Name: monitoring bacterial colonization and maintenance on arabidopsis thaliana roots in a floating hydroponic system
Uploaded: 2019-05-28T09:00:00
Description: Watch this Scientific Journal Video about Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System at JoVE.com

这项工作得到了能源生物和环境研究部(DOE-BER 0000217519至E.A.S.)、国家科学基金会(INSPIRE IOS-1343020至E.A.S)提供的研究基金的支持。SLH还得到了国家科学基金会研究生研究奖学金计划的支持。我们感谢Jeffery Dangl博士提供细菌菌株和宝贵的洞察力。我们感谢安德鲁·克莱因博士和马修·鲍尔斯的实验建议。最后,SLH感谢社交媒体上的联系提醒我们,传播科学是一种特权和责任,特别是通过创造性和无障碍手段。

注: 为了清楚起见,实验设置被描述,并用于生成本报告中包含的代表性结果,但条件可以根据需要修改。所有步骤都应使用 PPE 执行,并遵循机构及联邦安全建议,根据所用细菌的 BSL 状态。
1. 细菌特征
<ol><li>确定生长培养基琼脂板上细菌的形态。在近似 OD600 = 0.5 处重新悬浮细胞,并将 1 μL 体积板到选择的琼脂培养基上。将X-gal添加到琼脂板中,最终浓度为20mg/mL,以更好地区...

<ol>
	<li>Strange, R. N., Scott, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+disease:+a+threat+to+global+food+security.">Plant disease: a threat to global food security.</a> Annual Review of Phytopathology. 43, 83-116 (2005).</li><li>Cook, A. M., Grossenbacher, H., H&#252;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+microbes+with+biodegradative+potential.">Isolation and cultivation of microbes with biodegradative potential.</a> Experientia. 39 (11), 1191-1198 (1983).</li><li>Vacheron, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+growth-promoting+rhizobacteria+and+root+system+functioning.">Plant growth-promoting rhizobacteria and root system functioning.</a> Fronteirs in Plant Science. 4, 356 (2013).</li><li>Backer, 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=Plant+Growth-Promoting+Rhizobacteria:+Context,+Mechanisms+of+Action,+and+Roadmap+to+Commercialization+of+Biostimulants+for+Sustainable+Agriculture.">Plant Growth-Promoting Rhizobacteria: Context, Mechanisms of Action, and Roadmap to Commercialization of Biostimulants for Sustainable Agriculture.</a> Fronteirs in Plant Science. 9, 1473 (2018).</li><li>Zamioudis, C., Pieterse, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+host+immunity+by+beneficial+microbes.">Modulation of host immunity by beneficial microbes.</a> Molecular Plant-Microbe Interactions. 25 (2), 139-150 (2012).</li><li>Kr&#246;ber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+strain+Bacillus+amyloliquefaciens+FZB42+on+the+microbial+community+in+the+rhizosphere+of+lettuce+under+field+conditions+analyzed+by+whole+metagenome+sequencing.">Effect of the strain Bacillus amyloliquefaciens FZB42 on the microbial community in the rhizosphere of lettuce under field conditions analyzed by whole metagenome sequencing.</a> Frontiers in Microbiology. 5, 252 (2014).</li><li>Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van Themaat, E., Schulze-Lefert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functions+of+the+bacterial+microbiota+of+plants.">Structure and functions of the bacterial microbiota of plants.</a> Annual Review of Plant Biology. 64, 807-838 (2013).</li><li>Niu, B., Paulson, J. N., Zheng, X., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+and+representative+bacterial+community+of+maize+roots.">Simplified and representative bacterial community of maize roots.</a> Proceedings of the National Academy of Sciences USA. 114 (12), E2450-E2459 (2017).</li><li>Richter-Heitmann, T., Eickhorst, T., Knauth, S., Friedrich, M. W., Schmidt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Strategies+to+Separate+Root-Associated+Microbial+Communities:+A+Crucial+Choice+in+Rhizobiome+Research.">Evaluation of Strategies to Separate Root-Associated Microbial Communities: A Crucial Choice in Rhizobiome Research.</a> Frontiers in Microbiology. 7, 773 (2016).</li><li>Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+coculture+to+detect+chemically+mediated+interspecies+interactions.">Using coculture to detect chemically mediated interspecies interactions.</a> Journal of Visual Experiments. (80), e50863 (2013).</li><li>Woodward, A. W., Bartel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+in+Bloom:+A+Primer+on+the+Arabidopsis+thaliana+Model+System.">Biology in Bloom: A Primer on the Arabidopsis thaliana Model System.</a> Genetics. 208 (4), 1337-1349 (2018).</li><li>Alatorre-Cobos, F., 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=An+improved,+low-cost,+hydroponic+system+for+growing+Arabidopsis+and+other+plant+species+under+aseptic+conditions.">An improved, low-cost, hydroponic system for growing Arabidopsis and other plant species under aseptic conditions.</a> BMC Plant Biology. 14, 69 (2014).</li><li>Haney, C. H., Samuel, B. S., Bush, J., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+with+rhizosphere+bacteria+can+confer+an+adaptive+advantage+to+plants.">Associations with rhizosphere bacteria can confer an adaptive advantage to plants.</a> Nature Plants. 1 (6), (2015).</li><li>Massalha, H., Korenblum, E., Malitsky, S., Shapiro, O. H., Aharoni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+root-bacteria+interactions+in+a+microfluidics+setup.">Live imaging of root-bacteria interactions in a microfluidics setup.</a> Proceedings of the National Academy of Sciences USA. 114 (17), 4549-4554 (2017).</li><li>Townsley, L., Yannarell, S. M., Huynh, T. N., Woodward, J. J., Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+di-AMP+Acts+as+an+Extracellular+Signal+That+Impacts.">Cyclic di-AMP Acts as an Extracellular Signal That Impacts.</a> MBio. 9 (2), (2018).</li><li>Beauregard, P. B., Chai, Y., Vlamakis, H., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+biofilm+induction+by+plant+polysaccharides.">Bacillus subtilis biofilm induction by plant polysaccharides.</a> Proceedings of the National Academy of Sciences U S A. 110 (17), E1621-E1630 (2013).</li><li>Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherence+of+Bacteria+to+Plant+Surfaces+Measured+in+the+Laboratory.">Adherence of Bacteria to Plant Surfaces Measured in the Laboratory.</a> Journal of Visual Experiments. 136 (136), (2018).</li><li>Garcia-Betancur, J. C., Yepes, A., Schneider, J., Lopez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+Bacillus+subtilis+biofilms+using+fluorescence+microscopy+and+flow+cytometry.">Single-cell analysis of Bacillus subtilis biofilms using fluorescence microscopy and flow cytometry.</a> Journal of Visual Experiments. (60), (2012).</li><li>Cole, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+bacterial+plant+colonization+genes.">Genome-wide identification of bacterial plant colonization genes.</a> PLoS Biology. 15 (9), e2002860 (2017).</li><li>Lundberg, D. S., 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=Defining+the+core+Arabidopsis+thaliana+root+microbiome.">Defining the core Arabidopsis thaliana root microbiome.</a> Nature. 488 (7409), 86-90 (2012).</li><li>Grandchamp, G. M., Caro, L., Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pirated+Siderophores+Promote+Sporulation+in+Bacillus+subtilis.">Pirated Siderophores Promote Sporulation in Bacillus subtilis.</a> Applied Environmental Microbiology. 83 (10), (2017).</li><li>Gange, A. C., Gadhave, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+growth-promoting+rhizobacteria+promote+plant+size+inequality.">Plant growth-promoting rhizobacteria promote plant size inequality.</a> Science Reports. 8 (1), 13828 (2018).</li><li>Levy, 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=Genomic+features+of+bacterial+adaptation+to+plants.">Genomic features of bacterial adaptation to plants.</a> Nature Genetics. 50 (1), 138-150 (2018).</li><li>Mart&#237;nez-Hidalgo, P., Maymon, M., Pule-Meulenberg, F., Hirsch, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+root+microbiomes+for+healthier+crops+and+soils+using+beneficial,+environmentally+safe+bacteria.">Engineering root microbiomes for healthier crops and soils using beneficial, environmentally safe bacteria.</a> Canada Journal of Microbiology. , 1-14 (2018).</li><li>Niu, B., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+Composition+Dynamics+of+a+Maize+Root-associated+Simplified+Bacterial+Community+and+Evaluation+of+Its+Biological+Control+Effect.">Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect.</a> Bio Protocol. 8 (12), (2018).</li></ol>

所有环境中的植物与数千到数百万种不同的细菌和真菌相互作用5,7。这些相互作用可能对植物健康产生消极和积极的影响,对作物产量和粮食生产有潜在影响。最近的工作还表明,在田间试验中,PGPB对作物的可变殖民化可能是不可预测的植物规模和作物产量的原因。了解这些相互作用背后的机制可能使我们能够直接操纵植物相关的微生物?...

细菌和真菌疾病造成的作物破坏会导致粮食产量下降,并可能严重破坏全球稳定1。基于抑制土壤中的微生物对植物健康增加的发现2,科学家们询问植物微生物群落是否可以通过改变植物的存在和丰度来支持植物的生长。细菌种类3。发现有助于植物生长或发育的细菌统称为植物生长促进细菌 (PGPB)。最近,研究已从简单地识别潜在的PGPB转向了解土壤、根部周围或根圈(直接围绕和包括根表面的区域)的王国间相互作用如何影响PGPB活动4.
PGPB的根圈殖民化可以增加宿主植物的健康或发育,以应对相对于非殖民化植物的不同压力5。然而,结果在原生土壤条件下往往比在严格控制的温室和实验室环境中观察到的结果多变。这种差异的一个假设是,PGPB的生长或行为可能被原生土壤细菌或真菌在田地7,8抑制。根圈细菌的有益作用通常取决于细菌1)定位和向根部移动的能力,2)通过生物膜形成殖民根部,3)通过生产小分子与宿主植物或病原体相互作用代谢物7,9。任何这些殖民行为都可能受到邻近微生物10的存在和活动的影响。
我们设计了一个系统来量化和可视化根生球的这些独特的细菌殖民化阶段(图1)。这种方法将有助于研究为什么在植物转移到新环境(如接种前幼苗种植期间)后,有时无法观察到长期的PGPB维持。阿拉伯植物被选作植物模型,因为它在实验室研究中的广泛应用,以及关于其微生物相互作用的充分数据11。系统中有三个阶段:1) A. thaliana生长,2) 细菌殖民化,和 3) 细菌维护(参见图 1)。由于A.thaliana是陆地植物,因此必须确保它在水培系统12中不会遭受不必要的水压。受Haney等人13年使用的方法的启发,幼苗在塑料网中生长,将芽与液体生长介质分离。该系统似乎不会损害植物宿主的健康和发展,它提高了液体11的A.thaliana生长。当植物芽漂浮在表面之上时,根部完全暴露在细菌的殖民化中,这些细菌被接种到液体细菌生长培养基中。这允许对感兴趣的细菌进行殖民化,以在最有利于生长的营养物质中殖民化,然后改变条件,使植物在营养介质中继续生长,以支持其生长。两个阶段包括稳定摇动,以防止根13的缺氧。细菌可以从从殖民化介质或维护介质转移后从植物根进行可视化或量化。这种水培系统非常灵活,允许实验条件和应用应力根据研究人员的兴趣轻松改变。
在关于植物-微生物相互作用的文献中,这种描述的方法非常重要,因为它提供了一个强大的系统,用于研究根表面的这些相互作用,同时还可以根据植物-微生物的生长偏好进行定制。不同的细菌。植物生物学实验室经常在固体琼脂上进行植物微生物殖民化实验,只允许细菌的平面运动(如果是的话),同时要求在随后转移过程中对植物进行潜在的破坏性操作。相比之下,微生物实验室经常在实验中优先考虑细菌的健康,这不利于植物14、15。以植物和微生物为重点的实验室的这些不同优先级历来使得很难比较这些组之间的结果,因为每个组通常优化实验条件以优化其感兴趣的生物体。此处描述的浮网-植物生长系统可防止植物完全浸没,这是以往以微生物学为导向的研究的显著优势,同时还暂时优化了细菌的生长和存活,以促进殖民化。因此,我们在这里的测定可以解决植物生物学家的担忧(关于植物的过度水分和触觉操纵),同时满足微生物学家的标准(允许不同的细菌生长条件和多重物种的相互作用)7.该协议旨在适应各种细菌、植物和环境条件。

<table>
	<tbody>
		<tr>
			<td>Required Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL eppendorf tubes</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>BD Falcon</td>
			<td>1801343</td>
			<td></td>
		</tr>
		<tr>
			<td>Aeraseal</td>
			<td>Excel Scientific</td>
			<td>BE255A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteria of Interest</td>
			<td>any</td>
			<td>N/A</td>
			<td>Stored at -80&#730;C in 40% glycerol preferred</td>
		</tr>
		<tr>
			<td>BactoAgar</td>
			<td>BD</td>
			<td>2306428; REF 214010</td>
			<td></td>
		</tr>
		<tr>
			<td>bleach</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Conviron</td>
			<td>any</td>
			<td>N/A</td>
			<td>Short Day Light-Dark Cycles: 460-600 &#181;moles/m&#178;/s set at 9/15 hours light/dark at 18/21&#730;C, with inner power outlet</td>
		</tr>
		<tr>
			<td>Dessicator Jar: glass or heavy plastic</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Flame</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>any</td>
			<td>N/A</td>
			<td>At optimal temperature for growth of specified bacteria</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lennox LB Broth</td>
			<td>RPI</td>
			<td>L24066-1000.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipetters</td>
			<td>any</td>
			<td>N/A</td>
			<td>Volumes 5 &#181;L to 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Microscope (preferably fluorescence)</td>
			<td>any</td>
			<td>N/A</td>
			<td>Could be light if best definition not important</td>
		</tr>
		<tr>
			<td>MS Salts + MES</td>
			<td>RPI</td>
			<td>M70300-50.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Plate Shaker</td>
			<td>any</td>
			<td>N/A</td>
			<td>Capable of running at 220 rpm for at least 96 hours</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>any</td>
			<td>N/A</td>
			<td>50 mL total volume</td>
		</tr>
		<tr>
			<td>Reservoirs</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Hole Punch</td>
			<td>any</td>
			<td>N/A</td>
			<td>Approximately 7mm punch diameter</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Tape</td>
			<td>3M</td>
			<td>MMM1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon Mesh</td>
			<td>McMaster-Carr</td>
			<td>1100t41</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonicator</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>GoldBio</td>
			<td>x4281c</td>
			<td>other vendors available</td>
		</tr>
		<tr>
			<td>Suggested Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 Prong Ultrasonicator attachment</td>
			<td>any</td>
			<td>N/A</td>
			<td>For sonicating multiple samples at once. Can be done individually</td>
		</tr>
		<tr>
			<td>Alumaseal II</td>
			<td>Excel Scientific</td>
			<td>FE124F</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipetter/Repetter</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 96-well plates</td>
			<td>any</td>
			<td>N/A</td>
			<td>For serial dilutions. Can be replaced by eppendorf tubes</td>
		</tr>
		<tr>
			<td>Biological Materials Used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arabidopsis thaliana seeds</td>
			<td>any</td>
			<td>N/A</td>
			<td>We recommend Arabidopsis Biological Resource Center for seed stocks</td>
		</tr>
		<tr>
			<td>Arthrobacter nicotinovorans</td>
			<td></td>
			<td></td>
			<td>Levy, et al. 2018</td>
		</tr>
		<tr>
			<td>Curtobacterium oceanosedimentum</td>
			<td></td>
			<td></td>
			<td>Levy, et al. 2018</td>
		</tr>
		<tr>
			<td>Microbacterium oleivorans</td>
			<td></td>
			<td></td>
			<td>Levy, et al. 2018</td>
		</tr>
		<tr>
			<td>Pseudomonas simiae WCS417r</td>
			<td></td>
			<td></td>
			<td>Published in a similar system in Haney, et al. 2015. Strain used developed in Cole, et al. 2017</td>
		</tr>
	</tbody>
</table>

monitoring bacterial colonization and maintenance on arabidopsis thaliana roots in a floating hydroponic system

细菌形成复杂的根系层微生物群落,由相互作用的微生物、较大的生物体和非生物环境形成。在实验室条件下,植物生长促进细菌(PGPB)的根圈殖民化可以相对于非殖民化植物增加宿主植物的健康或发育。然而,在田间环境中,使用PGPB进行细菌处理通常不会给作物带来实质性的好处。一种解释是,这可能是由于在植物寿命期间与内源性土壤微生物相互作用时PGPB的丧失。这种可能性一直难以证实,因为大多数研究侧重于最初的殖民化,而不是根圈社区内PGPB的维持。这里假设细菌群落的组装、共存和维护是由根生圈微环境的确定性特征决定的,这些相互作用可能会影响PGPB在原生环境中的生存。为了研究这些行为,使用阿拉伯植物生长测定对水培植物生长测定进行了优化,以量化和可视化植物根系初始殖民化和转移到不同生长后的细菌空间分布环境。该系统的可重复性和实用性,然后验证与精心研究的PGPB伪多尼纳斯模拟。研究多种细菌物种的存在如何影响植物根系的殖民化和维护动力学,三种细菌菌株(一种阿尔氏杆菌、柯氏杆菌和微细菌)的模型群落物种)最初与A.thaliana根生球分离的构造。研究表明,这些不同的细菌物种的存在可以通过这种水培植物主量测定来测量,这为基于测序的细菌群落研究提供了替代方案。将来使用该系统的研究可以增进对多物种植物微生物群落中细菌行为的了解,以及随时间变化的环境条件。

著名的PGPB P. simiae WCS417r已知在水培文化中殖民A.thaliana的根。 这种天然荧光细菌在殖民化后可以很容易地使用显微镜在幼苗的根上进行可视化(图2)。虽然可以成像这些A.thaliana幼苗的全长(4⁄6毫米长度)根,这样做的许多植物将需要一段令人望而生畏的时间。由于不同时点和细菌物种的大多数变异可以通过成像根14?...

Watch this Scientific Journal Video about Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System at JoVE.com

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

在这里,我们描述了一种水培植物生长测定,用于量化物种存在,并可视化细菌在植物根系初始殖民化期间和转移到不同生长环境后的空间分布。

在浮动水培系统中监测阿拉伯菌的殖民化与维护

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

大多数实验通过关注殖民化来研究植物-微生物的相互作用，但我们的协议旨在同时研究殖民化和根部细菌的维持。我们可以改变实验，以适应不同的细菌和植物，我们甚至可以测试多个细菌在每个24井板。首先，使用标准打孔器创建塑料网格磁盘。将磁盘收集在玻璃容器中，并松散地盖住。使用设置为 20 分钟干燥循环的自动洗车进行灭菌。使用火焰消毒的钳子将大约 40 个灭菌网盘在先前准备的植物生长介质的红板表面的单层中分配。将两个先前经过消毒的种子放在每个网格的中心。用手术胶带封住盘子，在黑暗中四摄氏度下孵育两到六天，使种子发芽。然后，在21摄氏度的9小时光和15小时的黑暗下，在植物生长室中放置8至10天的板加糖侧，以发芽和生长幼苗。在无菌24井板的每个井中加入一毫升的细菌生长介质。要转移嵌入网状的发芽幼苗，使用火焰灭菌钳子轻轻剥落含有两个同一尺寸的、大小相同且未损坏的幼苗的网状。将一个浮子与幼苗转移到每个井的细菌生长液，根侧向下。接下来，在液体细菌生长介质的阿加板上隔夜生长的再暂停细菌。在每个油井中加入10微升的细菌悬浮液，但仅介质控制井除外，每口井最终浓度为100万菌群形成单位。要密封板以进行无菌生长，请小心地将气体渗透膜压在板上，而不接触粘性面。在井的每个环周围施加压力，以确保每口油井都经过单独密封。然后，将塑料盖紧贴在气体渗透膜上，将板紧贴在盖子上。将板放在植物生长室中，放在设置为 220 rpm 的轨道板摇床上，并在与幼苗最初发芽的相同条件下孵育 18 小时。要用植物冲洗所有浮子，请向新24井板的每个井中加入一毫升无菌水。用植物从板中去除气体渗透膜，使用无菌钳子将浮子转移到有水的水井中，并在室温下孵育10分钟，不搅拌。然后，用一毫升的植物生长介质填充新24井板的每个井。将一个网眼转移到每口井，并盖上气体渗透密封。在植物生长室220转/分的轨道板摇床上孵育72小时。孵育后，像以前一样冲洗植物。冲洗后，将幼苗从网状物中去除，轻轻将火焰消毒钳放在网片叶面的叶子下方。轻轻捏茎，晃动幼苗，远离网状物，在不破坏根部的情况下将根部开走。为了去除植物根部中的细菌，将幼苗从每个网眼转移到含有一毫升双蒸馏水的24孔声波板的单个井中。使用手稿中描述的多管齐下声波器一次对板内的所有样品进行声波化。为了量化细菌，在细菌生长介质中对声波样品进行连续10倍的稀释。使用无菌玻璃珠传播，并在细菌的最佳温度下孵育，直到单个菌落可计数。要收集完整的植物根，请使用钳子从网格中去除幼苗。然后，通过将根尖放在幻灯片上并将其从滑头上拖离，将拍摄与幻灯片齐平，将每个植物转移到显微镜幻灯片上，确保拉直根部以获得最佳成像效果。在每个样品中添加一滴水或无菌植物生长介质，使盖玻片和滑梯之间的界面水合。将玻璃盖玻片放在根冠的上方和射片叶下方，以避免表盖唇倾斜，然后轻轻按下。然后，使用适当的激发/发射过滤器对细菌进行图像图像，以区分细菌彼此和植物根。伪多多多丝，天然荧光细菌，殖民的阿拉伯多普西沙利亚纳根，并在转移到植物生长介质后保留在根部。根冠，中长，和尖端显示荧光由于殖民化伪莫纳斯模拟。无细菌阴性对照显示没有殖民化。当在18小时的殖民化或72小时的维护后对阿拉伯沙皮萨利亚纳根的伪多莫纳斯的模拟被量化时，两个时间点每个幼苗的成群单位总数在不同日子进行的生物复制中显示出良好的可重复性。与殖民化后18小时时间点观察到的数字相比，每种幼苗在72小时后形成菌落的单位数量增加，表明在维持阶段，植物根部上菌群的活性生长。在实验室条件下生长在天然土壤中的阿拉伯多普西沙利亚纳分离出的三种细菌被用来监测植物根部上多种物种的关联。由于菌落形态和颜色的差异，它们在含有X-gal的介质上明显有区别，这使得它们能够计算每个物种的幼苗的成菌单位，而不用选择抗生素，即使在多物种共培养中。这三种细菌都是殖民的，并维持在根部，无论是单独还是细菌共培养。每种物种都显示出不同生物和技术复制的相似趋势，表明该协议可用于测量每个物种的根的相对或总群落形成单位。单独生长时，在维持阶段没有个别物种的丰度显著增加，但联合群落每个根的群落形成单位在联合栽培中增加，表明这些细菌并不禁止其他菌株的殖民化。最困难的步骤是那些需要从网盘中删除完好无损的植物的步骤。耐心和温柔会使这一切成为可能。如果细菌细胞是荧光的，样品可以通过流式细胞学进行排序，以确定细胞的相对数量。

Research

JoVE Journal

Immunology and Infection

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

浆液性卵巢癌原位模型在免疫小鼠，为在体内肿瘤显像和肿瘤免疫反应的监测

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

科研

免疫与感染

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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

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

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 ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

模式触发的氧化爆裂和幼苗生长抑制测定在阿拉伯拉多普西斯塔利亚纳

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

高通量化学化合物筛选，以阐明其对细菌持久性的影响

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...