洗净切片三次，每次1分钟，在杂交缓冲液。 </li><li>杂交的部分用500μl的杂交缓冲液中添加有10皮摩尔的荧光寡核苷酸探针的Cy3-5&#39;-GGAACATCAGGGCTTCGATA-3&#39;，它互补于TYLCV外壳蛋白基因的序列组成。 </li><li>冲洗切片3次，每次1分钟，在杂交缓冲液中。 </li><li>山路段全在杂交缓冲液补充用DAPI（0.1 mg / ml的1×PBS中），并包含有液体阻断剂，盖上盖玻片，用密封在荧光或共聚焦显微镜指甲油和看法。 </li></ol>

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作者什么都没有透露。

此处描述的植物病毒在其宿主植物和昆虫载体的本地化和共生细菌在其特定粉虱主机协议，可适于其它病毒在植物中，甚至在动物组织中的定位。此外，该协议可以用于本地化共生和致病性细菌和在植物和动物的系统的其他微生物。所描述的方法依赖于杂交的一个短的，荧光标记的寡核苷酸DNA探针与靶DNA或RNA分子在细胞之间简单的概念。的结果是一个特定的杂交，以最小的背景，并使用荧光或共?...

当研究其感染的植物宿主植物病毒和其他病原体之间的相互作用，它以可视化的病原体和它们各自的核酸在原位 ，而不论它们是否会造成对寄主的负面影响是非常重要的。内部和之间的植物细胞研究病原体的运动时，这是最重要的。 在病原体的基因产物原位定位是补充其他方法为研究致病过程中的重要一步。许多植物病原菌，尤其是病毒，是由昆虫传播的，有其复杂的向量和亲密的互动。这些病毒在它们的矢量定位，是研究的路径进行传输重要的，并且该载体内的可能的相互作用位点。某些昆虫向量植物病毒的传播是通过驻留这些昆虫内共生菌资助。为了更好地研究这些植物病毒B中的传输Ÿ他们的向量，它也是必不可少的，以可视化的一般内共生菌，以及参与病毒传播，尤其如此。因此，病毒和内共生细菌的共存需要调查在其昆虫宿主这些生物之间的可能关系。除了病毒传播，内共生细菌的影响昆虫媒介生物学等几个方面，这些细菌内昆虫因而空间定位较高的兴趣和重视的。 番茄黄化曲叶病毒 （TYLCV）（菜豆金色花叶病毒，Geminiviridae）是栽培番茄的1-4全世界最重要的病毒性疾病复杂。 TYLCV是一种韧皮部限制病毒和专门向量由粉虱 5,6。模型描述其向量粉虱双生的易位已经提出了6-9。两个具体的障碍正在积极越过DURing这个循环式传输：中肠/血淋巴和血淋巴/唾液腺障碍。这个传输过程中被假设由未知的受体能识别病毒衣壳来介导的。 TYLCV被认为是穿越B。粉虱肠6,10，并通过主唾液腺6吸收之前它被注入到植物。在粉虱血淋巴，TYLCV与由昆虫次级内共生菌Hamiltonella产生的GroEL的蛋白相互作用。这种相互作用保证了在血淋巴TYLCV的安全传输，并且由昆虫免疫系统11。Hamiltonella和Portiera，初级共生细菌免受攻击保护它粉虱的，都装在bacteriocytes，血淋巴和房子内共生菌11。B.发现昆虫细胞粉虱怀有额外的内共生菌，包括立克次氏体，Arsenophonus，沃尔巴克氏体和弗里奇EA，它可以内部或外部的bacteriocytes是局部的，并且对昆虫的生物学12不同的影响。 若干报告已经尝试通过使用费时和昂贵的协议，如透射电子显微镜（TEM），抗体和RNA 原位上微观厚部10,13来研究TYLCV的植物和粉虱定位，一项最近的研究中所述的定位通过使用一个简单的协议，14他们的工厂和矢量主机内的植物病毒。在这里，我们描述了一个简单的协议TYLCV在B中的定位烟粉虱解剖肠和唾液腺，并在部分从TYLCV感染的植物编制。我们进一步描述Portiera，B的主要共生细菌的本地化烟粉虱，其二次内共生菌Hamitonella，立克次氏体和Arsenophonus，该协议是基于使用短的DNA探针，即荧光标记在其5&#39;末端，并特异性杂交​​中病毒或细菌的基因序列互补的序列。样品处理是比较容易和得到的信号是高度特异性的。所描述的协议可以用来在他们的植物，动物和昆虫宿主本地化的病毒，细菌和其它病原体，并能进一步用于在任何给定的组织定位的mRNA。

<table border="0" cellpadding="0" cellspacing="0" width="653">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fluorescently labeled&#160; Probes</td>
			<td style="width:95px;">Metabion</td>
			<td style="width:129px;">20 bp HPLC purified</td>
			<td style="width:212px;">Sequence designed by customer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Toluidine blue</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">89640</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">sodium dodecyl sulfate</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">L3771</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Formamide</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">F9037</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Tris-HCl</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">T5941</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Glacial Acetic Acid</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">320099</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Blocker</td>
			<td>Ted Pella Inc.</td>
			<td>22309</td>
			<td></td>
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fluorescence in situ hybridizations (fish) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues

荧光原位杂交（FISH）是考虑到了各种常用的用于可视化基因转录物在真核细胞中，并且可以被进一步修饰以可视化单元中的其他组分，如感染病毒和细菌的方法的名称。空间定位和感染过程中病毒和细菌的可视化的是，在应对不同的刺激补充表达谱实验，如芯片和RNAseq一个必不可少的步骤。了解时空感染这些药物补充生物实验旨在了解与细胞成分及其相互关系。几种技术用于可视化病毒和细菌等的报告基因系统或免疫组织化学的方法是耗时的，并且一些被限制与模型生物体的工作，并涉及复杂的方法。 FISH靶向的RNA或DNA的物种的细胞是相对容易和快速的我的ThOD用于研究基因的时空定位和诊断的目的。此法可强健和相对容易实现，当协议使用短的杂交，商业购买的探头，它并不昂贵。这是特别可靠时的样品制备，固定，杂交和显微可视化不涉及复杂的步骤。在这里，我们描述了一个协议，用于细菌和病毒在昆虫和植物组织定位。该方法是基于简单的制剂，固定，和昆虫整个支架并解剖器官或手工制作的植物部分杂交，用20个碱基对缀合的荧光染料在其5&#39;或3&#39;短的DNA探针末端。这个协议已经被成功地应用于许多昆虫和植物组织，可用于分析在细胞中的mRNA或其它RNA或DNA的物种中的表达。

研究了在该手稿的系统示于图1，并包括一个受感染的植株用TYLCV，成年人和粉虱B的若虫粉虱和白粉虱示出了用于在昆虫TYLCV易位路径的内部解剖结构， 图2示出了双FISH在成年烟粉虱的初级共生Portiera和次级共生Arsenophonus。 图3显示了双FISH为Portiera和Hamiltonella，并图4显示了双鱼的Portiera和立克次体，...

Watch this Scientific Journal Video about Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues at JoVE.com

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues | Immunology and Infection | Video

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

我们在这里描述原位杂交简单的荧光（FISH）方法对病毒和细菌的昆虫和植物组织的定位。这个协议可以延长mRNA的整装可视化和显微切片。

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

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Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

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23.2K 次观看.  Volcani Center. 我们在这里描述原位杂交简单的荧光（FISH）方法对病毒和细菌的昆虫和植物组织的定位。这个协议可以延长mRNA的整装可视化和显微切片。 

视频: Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

科研

免疫与感染

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

免疫荧光和定量PCR对白蝇组织中白蝇病毒的定位和定量

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...