Name: screening and identification of rna silencing suppressors from secreted effectors of plant pathogens
Uploaded: 2020-02-03T14:00:00
Description: Watch this Scientific Journal Video about Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens at JoVE.com

这项工作得到了上海市教育发展基金会"曙光计划"和上海市教委、国家重点研发项目国家重点研发项目的资助。2018YFD0201500），国家自然科学基金（第31571696号，31660510号），中国青年专业人才千人计划，上海市科委（18DZ2260500）。

注：程序的所有步骤应在室温 （RT） 下执行。
注意：在丢弃之前，将所有含有致病微生物的介质以及测定中使用的植物和植物组织放入适当的废物容器和高压灭菌器中。
1. 制备含有假定效应器的质粒结构
<ol><li>选择在感染过程中高度表达的假定分泌效应器，由RNA测序（RNA-Seq）确定，并通过定量实时PCR（qRT-PCR）技术进一步测定。</li>
	<li>使用信号<sup cla...

<ol>
	<li>Waterfield, N. R., 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=Rapid+Virulence+Annotation+(RVA):+identification+of+virulence+factors+using+a+bacterial+genome+library+and+multiple+invertebrate+hosts.">Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (41), 15967-15972 (2008).</li><li>Rovenich, H., Boshoven, J. C., Thomma, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+pathogen+effector+functions:+of+pathogens,+hosts+and+microbiomes.">Filamentous pathogen effector functions: of pathogens, hosts and microbiomes.</a> Current Opinion in Plant Biology. 20, 96-103 (2014).</li><li>Varden, F. A., De la Concepcion, J. C., Maidment, J. H., Banfield, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+the+stage:+effectors+in+the+spotlight.">Taking the stage: effectors in the spotlight.</a> Current Opinion in Plant Biology. 38, 25-33 (2017).</li><li>Lee, A. 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=Identifying+Pseudomonas+syringae+Type+III+Secreted+Effector+Function+via+a+Yeast+Genomic+Screen.">Identifying Pseudomonas syringae Type III Secreted Effector Function via a Yeast Genomic Screen.</a> G3: Genes, Genomes, Genetics. 9 (2), 535-547 (2019).</li><li>Toruno, T. Y., Stergiopoulos, I., Coaker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-Pathogen+Effectors:+Cellular+Probes+Interfering+with+Plant+Defenses+in+Spatial+and+Temporal+Manners.">Plant-Pathogen Effectors: Cellular Probes Interfering with Plant Defenses in Spatial and Temporal Manners.</a> Annual Review of Phytopathology. 54, 419-441 (2016).</li><li>Baulcombe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+in+plants.">RNA silencing in plants.</a> Nature. 431 (7006), 356-363 (2004).</li><li>Pumplin, N., Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+suppression+by+plant+pathogens:+defence,+counter-defence+and+counter-counter-defence.">RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence.</a> Nature Reviews Microbiology. 11 (11), 745-760 (2013).</li><li>Csorba, T., Kontra, L., Burgyan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+silencing+suppressors:+Tools+forged+to+fine-tune+host-pathogen+coexistence.">Viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence.</a> Virology. 479-480, 85-103 (2015).</li><li>Johansen, L. K., Carrington, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+on+the+spot.+Induction+and+suppression+of+RNA+silencing+in+the+Agrobacterium-mediated+transient+expression+system.">Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system.</a> Plant Physiology. 126 (3), 930-938 (2001).</li><li>Powers, J. G., 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=A+versatile+assay+for+the+identification+of+RNA+silencing+suppressors+based+on+complementation+of+viral+movement.">A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.</a> Molecular Plant-Microbe Interactions. 21 (7), 879-890 (2008).</li><li>Voinnet, O., Lederer, C., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+movement+protein+prevents+spread+of+the+gene+silencing+signal+in+Nicotiana+benthamiana.">A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana.</a> Cell. 103 (1), 157-167 (2000).</li><li>Deleris, 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=Hierarchical+action+and+inhibition+of+plant+Dicer-like+proteins+in+antiviral+defense.">Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense.</a> Science. 313 (5783), 68-71 (2006).</li><li>Li, W. X., 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=Interferon+antagonist+proteins+of+influenza+and+vaccinia+viruses+are+suppressors+of+RNA+silencing.">Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (5), 1350-1355 (2004).</li><li>Navarro, 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=A+plant+miRNA+contributes+to+antibacterial+resistance+by+repressing+auxin+signaling.">A plant miRNA contributes to antibacterial resistance by repressing auxin signaling.</a> Science. 312 (5772), 436-439 (2006).</li><li>Qiao, Y., 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=Oomycete+pathogens+encode+RNA+silencing+suppressors.">Oomycete pathogens encode RNA silencing suppressors.</a> Nature Genetics. 45 (3), 330-333 (2013).</li><li>Yin, C., 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=A+novel+fungal+effector+from+Puccinia+graminis+suppressing+RNA+silencing+and+plant+defense+responses.">A novel fungal effector from Puccinia graminis suppressing RNA silencing and plant defense responses.</a> New Phytologist. 222 (3), 1561-1572 (2019).</li><li>Zhang, P., 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+WY+domain+in+the+Phytophthora+effector+PSR1+is+required+for+infection+and+RNA+silencing+suppression+activity.">The WY domain in the Phytophthora effector PSR1 is required for infection and RNA silencing suppression activity.</a> New Phytologist. 223 (2), 839-852 (2019).</li><li>Petersen, T. N., Brunak, S., von Heijne, G., Nielsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SignalP+4.0:+discriminating+signal+peptides+from+transmembrane+regions.">SignalP 4.0: discriminating signal peptides from transmembrane regions.</a> Nature Methods. 8 (10), 785-786 (2011).</li><li>Earley, K. W., 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=Gateway-compatible+vectors+for+plant+functional+genomics+and+proteomics.">Gateway-compatible vectors for plant functional genomics and proteomics.</a> Plant Journal. 45 (4), 616-629 (2006).</li><li>Woodman, M. E., Savage, C. R., Arnold, W. K., Stevenson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+PCR+of+Intact+Bacteria+(Colony+PCR).">Direct PCR of Intact Bacteria (Colony PCR).</a> Current Protocols in Microbiology. 42 (1), 1-7 (2016).</li><li>Cao, X., 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=Identification+of+an+RNA+silencing+suppressor+from+a+plant+double-stranded+RNA+virus.">Identification of an RNA silencing suppressor from a plant double-stranded RNA virus.</a> Journal of Virology. 79 (20), 13018-13027 (2005).</li><li>Burgyan, J., Havelda, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+suppressors+of+RNA+silencing.">Viral suppressors of RNA silencing.</a> Trends in Plant Science. 16 (5), 265-272 (2011).</li><li>Incarbone, M., Dunoyer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+and+its+suppression:+novel+insights+from+in+planta+analyses.">RNA silencing and its suppression: novel insights from in planta analyses.</a> Trends in Plant Science. 18 (7), 382-392 (2013).</li><li>Martinez-Priego, L., Donaire, L., Barajas, D., Llave, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+suppressor+activity+of+the+Tobacco+rattle+virus-encoded+16-kDa+protein+and+interference+with+endogenous+small+RNA-guided+regulatory+pathways.">Silencing suppressor activity of the Tobacco rattle virus-encoded 16-kDa protein and interference with endogenous small RNA-guided regulatory pathways.</a> Virology. 376 (2), 346-356 (2008).</li></ol>

RNA沉默是植物对抗病毒、细菌、卵母和真菌病原体的关键防御机制。反过来，这些微生物已经进化了沉默抑制蛋白，以抵消抗病毒沉默，这些RSS干扰RNA沉默途径22，23的不同步骤。已经开发了几种筛选检测，以识别RSS10。
在这里，我们描述了一种改进的协议，用于筛选P.sojae在感染时分泌到宿主细胞中的有效?...

在过去的二十年里，导致植物疾病的微生物基因组测序的加速导致了序列信息与编码蛋白1的生物功能之间的知识差距越来越大。在测序项目揭示的分子中，有抑制先天免疫和促进宿主殖民化的效应分子;这些因素由破坏性的植物病原体分泌，包括细菌、线虫和丝状微生物。为了应对这些威胁，宿主植物已经进化出新的受体来识别这些效应者，从而恢复免疫反应。因此，效应器受到各种选择性压力，导致病原体谱系中的效应器谱系多样化。近年来，植物病原体的假定效应器已被证明会破坏植物先天免疫，阻碍宿主细胞过程，使微生物以各种方式受益，包括信号通路调节失调、转录、细胞内迁移、细胞骨架稳定性、囊泡贩运和RNA沉默3、4、5。然而，绝大多数病原体效应者，特别是来自丝状病原体的病原体，仍然神秘莫测。
RNA沉默是一种同源介导的基因失活机制，在真核生物中保存。这个过程由长双链RNA（dsRNA）触发，以序列特异性的方式瞄准同源单链RNA（ssRNA），并操纵广泛的生物过程，包括抗病毒防御6。为了克服宿主的先天免疫反应，一些病毒已经进化，以抵消RNA沉默，包括能够在细胞内腔内复制或从沉默重组信号中逃脱的能力。然而，病毒保护其基因组免受RNA沉默依赖基因功能丧失的最普遍策略是编码抑制RNA沉默的特异性蛋白质。已经建立了几种机械不同的方法来筛选和表征RNA沉默（VSRs）的病毒抑制剂，包括共同渗透的农业细菌，表达假定抑制剂的转基因植物，移植和细胞培养9，10，11，12，13。
每种检测都有优缺点，并且以自己独特的方式识别 VSR。最常见的方法之一是基于个体A.tmuefaes培养物的共同渗透，这种培养物蕴藏着潜在的病毒蛋白和报告基因（通常是绿色荧光蛋白[GFP]）在尼科蒂亚纳本查米亚纳16c植物的构成下在花椰菜马赛克病毒35S启动子的控制下表达GFP。在没有活性病毒沉默抑制器的情况下，GFP被宿主细胞识别为外源性，并在渗透后3天内沉默（dpi）。相比之下，如果病毒蛋白具有抑制活性，GFP的表达水平稳定在3~9 dpi9以上。这种共渗透测定简单快捷;然而，它既不高度稳定，也不敏感。尽管如此，该测定已经鉴定出许多VSR具有不同的蛋白质序列和结构在许多RNA病毒7，8。
最近，几种能抑制细胞RNA沉默活性的效应蛋白已被细菌、卵母细胞和真菌植物病原体14、15、16所制成。这些发现表明，RNA沉默抑制是大多数王国的病原体使用的常见策略，促进感染。从理论上讲，许多（如果不是全部）效应器可能编码RNA沉默抑制器（RSS）;然而，迄今为止，只确定了少数，主要原因是缺乏可靠和一般性的筛查战略。此外，在绝大多数植物病原体中，尚未对RNA沉默抑制器进行研究。
在本报告中，我们提出了一个优化的一般方案，用于识别植物病原体效应器，该效应器可以使用农业渗透测定抑制局部和系统性RNA沉默。本研究的首要目标是强调协议的关键方面，并详细描述步骤，从而提供适合几乎所有植物病原体效应物的筛选测定。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Morpholinoethanesulfonic Acid (MES)</td>
			<td>Biofroxx</td>
			<td>1086GR500</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>2xTaq Master Mix</td>
			<td>Vazyme Biotech</td>
			<td>P112-AA</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>3-(N-morpholino) propanesulfonic acid (MOPS)</td>
			<td>Amresco</td>
			<td>0264C507-1KG</td>
			<td>MOPS Buffer</td>
		</tr>
		<tr>
			<td>Acetosyringone (AS)</td>
			<td>Sigma-Aldrich</td>
			<td>D134406-5G</td>
			<td>Induction of Agrobacterium</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296-1KG</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biofroxx</td>
			<td>1110GR100</td>
			<td>Electrophoresis</td>
		</tr>
		<tr>
			<td>Amersham Hybond -N+</td>
			<td>GE Healthcare</td>
			<td>RPN303 B</td>
			<td>Nothern blot</td>
		</tr>
		<tr>
			<td>Amersham Imager</td>
			<td>GE Healthcare</td>
			<td>Amersham Imager 600</td>
			<td>Image</td>
		</tr>
		<tr>
			<td>Bacto Tryptone</td>
			<td>BD Biosciences</td>
			<td>211705</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Bacto Yeast Extraction</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Nikon</td>
			<td>D5100</td>
			<td>Photography</td>
		</tr>
		<tr>
			<td>ChemiDoc MP Imaging System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemiluminescent detection module component of dafa kits</td>
			<td>Thermo Fisher Scientific</td>
			<td>89880</td>
			<td>Luminescence detection</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Amresco</td>
			<td>0230-100G</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>ClonExpress II One Step Cloning Kit</td>
			<td>Vazyme Biotech</td>
			<td>C112-01</td>
			<td>Ligation</td>
		</tr>
		<tr>
			<td>DIG Easy Hyb</td>
			<td>Sigma-Aldrich</td>
			<td>11603558001</td>
			<td>Hybridization buffer</td>
		</tr>
		<tr>
			<td>Easypure Plasmid Miniprep kit</td>
			<td>TransGen Biotech</td>
			<td>EM101-02</td>
			<td>Plasmid Extraction</td>
		</tr>
		<tr>
			<td>EasyPure Quick Gel Extraction Kit</td>
			<td>TransGen Biotech</td>
			<td>EG101-02</td>
			<td>Gel Extraction</td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate</td>
			<td>Amresco/VWR</td>
			<td>0105-1KG</td>
			<td>MOPS Buffer</td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply</td>
			<td>LiuYi</td>
			<td>DYY6D</td>
			<td>Nucleic acid electrophoresis.</td>
		</tr>
		<tr>
			<td>FastDigest EcoRV</td>
			<td>Thermo Fisher Scientific</td>
			<td>FD0304</td>
			<td>Vector digestion</td>
		</tr>
		<tr>
			<td>Gel Image System</td>
			<td>Tanon</td>
			<td>Tanon3500</td>
			<td>Image</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Amresco</td>
			<td>0304-5G</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>K1914</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>LR Clonase II enzyme</td>
			<td>Invitrogen</td>
			<td>11791020</td>
			<td>LR reaction</td>
		</tr>
		<tr>
			<td>Nitrocellulose Blotting membrane 0.45um</td>
			<td>GE Healthcare</td>
			<td>10600002</td>
			<td>Northern</td>
		</tr>
		<tr>
			<td>NORTH2south biotin random prime dna labeling kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>17075</td>
			<td>Biotin labeling</td>
		</tr>
		<tr>
			<td>PCR Thermal Cyclers</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Peat moss</td>
			<td>PINDSTRUP</td>
			<td>Dark Gold + clay</td>
			<td>Plants</td>
		</tr>
		<tr>
			<td>Peters Water-Soluble Fertilizer</td>
			<td>ICE</td>
			<td>Peter Professional 20-20-20</td>
			<td>Fertilizer</td>
		</tr>
		<tr>
			<td>Phanta Max Super-Fidelity DNA Polymerase</td>
			<td>Vazyme Biotech</td>
			<td>P505-d1</td>
			<td>Enzyme</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>MP Biomedicals</td>
			<td>219549005</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>RNA Gel Loading Dye (2X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0641</td>
			<td>RNA Gel Loading Dye</td>
		</tr>
		<tr>
			<td>Sodium Acetate Hydrate</td>
			<td>Amresco/VWR</td>
			<td>0530-1KG</td>
			<td>MOPS Buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Amresco</td>
			<td>0241-10KG</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Tri-Sodium citrate</td>
			<td>Amresco</td>
			<td>0101-1KG</td>
			<td>SSC Buffer</td>
		</tr>
		<tr>
			<td>Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>UV lamp</td>
			<td>Analytik Jena</td>
			<td>UVP B-100AP</td>
			<td>Observation</td>
		</tr>
		<tr>
			<td>UVP Hybrilinker Oven</td>
			<td>Analytik Jena</td>
			<td>OV2000</td>
			<td>Northern</td>
		</tr>
	</tbody>
</table>

screening and identification of rna silencing suppressors from secreted effectors of plant pathogens

RNA沉默是真核生物中一种进化保存的序列特异性基因调控机制。几种植物病原体已经进化出蛋白质，能够抑制宿主植物RNA沉默途径。与病毒效应蛋白不同，只有几种分泌效应蛋白显示出抑制细菌、卵母细胞和真菌病原体中RNA沉默的能力，大多数效应器的分子功能在很大程度上仍不得而知。在这里，我们详细介绍了共渗透测定的稍作修改版本，可作为观察RNA沉默和描述植物病原体分泌的效应蛋白的通用方法。该方法的关键步骤是选择健康且完全发育的叶子，在 600 nm 处将细菌培养值调整为适当的光学密度 （OD），并在渗透的最佳时间观察绿色荧光蛋白 （GFP） 荧光叶，以避免省略抑制活性较弱的效应器。这种改进的修饰方案将有助于快速、准确和广泛的RNA沉默抑制剂的筛选，并作为研究这些蛋白质的分子功能的一个极好的起点。

在上面，我们描述了用于改进筛选检测的分步程序，以评估P. sojae RxLR 效应器的 RSS 活性。实验总共需要5到6周。随后，测定所识别的RSS可以在功能和分子机制方面进一步描述。作为我们方法的一个示例，我们使用RNA沉默1（PSR1）的P.sojae RxLR效应抑制剂，在感染期间通过haustoria分泌并输送到宿主细胞中。
为了确认PSR...

Watch this Scientific Journal Video about Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens at JoVE.com

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

在这里，我们提出了一种经过改良的筛选方法，该方法可广泛用于快速筛选植物病原体中的RNA沉默抑制剂。

植物病原体分泌效应器中RNA沉默抑制器的筛选和鉴定

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

此方法可以帮助回答有关如何筛选植物病原体分泌的RNA沉默抑制剂的关键问题。该技术的主要优点是提供快速、准确和广泛的筛选检测，用于RNA沉默抑制器的识别。准备盆栽土壤混合物，包括50%的泥炭苔，30%的石，20%的紫土和120摄氏度的自动杀菌20分钟。将自洗土壤与植物肥料溶液混合，以每升一克的速度混合，然后将它们分包装成储存在较大托盘中的较小锅中。将尼科蒂亚娜·贝塔米亚娜16c的一两个种子播种到每个锅的土壤表面。用塑料圆顶盖住托盘，让种子发芽。将托盘放在光和温度控制的生长室下，温度为 23 至 25 摄氏度，相对湿度为 50 至 60%，且全天为长。三到四天后，种子发芽。关闭塑料圆顶，让幼苗在用于发芽步骤的相同条件下生长。每两到三天，加入适当的水分，保持土壤湿润，但不浸泡。每10天，添加肥料，以促进进一步生长。保持尼科蒂亚娜贝thamiana 16c植物在正常条件下，直到植物有至少五个完全发达的真叶，没有可见的轴或花蕾，叶子有一个健康的绿色外观。使用 10 至 14 天大的尼科蒂亚娜贝thamiana 16c 植物进行全身RNA沉默测定，使用3至4周大的尼科蒂亚娜贝thamiana 16c叶进行局部RNA沉默测定。使用尖端从LB板中挑选一个阳性菌落，将细胞接种到含有5毫升LB介质的玻璃管中，并辅以每毫升卡纳霉素50微克和每毫升利法霉素50微克。在 30 摄氏度下生长细胞，在 200 RPM 下摇晃 24 至 48 小时。将培养的100微升转移到5毫升的LB介质中，并辅以相同的抗生素、10毫摩尔 MES，在pH 5.6，和20微摩尔A。在 30 摄氏度下生长细菌，在 200 RPM 下摇晃 16 至 20 小时。将细胞以4000倍g离心10分钟。丢弃上流液，将颗粒重新在两毫升10毫摩尔氯化镁缓冲液中。重复洗涤，以确保完全去除抗生素。通过测量 600 纳米的光学密度，确定农业细菌培养的密度。使用 10 毫摩尔氯化镁缓冲液调整细胞培养，使 1.5 至 2.0 的 OD 600。在pH 5.6和150微摩尔S下加入10毫摩尔 MES，加入最终悬浮培养，并在室温下孵育细胞至少3小时，不摇晃。混合含有35S绿色荧光蛋白的农业细菌培养物与含有35S黄瓜马赛克病毒抑制剂2b、作用器或空载体的农业细菌培养物的等量。使用一毫升无针注射器，小心和缓慢地渗透到尼科蒂亚娜贝thamiana 16c叶的亚基亚叶的亚基亚侧的混合农业细菌悬浮液。用软组织湿巾从叶子上去除剩余的细菌悬浮液，然后用记号笔圈出渗透斑块的边缘。渗透后三到四天，使用长波紫外灯直观地检测出渗透的树叶片中的GGFP荧光。渗透后两周，使用灯检测整个植物新生长的叶子，进行系统RNA沉默。为了在渗透后四到七天内从叶组织中分离出总RNA，请从渗入的尼科蒂亚娜贝thamiana 16c斑块中收集叶组织，将其收集到迫击炮中。将液氮放入砂浆中，用研磨棒将组织研磨成细粉末，然后将粉末转移到无菌的两毫升管中。立即将每 100 毫克组织一毫升的 RNA 分离试剂添加到机罩中的样品管中，用力将涡流加入均质，并在室温下孵育五分钟。每毫升RNA分离试剂的体积为200微升加入氯仿，加入到机罩中每个管中，大力摇动15秒，并在室温下孵育5分钟。在4摄氏度下以12，000倍g离心15分钟。将上一液转移到新的无 RNase 管中，然后丢弃颗粒。在上流液中加入0.7体积异丙醇，轻轻反转数次，并在室温下孵育混合物10分钟。在4摄氏度下，以12，000次g的离心将RNA颗粒沉淀15分钟。丢弃上一杯。用 70%乙醇清洗颗粒，将颗粒在罩中风干。在65摄氏度的水浴中孵育10至20分钟，将RNA溶解在二乙基热碳酸盐处理水中。要对GP信使RNA水平进行北方印迹分析，在1X MOPS运行缓冲液中准备1.2%甲醛变性甲糖凝胶。将RNA一对一地与RNA加载染料混合，在65摄氏度下孵育RNA10分钟。立即将变性样品冷却在冰上一分钟。使用移液器，将样品以 100 伏电压将样品加载到凝胶和电泳的孔中 50 分钟，直到 RNA 分离良好。将凝胶冲洗成20X盐水柠檬酸钠缓冲液，去除甲醛。通过设置20X盐水柠檬酸钠缓冲液、一层纸巾、两层湿什么纸、一层凝胶、一层尼龙膜、两层湿 Whatman 纸、两层干 Whatman 纸、一块玻璃板和一个重量，在一夜之间执行毛细管转移。早晨，凝胶中的RNA被转移到尼龙膜上。将膜浸泡在2X盐水柠檬酸钠中，通过将湿膜暴露在UV交联中，将RNA固定到膜上。按照手稿进行。三到四周大的尼科蒂亚娜贝thamiana 16c植物的完全发育的叶子与携带 35S GFP 的农业细菌混合物共同渗透在补丁中。渗透后四天，渗透区域的GP荧光在自然光和长波紫外光下被成像。北方印迹显示，GFP信使RNA在表达35S GFP加35S CMV2b或35S GFP加35S PSR1的叶子中积累高于表达35S GFP加EV的叶子。进行了农业渗透测定，以评估沉默信号在两周大的尼科蒂亚娜贝thamiana16c幼苗的叶子上的传播。在渗透后14天，超过98%的EV在系统叶子中没有明显的GP信号。而CMV2b和PSR1都有效地抑制了沉默信号的系统性扩散。在约80%的共渗透植物中观察到GP荧光，在其余20%的渗透植物中，只有几条红静脉出现在新出现的叶子中。这项技术为研究人员识别许多植物病原体分泌的新的RNA沉默抑制剂铺平了道路。植物和适当的OD的组合是尝试此过程时要记住的最重要的事情。按照此程序，可以进行RNA沉默抑制器的功能表征，以探索这些抑制器如何压按宿主RNA沉默的机制以及输出是什么。当您渗入工厂时，请戴上安全护目镜，并在所有实验步骤中戴上乳胶手套。

Research

JoVE Journal

Genetics

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Identification of Circular RNAs using RNA Sequencing

使用RNA测序识别循环RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...