Name: plant-microbe interaction: transcriptional response of bacillus mycoides to potato root exudates
Uploaded: 2018-07-02T13:00:00.000+00:00
Duration: 8 min 59 s
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, 放入含湿蛭石的蒸压锅中。</li>
	<li>把锅放在24摄氏度的气候室里三周。设置它的周期16小时的光 (120 µmol m-2s-1) 和 8 h 的黑暗。为了避免微生物污染, 用一个大的玻璃纤维盒盖住锅。</li>
</ol>2. 马铃薯根系分泌物的收集
<ol><li>当芽苗发芽时, 将篮子与整个马铃薯转移到一个被消毒的烧杯中, 装满150毫升的蒸压去离子水, 土豆块茎放在水面上, 根埋在水中 (见图 1)。保持幼苗在气候室, 并使用相同的设置之前 [24 °c; 周期的16小时的光 (120 µmol m-2s-1), 8 h 的黑暗]。</li>
	<li>从第二天起, 收集含有分泌物的水, 用无菌的去离子水将烧杯填满。每天进行取样, 直到转移苗后的第七天。</li>
	<li>将每个样品分别存放在4摄氏度。对于每个样品, 在 Luria Bertani (磅; 1% 胰蛋白胨, 0.5% 酵母萃取物, 0.5% NaCl) 琼脂板上传播100µL, 以检查微生物污染物。丢弃被污染的样品。</li>
	<li>将所采集的样品组合成一个幼苗, 并将其浓缩为-40 °c, 最后体积为150毫升。</li>
</ol>3. 细菌生长
<ol><li>将 a 的丝状菌株从-80 摄氏度甘油储存到 LB 琼脂板上, 并在30摄氏度的一夜内孵化出盘子。</li>
	<li>接种一个磅液体培养基与一个单一的殖民地从板块, 并种植的文化在一个颤抖的孵化器在 200 rpm 隔夜在30摄氏度。</li>
</ol>4. 细菌的治疗和取样
<ol><li>将根分泌物处理的细菌生长曲线与阴性对照进行比较, 制备出含有50毫升 LB 液培养基的一系列烧瓶。在所有烧瓶中加入0.5 毫升的隔夜细菌培养, 并分别将0%、5%、10% 或 15% (v/v) 根渗出物或无菌去离子水添加到培养基中。</li>
	<li>使用具有去离子水的文化代替根分泌物作为控制。测量光学密度在600毫微米 (OD600) 以后每 1 h。通过绘制 OD600值与时间来生成增长曲线。 注: 我们看到10% 的根分泌物不影响B. 丝状的生长 (见图 2);该比值用于 RNA 序列实验。</li>
	<li>用90毫升预热的 LB 培养基, 将一夜之间的丝状文化稀释到600毫升0.05 的最初 OD。添加10毫升的根分泌物的培养和孵化它在30°c 1 小时。</li>
	<li>使用10毫升无菌去离子水处理作为控制。以 9000 x g 为2分钟, 在4摄氏度时, 从养殖中收集细胞。丢弃上清液, 立即将颗粒冻结在液氮中, 然后将其贮存在-80 摄氏度, 直至使用。</li>
</ol>5. RNA 分离
注意: 在开始隔离之前, 准备工作台、机架和吸管, 方法是用 RNase 净化液清洗它们 (见材料表)。随时戴上手套, 确保所有的管子、小贴士和解决方案都是 RNase 的。尽可能把样品放在冰上。
<ol><li>添加 0.1% (v/v) 的焦碳酸二乙基 (DEPC) 到超纯水 (100 µL DEPC 每100毫升溶液), 搅拌好, 并保持在室温下过夜。在隔夜治疗后, 将混合物 DEPC 灭菌。使用此 DEPC 处理的超纯水制备 TE 缓冲器 [10 毫米三盐酸 (pH = 8); 1 毫米 EDTA] 和 10% SDS (w/v) 溶液。</li>
	<li>在冰上解冻细胞颗粒, 在400µL (DEPC) 缓冲中悬浮。将悬浮液转移到2毫升螺钉盖管中。</li>
	<li>预混料300µL 氯仿-异戊醇 (24:1 伏/五) 和300µL 苯酚 (酸苯酚, RNA 级), 并允许混合物站立5分钟。用500µL 的有机上相添加到悬浮细胞。然后加入50µL 10% SDS 和0.5 克玻璃珠 (0.5 µm) 入管。</li>
	<li>牢固地关闭盖子并且安置管子在一个珠磨房均质机 (参见材料表)。执行3×四十五年代脉冲均匀化与1分钟间隔在冰上。</li>
	<li>离心样品为10分钟在 1.1万 x g (4 °c), 并且转移上部阶段到新的管子。</li>
	<li>加入500µL 氯仿-异戊醇 (24:1) 到上阶段从步骤5.5 和离心机混合物5分钟在 1.1万 x g (4 °c)。 注: 以下步骤是从制造商的说明中修改的, 它是基于商用玻璃纤维过滤器的 RNA 隔离套件 (见材料表)。</li>
	<li>将上部相的500µL 转移到一个新鲜的管子上, 加入2卷 (1 毫升) 的裂解/结合缓冲液, 并将其与吹打上下混合。</li>
	<li>结合过滤器和收集管 (从 RNA 隔离套件) 和吸管的混合物从步骤5.7 到过滤管。离心管为十五年代在 8000 x g 和放弃流动通过。</li>
	<li>准备1.5 毫升离心管, 100 µL DNase 缓冲器, 10 µL DNase I, µL 抑制剂 5 RNase (见材料表)。在过滤管的过滤器上添加准备好的溶液组合, 在15-25 摄氏度时孵化 20-30 分钟。</li>
	<li>按照制造商的指示执行洗涤步骤。使用50µL 的洗脱缓冲洗脱 RNA。</li>
	<li>保存洗脱 RNA 在-80 摄氏度, 并在它周围放置一个塑料石蜡膜。同时, 将一些微升的样品转移到一个1.5 毫升的离心管上进行质量检查。</li>
</ol>6. RNA 质量检查和排序
<ol><li>用 microvolume 分光光度计检查 RNA。 注: 吸光度率在260/280 以上应高于 1.8, 比例为260/230 以上至少 1.8, 最好在2.0 以上。</li>
	<li>使用推荐的 rna 分析试剂盒在自动电泳仪器中运行纯化 rna (见材料表);RNA 完整性号 (玲) 必须至少在7以上才能继续。</li>
	<li>根据制造商的建议, 每样使用3µg 的 rna, 生成具有 rna 序列库准备套件 (见材料表) 的库。 注: 在高通量测序平台上对库的准备工作进行了排序, 生成了配对端读取。</li>
</ol>7. 使用基于 Web 的管道 T 雷克斯的数据分析
<ol><li>使用 FASTX 工具包版本 0.0.13 (Phred 质量分数 > 20) 执行质量筛选。用 Trimmomatic 工具修剪来自适配器序列的原始 RNA 顺序读取。 注: 所有下游分析都是以高质量的干净数据为基础的。</li>
	<li>下载B. 丝状ATCC 6462 NCBI 基因组数据库 (NCBI 加入号: CP009692.1), 并将其作为参考基因组, 用于映射使用 Bowtie2/2.2. 3 的清洁读数。</li>
	<li>使用 HTSeq v0.6.1 计数映射到每个基因的读取数字和每 kilobase 每百万的转录读数 (RPKM) 的每个基因根据该基因的长度和读取计数映射到这个基因。</li>
	<li>为了分析 T 雷克斯管道的数据, 请使用 RPKM 表作为输入, 以及三描述性文件: (i) 一个因素文件来描述实验的因素, (ii) 对比文件, 以确定哪些因素将比较的差异基因表达和 (iii) 一个类文件来描述感兴趣的基因组。 注意: 描述性文件是 in.txt 格式。例子可以在 T-雷克斯 (http://genome2d.molgenrug.nl/) 的网页上找到。一个详细的数据分析指令, 使用 T 雷克斯可以在以前的出版物中找到. 14。</li>
</ol>

<ol>
	<li>Haichar, F. e. Z., 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=Plant+host+habitat+and+root+exudates+shape+soil+bacterial+community+structure.">Plant host habitat and root exudates shape soil bacterial community structure.</a> The ISME Journal: Multidisciplinary Journal of Microbial Ecology. 2 (12), 1221-1230 (2008).</li><li>Badri, D. V., Vivanco, J. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corn+(Zea+mays+L.)+root+exudates+and+their+impact+on+14C-pyrene+mineralization.">Corn (Zea mays L.) root exudates and their impact on 14C-pyrene mineralization.</a> Soil Biology and Biochemistry. 33 (12), 1769-1776 (2001).</li><li>Lambert, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clover+root+exudate+produces+male-biased+sex+ratios+and+accelerates+male+metamorphic+timing+in+wood+frogs.">Clover root exudate produces male-biased sex ratios and accelerates male metamorphic timing in wood frogs.</a> Royal Society Open Science. 2 (12), (2015).</li><li>Tuason, M. M. S., Arocena, J. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+the+Swiss-army+tool+for+genome+feature+analysis.">BEDTools: the Swiss-army tool for genome feature analysis.</a> Current Protocols in Bioinformatics. , (2014).</li><li>Boria, I., Boatti, L., Pesole, G., Mignone, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGS-trex:+next+generation+sequencing+transcriptome+profile+explorer.">NGS-trex: next generation sequencing transcriptome profile explorer.</a> BMC Bioinformatics. 14 (7), S10 (2013).</li></ol>

作者声明他们没有竞争的财政利益。

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

plant-microbe-interaction-transcriptional-response-bacillus-mycoides

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

12.5K 次观看.  University of Groningen. 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。

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

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

农杆菌和根瘤菌--马铃薯介导的马铃薯转化与葡萄染色诱导素基因的启动子活性

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

科研

环境科学

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

马铃薯病毒基于马铃薯的X型微RNA沉默（VbMS）。

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

用土壤微生物感染植物根系的接种策略

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

细菌性叶渗透试验使用的植物防御反应精细表征<em&gt;拟南芥，丁香假单胞菌</em&gt;病害系统

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

免疫与感染

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

生物工程

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 ...

生物学

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

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

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. ...

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

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

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 ...

半水培玻璃罐系统，用于多种植物物种的渗出物分析

A Versatile Glass Jar System for Semihydroponic Root Exudate Profiling

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are dynamic and shaped by biological, environmental, and experimental conditions. Due to their wide diversity and low concentrations, accurate exudate profiles are challenging to determine, even more so in natural environments where other organisms are present, turning over plant-derived compounds and producing additional compounds themselves. The semihydroponic glass jar experimental system introduced here allows control over biological, environmental, and experimental factors. It allows the growth of various phylogenetically distinct plant species for up to several months with or without microbes, in a variety of different growth media. The glass-based design offers a low metabolite background for high sensitivity and low environmental impact as it can be reused. Exudates can be sampled nondestructively, and conditions can be altered over the course of an experiment if desired. The setup is compatible with mass spectrometry analytics and other downstream analytical procedures. In summary, we present a versatile growth system suited for sensitive root exudate analysis in a variety of conditions.

作者聚焦:在新型生长系统中探索植物与微生物通过根系分泌物的相互作用 

We present a protocol for a glass-based, semihydroponic experimental system supporting the growth of a variety of phylogenetically distinct plants with or without microbes. The system is compatible with different growth media and permits nondestructive root exudate sampling for downstream analysis.

用于半水培根系渗出物分析的多功能玻璃罐系统

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are ...