Name: plant-microbe interaction: transcriptional response of bacillus mycoides to potato root exudates
Uploaded: 2018-07-02T13:00:00
Description: Watch this Scientific Journal Video about Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates at JoVE.com

我们感谢雅各布维尔为他提供了有益的意见和建议。我们还感谢安妮在生物信息学分析方面的帮助。Yanglei 和智博李得到中国奖学金委员会 (CSC) 的支持。我们感谢 NWO-TTW Perspectief Programma Back2Roots (TKI-AF-15510) 对 OPK 的财政支持。

1. 杀菌条件下的马铃薯发芽
<ol><li>用无菌水冲洗马铃薯表面。将马铃薯浸泡在70% 乙醇中, 然后在3% 次氯酸钠中沐浴, 每人5分钟。再用无菌水冲洗, 除去剩下的次氯酸钠。</li>
	<li>准备发芽和生长马铃薯块茎所需的材料;将塑料罐、植入篮子、蛭石和水热处理121摄氏度以20分钟消毒。 注: 确保所有使用的材料都耐高压。否则, 使用其他杀菌方法。</li>
	<li>将表面杀菌的马铃薯放入植入篮中, 放入含湿?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+root+exudates.">Regulation and function of root exudates.</a> Plant, Cell &amp; Environment. 32 (6), 666-681 (2009).</li><li>Rohrbacher, F., St-Arnaud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+exudation:+the+ecological+driver+of+hydrocarbon+rhizoremediation.">Root exudation: the ecological driver of hydrocarbon rhizoremediation.</a> Agronomy Journal. 6 (1), 19 (2016).</li><li>Mark, G. 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=Transcriptome+profiling+of+bacterial+responses+to+root+exudates+identifies+genes+involved+in+microbe-plant+interactions.">Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (48), 17454-17459 (2005).</li><li>Fan, 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=Transcriptomic+profiling+of+Bacillus+amyloliquefaciens+FZB42+in+response+to+maize+root+exudates.">Transcriptomic profiling of Bacillus amyloliquefaciens FZB42 in response to maize root exudates.</a> BMC Microbiology. 12 (1), 116 (2012).</li><li>Yoshitomi, K. J., Shann, J. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+organic+acid+exudates+and+properties+of+rhizosphere+soils+of+white+spruce+(Picea+glauca)+and+subalpine+fir+(Abies+lasiocarpa).">Root organic acid exudates and properties of rhizosphere soils of white spruce (Picea glauca) and subalpine fir (Abies lasiocarpa).</a> Canadian Journal of Soil Science. 89 (3), 287-300 (2009).</li><li>Grayston, S. J., Vaughan, D., Jones, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhizosphere+carbon+flow+in+trees,+in+comparison+with+annual+plants:+the+importance+of+root+exudation+and+its+impact+on+microbial+activity+and+nutrient+availability.">Rhizosphere carbon flow in trees, in comparison with annual plants: the importance of root exudation and its impact on microbial activity and nutrient availability.</a> Applied Soil Ecology. 5 (1), 29-56 (1997).</li><li>Rovira, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+root+exudates.">Plant root exudates.</a> Botanical Review. 35 (1), 35-57 (1969).</li><li>Bargabus, R. L., Zidack, N. K., Sherwood, J. E., Jacobsen, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+systemic+resistance+in+sugar+beet+elicited+by+a+non-pathogenic,+phyllosphere-colonizing+Bacillus+mycoides,+biological+control+agent.">Characterisation of systemic resistance in sugar beet elicited by a non-pathogenic, phyllosphere-colonizing Bacillus mycoides, biological control agent.</a> Physiological and Molecular Plant Pathology. 61 (5), 289-298 (2002).</li><li>Peng, Y. -. 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=Inhibition+of+cucumber+Pythium+damping-off+pathogen+with+zoosporicidal+biosurfactants+produced+by+Bacillus+mycoides.">Inhibition of cucumber Pythium damping-off pathogen with zoosporicidal biosurfactants produced by Bacillus mycoides.</a> Journal of Plant Diseases and Protection. 124 (5), 481-491 (2017).</li><li>Ambrosini, 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=Diazotrophic+bacilli+isolated+from+the+sunflower+rhizosphere+and+the+potential+of+Bacillus+mycoides+B38V+as+biofertiliser.">Diazotrophic bacilli isolated from the sunflower rhizosphere and the potential of Bacillus mycoides B38V as biofertiliser.</a> Annals of Applied Biology. 168 (1), 93-110 (2016).</li><li>de Jong, A., van der Meulen, S., Kuipers, O. P., Kok, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-REx:+transcriptome+analysis+webserver+for+RNA-seq+expression+data.">T-REx: transcriptome analysis webserver for RNA-seq expression data.</a> BMC Genomics. 16 (1), 663 (2015).</li><li>Yi, Y., de Jong, A., Frenzel, E., Kuipers, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transcriptomics+of+Bacillus+mycoides+strains+in+response+to+potato-root+exudates+reveals+different+genetic+adaptation+of+endophytic+and+soil+isolates.">Comparative transcriptomics of Bacillus mycoides strains in response to potato-root exudates reveals different genetic adaptation of endophytic and soil isolates.</a> Frontiers in Microbiology. 8, 1487 (2017).</li><li>Nwokeoji, A. O., Kilby, P. M., Portwood, D. E., Dickman, 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=RNASwift:+a+rapid,+versatile+RNA+extraction+method+free+from+phenol+and+chloroform.">RNASwift: a rapid, versatile RNA extraction method free from phenol and chloroform.</a> Analytical Biochemistry. 512, 36-46 (2016).</li><li>Ramirez-Gonzalez, R. H., Bonnal, R., Caccamo, M., MacLean, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-samtools:+Ruby+bindings+for+SAMtools,+a+library+for+accessing+BAM+files+containing+high-throughput+sequence+alignments.">Bio-samtools: Ruby bindings for SAMtools, a library for accessing BAM files containing high-throughput sequence alignments.</a> Source Code for Biology and Medicine. 7 (1), 6 (2012).</li><li>Quinlan, A. 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植物-微生物相互作用被假设是由一个微妙地调整的平衡在细菌和植物之间决定。这种相互作用是高度复杂和难以研究的自然系统, 其中包括多种微生物物种, 可能作为联营集团。本文介绍了一种简化的协议, 以研究在良好的控制条件下, 根分泌物的细菌反应。在暴露于根分泌物时, 细菌的转录剖面提供了细菌对根际生态位的适应的详细信息。这种根分泌物收集协议不需要复杂的程序和专用设备。然?...

植物可能渗出多达20% 的碳固定在光合作用中通过根进入根际1,即, 土壤狭窄的区域附近的根。由于养分的可用性较高, 根际是一种适合多种微生物的栖息地, 包括植物生长促进细菌。根分泌物含有一系列无机化合物, 如离子、无机酸、氧和水。然而, 大多数根系分泌物是由有机物质形成的, 可分为低分子量化合物和高分子量化合物。低分子量化合物包括氨基酸, 有机酸, 糖, 酚类化合物, 脂肪酸, 和一系列次生代谢物。高分子量化合物包括粘液和蛋白质2,3。根际微生物可以将其中一些化合物用作生长和发育的能量来源。根系分泌物在 rhizobacterial 群落的形成过程中起着重要的作用, 因为植物产生的化合物能通过影响特异基因的表达影响根际相关细菌的行为。
了解细菌对根分泌物的反应是破译植物-微生物相互作用机制的关键步骤。由于细菌对植物-微生物相互作用的反应是差异基因表达的产物, 可以通过转录分析来研究。使用这种方法, 以前的研究发现了几个重要的基因参与植物-微生物的相互作用。在铜绿假单胞菌中, 参与代谢、趋化和 II. 型分泌的基因对甜菜根分泌物4有反应。风扇等。5研究了amyloliquefaciens FZB42 对玉米根系分泌物的 transcriptomic 分析。结果表明, 在根分泌物强烈诱发的基因中, 有几个组参与了与养分利用、趋化、运动和非核糖体合成抗菌肽和 polyketides 有关的代谢途径。
这些研究的准确性依赖于根系分泌物的收集。虽然有几种方法描述了用于不同目的的根分泌物的收集, 但它们要么要求尖端仪器, 要么在受控条件6、7、8中不执行。此外, 根际抑制微生物可以影响植物细胞膜通透性和损害根组织, 特别是在微生物联合体9的情况下, 对根分泌物的组成有很显著的作用。在调查微生物对根分泌物的反应时, 重要的是使用明确的条件, 以避免其他微生物对化合物的改变10。此外, 基于 rna 序列的转录研究需要高质量的 rna。然而, 当处理非模型细菌菌株时, 标准协议或商业套件通常由于未知因素或特殊生长特性而效率低下。
这里描述的协议是使用丝状, 这是一个革兰阳性, 孢子形成细菌的 Firmicute。它在各种植物的根际中普遍存在。本文报道了几种植物生长促进特性, 包括甜菜11的系统抗性 (ISR) 的诱导, 对黄瓜12的禾草病原菌的抑制作用, 以及氮固定在向日葵根际13。然而, 其与寄主植物相互作用的分子机制还没有得到很好的研究。
本文的实验目的是研究 endosphere 分离的丝状对马铃薯根系分泌物的 transcriptomic 反应。简而言之, 该议定书由以下步骤组成: 一是在无菌条件下收集马铃薯根系分泌物。然后, 从细菌细胞中提取高质量的 RNA, 用根分泌物处理。最后一步是使用基于 web 的 T 雷克斯管道14的数据分析。该协议用于识别丝状基因, 表明在接触到根系分泌物时表达水平的变化, 从而可能在植物微生物相互作用中发挥重要作用。

<table border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">sodium hypochlorite</td>
			<td style="width:236px;">Sigma</td>
			<td style="width:152px;">&#160;CAS: 7681-52-9&#160;</td>
			<td style="width:339px;">10-15%&#160; active chlorine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Luria-Bertani (LB) broth</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">incubater</td>
			<td style="width:236px;">New Brunswick Scientific</td>
			<td style="width:152px;">Innova 4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spectrophotometer</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">Genesys 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">liquid nitrogen</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">glass beads</td>
			<td style="width:236px;">Sigma</td>
			<td style="width:152px;">G8893</td>
			<td>0.5 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">2.0 ml tube with screw cap</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1.5 ml and 2.0 ml eppendorf tube</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead mill homogenizer</td>
			<td>BioSpec</td>
			<td style="width:152px;">607</td>
			<td>Mini_beadbeater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">centrifuge</td>
			<td style="width:236px;">Eppendorf</td>
			<td style="width:152px;">5430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Diethyl pyrocarbonate (DEPC)</td>
			<td style="width:236px;">sigma</td>
			<td style="width:152px;">CAS: 1609-47-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:236px;">sigma</td>
			<td style="width:152px;">CAS: 151-21-3&#160;</td>
			<td>10% solution prepared with DEPC treated MQ water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">TE buffer</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>10 mM Tris-HCl; 1 mM EDTA, pH=8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">phenol</td>
			<td>Sigma</td>
			<td style="width:152px;"></td>
			<td>RNA grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">chloroform-isoamyl alcohol&#160;</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>prepare 24:1 of chloroform:isoamyl alcohol, store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">High pure RNA isolation kit</td>
			<td style="width:236px;">Roche</td>
			<td style="width:152px;">11828665001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase Decontamination Solution</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:152px;">AM9780</td>
			<td>RNase-Zap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Automated electrophoresis instrument</td>
			<td style="width:236px;">Agilent</td>
			<td style="width:152px;">2100</td>
			<td>Bioanalyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microvolume spectrophotometer</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">Nanodrop ND-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNA quality analysis kit</td>
			<td style="width:236px;">Agilent</td>
			<td style="width:152px;">RNA 6000 Nano kit&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase inhibitor</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">RiboLock</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Directional RNA library Prep kit</td>
			<td style="width:236px;">NEB</td>
			<td style="width:152px;">Ultra</td>
			<td style="width:339px;">For Illumina</td>
		</tr>
	</tbody>
</table>

plant-microbe interaction: transcriptional response of bacillus mycoides to potato root exudates

有益植物相关的细菌在促进植物生长和预防疾病中起着重要作用。应用植物生长促进细菌 (PGPR) 作为生物肥料或生防剂已成为使用常规肥料的有效替代品, 并能以较低的成本提高作物生产率。植物-微生物的相互作用取决于寄主植物分泌的信号以及它们伴生细菌的反应。然而, 分子机制的有益细菌如何响应其相关的植物衍生信号是没有充分了解。评估细菌对根分泌物的 transcriptomic 反应是确定际条件下细菌基因表达和调控的有力途径。这种知识对于了解植物-微生物相互作用所涉及的基本机制是必要的。本文描述了一个详细的协议, 以研究丝状EC18 的 transcriptomic 反应, 从马铃薯 endosphere 的菌株, 马铃薯根系分泌物。在最近的高通量测序技术的帮助下, 该协议可以在几个星期内执行, 并产生大量的数据集。首先, 我们收集在不育条件下的根分泌物, 然后将它们添加到B. 丝状文化中。这些文化中的 RNA 是用苯酚/氯仿方法与商业试剂盒相结合, 并通过自动电泳仪器进行质量控制而分离出来的。在测序后, 采用基于 web 的 T 型雷克斯管道进行数据分析, 确定了一组差异表达基因。这种方法是一个有用的工具, 以促进新发现的细菌基因所涉及的植物-微生物相互作用。

植物相关的微生物可以积极地影响植物的生长和健康。然而, 植物与微生物共生的复杂相互作用机制还没有得到充分的理解。根系分泌物在调节 rhizobacterial 活动和行为方面起着重要作用, 一般认为根系的微生物定植与微生物对根系分泌物的吸引有关。本研究的目的是探讨 rhizobacterial B 丝状对马铃薯根系分泌物的 transcriptomic 反应。为实现这一目的, 马铃薯块茎在蒸压?...

Watch this Scientific Journal Video about Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates at JoVE.com

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

本议定书的目的是研究 endosphere 分离芽孢杆菌丝状对马铃薯根系分泌物的 transcriptomic 反应。这种方法有助于鉴定与植物微生物相互作用有关的重要细菌基因, 并原则上适用于其他菌和植物, 并作小的调整。

植物-微生物相互作用:丝状芽孢杆菌对马铃薯根系分泌物的转录反应

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

Research

JoVE Journal

Biology

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

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

Virus-induced Gene Silencing VIGS 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

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

收集和分析<em&gt;拟南芥</em&gt;韧皮部分泌物使用EDTA促进方法

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

方法发现小鼠BCRP1的另类用法子和转录调控

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

用于植物/微生物分子相互作用和信号的同时系统分析的水培共培养系统

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Single-cell Microfluidic Analysis of Bacillus subtilis

单细胞微流控芯片分析枯草芽孢杆菌

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

一种用于快速鉴定根系分泌物中根细菌化学引诱剂的改进趋化性测定

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...