Name: an easy and flexible inoculation method for accurately assessing powdery mildew-infection phenotypes of arabidopsis and other plants
Uploaded: 2021-03-09T14:00:00
Description: Watch this Scientific Journal Video about An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants at JoVE.com

这项工作得到了美国国家科学基金会（IOS-1901566）对S. Xiao的支持。作者要感谢F. Coker和C. Hooks维护植物生长设施，并感谢Jorge Zamora在制造接种盒和接种室方面提供的技术支持。

1. 制作标准接种盒，带有可拆卸的顶盖，并安装有网眼
<ol><li>从商店购买一卷50 μm尼龙膜网或48 μm不锈钢网（推荐）。确保订购足够的量，以切割成多块 14 英寸 x 26 英寸的网眼，以更换磨损的网眼。</li>
	<li>购买一块 1/4 英寸 x 2 英尺 x 4 英尺的中密度纤维板或胶合板，并切下两个 24-1/2 英寸 x 10 英寸的碎片和两个 12 英寸 x 10 英寸的碎片，用于制作接种盒。使用 8 个角夹支撑四个侧面。 注：如?...

<ol>
	<li>Huckelhoven, R., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+bi+ology+of+the+plant-powdery+mildew+interaction.">Cell bi ology of the plant-powdery mildew interaction.</a> Current Opinion in Plant Biology. 14 (6), 738-746 (2011).</li><li>Dean, 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=The+top+10+fungal+pathogens+in+molecular+plant+pathology.">The top 10 fungal pathogens in molecular plant pathology.</a> Molecular Plant Pathology. 13 (4), 414-430 (2012).</li><li>Sakurai, H., Hirata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+the+relation+between+the+penetration+hypha+and+haustorium+of+barley+powdery+mildew+and+host+cell.+V.+Influence+of+water+spray+on+the+pathogen+and+host+tissue.">Some observations on the relation between the penetration hypha and haustorium of barley powdery mildew and host cell. V. Influence of water spray on the pathogen and host tissue.</a> Annual Phytopathology Society of Japan. 24, 239-245 (1959).</li><li>Shomari, S. H., Kennedy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+Oidium+anacardii+on+cashew+(Anacardium+occidentale)+in+southern+Tanzania.">Survival of Oidium anacardii on cashew (Anacardium occidentale) in southern Tanzania.</a> Plant Pathology. 48 (4), 505-513 (1999).</li><li>Sitterly, W. R., Spencer, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Powdery Mildews. , 369 (1978).</li><li>Reuveni, M., Agapov, V., Reuveni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+systemic+resistance+to+powdery+mildew+and+growth+increase+in+cucumber+by+phosphates.">Induction of systemic resistance to powdery mildew and growth increase in cucumber by phosphates.</a> Biological Agriculture &amp; Horticulture. 9 (4), 305-315 (1993).</li><li>Reifschneider, F. J. B., Boiteux, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vacuum-operated+settling+tower+for+inoculation+of+powdery+mildew+fungi.">A vacuum-operated settling tower for inoculation of powdery mildew fungi.</a> Phytopathology. 78 (11), 1463-1465 (1988).</li><li>Chowdhury, A., Bremer, G. B., Salt, D. W., Miller, P., Ford, M. G. <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+method+of+delivering+Blumeria+graminis+f.+sp+hordei+spores+for+laboratory+experiments.">A novel method of delivering Blumeria graminis f. sp hordei spores for laboratory experiments.</a> Crop Protection. 22 (7), 917-922 (2003).</li><li>Xiao, 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=Broad-spectrum+mildew+resistance+in+Arabidopsis+thaliana+mediated+by+RPW8.">Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8.</a> Science. 291 (5501), 118-120 (2001).</li><li>Xiao, S., Ellwood, S., Findlay, K., Oliver, R. P., Turner, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+three+loci+controlling+resistance+of+Arabidopsis+thaliana+accession+Ms-0+to+two+powdery+mildew+diseases.">Characterization of three loci controlling resistance of Arabidopsis thaliana accession Ms-0 to two powdery mildew diseases.</a> The Plant Journal. 12 (4), 757-768 (1997).</li><li>Reuber, T. 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=Correlation+of+defense+gene+induction+defects+with+powdery+mildew+susceptibility+in+Arabidopsis+enhanced+disease+susceptibility+mutants.">Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsis enhanced disease susceptibility mutants.</a> The Plant Journal. 16 (4), 473-485 (1998).</li><li>Xiao, 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=The+atypical+resistance+gene,+RPW8,+recruits+components+of+basal+defence+for+powdery+mildew+resistance+in+Arabidopsis.">The atypical resistance gene, RPW8, recruits components of basal defence for powdery mildew resistance in Arabidopsis.</a> The Plant Journal. 42 (1), 95-110 (2005).</li></ol>

与其他接种方法相比，我们基于网状盒的接种方法具有几个优点。首先，如果操作得当，可以实现孢子的均匀分布，如图 5所示。其次，使用~50μm网眼，加上通过轻轻摇动受感染的叶片来去除孢子，可以减少源植物中存在的蓟马或其他植物感染昆虫的植物感染。第三，使用不同大小的接种盒在塑料室内接种植物或分离的叶子（两者都可以通过喷洒75%乙醇轻松清洁）可以更有?...

白粉病是众多粮食作物和观赏植物中最常见和最重要的疾病之一1。对白粉病的研究非常受欢迎，Web of Science 的搜索结果中有超过 10，500 篇出版物以“白粉病”为关键词（截至 2020 年 11 月）。事实上，白粉病（以灰褐色白粉病为代表）被《分子植物病理学杂志》杂志2认为是十大真菌病原体之一。疾病易感性的定量是表征有助于抗病性或易感性的植物基因或功能鉴定白粉病候选效应基因的必要步骤。然而，与大多数其他真菌病原体相比，白粉病的可靠疾病表型更具挑战性，部分原因是，与后者的孢子不同，白粉病物种的孢子（例如基于我们的实验室经验的Golovinomyces cichoracearum UCSC1）在经历水悬浮过程后显示出生存能力降低3，4.用特定的白粉病病原体接种试验植物的不足和/或不均匀可能导致表型结果不准确。
报告了许多用于白粉病研究的接种方法。这些包括（i）直接从受感染的叶子中刷孢子以测试植物5，（ii）喷洒孢子悬浮液以测试植物6，（iii）使用真空操作的沉降塔将孢子吹到塔7底部的植物，以及（iv）通过组合使用尼龙网膜和基于声音的振动8来输送孢子.孢子刷（或除尘）方法易于执行，但本质上不均匀，因此可能无法准确进行定量评估。孢子喷洒方便均匀，但如上所述可能导致孢子萌发不良4.后两种（即iii-iv）是能够实现均匀接种的改进方法;然而，两者都不能灵活地根据单个事件中要接种的植物数量来调整其接种能力，这使得任何一种设备都不是微不足道的，并且它们的操作仅限于有真空和/或电源的实验室区域。
我们的实验室从事植物-白粉病相互作用已有20多年的历史9，10。在过去的十年中，我们测试了许多接种方法，最近开发了一种简单而有效的白粉病接种方法。这种基于网眼的孢子刷法可以确保均匀接种，并且简单且可扩展，因此任何处理白粉病的实验室都应该很容易采用。

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			<td >&#160;48 &#181;m stainless steel grid mesh screen; Size: 24&#34; X 48&#34;&#160;</td>
			<td >Amazon</td>
			<td >NA</td>
			<td >For making the lid of an inoculation box</td>
		</tr>
		<tr >
			<td >#6-32 x &#190;&#34; machine screws, flat washers and nuts&#160;</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >#6-32 zinc plated nylon lock nut (4-Pack)</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >#6-32x3/8&#8221; Phillips flat head machine screws, flat washers and nuts&#160;</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For securing&#160; magnet door catch plates</td>
		</tr>
		<tr >
			<td >#8-32x1/2&#34; machine screws, flat washers and nuts</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For securing corner braces and door hinge</td>
		</tr>
		<tr >
			<td >0.250 thick clear extruded acrylic film-masked sheet;&#160; Size: 17 &#189;&#34; X 20&#34;</td>
			<td >Professional Plastics</td>
			<td >SACR.250CEF</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >0.250 thick clear extruded acrylic film-masked sheet; Size: 18&#34; X 20&#34;&#160;&#160;</td>
			<td >Professional Plastics</td>
			<td >&#160;SACR.250CEF</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >0.250 thick clear extruded acrylic film-masked sheet; Size: 18&#34; X 30&#34;&#160;</td>
			<td >Professional Plastics</td>
			<td >SACR.250CEF</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >0.250 thick clear extruded acrylic film-masked sheet; Size: 20&#34; X 29 &#189; &#34;</td>
			<td >Professional Plastics</td>
			<td >SACR.250CEF</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >1-5/8&#34; cabinet door magnetic catch white</td>
			<td >Home Depot</td>
			<td >Model #P110-W</td>
			<td>For making an inoculation chamber</td>
		</tr>
		<tr >
			<td >2&#34; steel zinc-plated corner brace (8-Pack)&#160;</td>
			<td >Home Depot</td>
			<td >&#160;Model #13611&#160;</td>
			<td>For making an inoculation box &#38; chamber</td>
		</tr>
		<tr >
			<td >3&#34; Corner Clamp</td>
			<td>Harbor Freight Tools</td>
			<td>SKU 63653, 1852, 60589</td>
			<td>For making inoculation chamber</td>
		</tr>
		<tr >
			<td >3/4&#34;&#160; steel zinc plated corner brace (4-Pack)</td>
			<td >Home Depot</td>
			<td >Model #13542</td>
			<td>For making an inoculation box &#38; chamber</td>
		</tr>
		<tr >
			<td >4-7/8&#34; zinc-plated light duty door pull handles</td>
			<td >Home Depot</td>
			<td >Model #15184</td>
			<td>For making an inoculation box</td>
		</tr>
		<tr >
			<td >Fine fan-blender brushes</td>
			<td>Michaels Store</td>
			<td>M10472846&#160;</td>
			<td>For inoculation</td>
		</tr>
		<tr >
			<td >Kelleher 3/4&#34; x 3/4&#34; x 36&#34; wood square dowel&#160;</td>
			<td >Home Depot</td>
			<td >NA</td>
			<td>For making the lid of an inoculation box</td>
		</tr>
		<tr >
			<td >Medium density fiberboard (1/4&#34; x 2&#39; x 4&#39;);&#160;</td>
			<td >Home Depot</td>
			<td >Model# 1508104</td>
			<td>For making an inoculation box</td>
		</tr>
		<tr >
			<td >Round glass coverslips with a 500 &#181;m grid</td>
			<td >ibidi USA Inc.</td>
			<td >10816</td>
			<td>For determining&#160; spore density</td>
		</tr>
	</tbody>
</table>

an easy and flexible inoculation method for accurately assessing powdery mildew-infection phenotypes of arabidopsis and other plants

减少真菌病害造成的作物损失需要更好地了解控制植物免疫和真菌发病机制的机制，这反过来又需要准确确定植物在感染特定真菌病原体时的疾病表型。然而，使用不可培养的生物营养真菌病原体（如白粉病）进行准确的疾病表型并不容易实现，并且可能是研究项目的限速步骤。在这里，我们以拟南芥-白粉病相互作用为例，开发了一种安全、高效且易于操作的疾病表型系统。该系统主要由三个部分组成：（i）一个木制接种盒，配有一个可拆卸的盖子，该盖子装有~50μm孔的不锈钢或尼龙网，用于用真菌孢子接种植物，（ii）带有小前开口的透明塑料室，用于在内部进行接种时最大限度地减少孢子逃逸， 以及（iii）孢子移位和分配方法，以实现均匀有效的接种。 此处描述的方案包括以低成本制作接种盒和塑料室的步骤和参数，以及如何使用该系统均匀接种白粉病孢子的视频演示，从而提高疾病表型的准确性和可重复性。

在这里，我们提出了一种新的白粉病孢子接种方法，该方法易于制备，操作和调整。 图1 显示了标准接种盒的组装，重点是用50μm膜网安装的可拆卸盖子的制造。 图2 显示了接种室的组装。 图3 说明了使用该系统进行接种过程的关键步骤。 图4 显示了可用于接种整个扁平或更少的植物，或在MS琼脂培养基上接...

Watch this Scientific Journal Video about An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants at JoVE.com

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

我们提出了一种用于构建简单孢子分布系统的协议，该系统由具有~50μm网格的接种盒和透明塑料室组成。这可用于用白粉病孢子均匀地接种植物，从而能够准确和可重复地评估所研究植物的疾病表型。

一种简单灵活的接种方法，用于准确评估拟南芥和其他植物的白粉病感染表型

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...

白粉病是一种重要的植物病害。准确测定疾病表型甚至需要接种病原体。由于现有方法效率低下，我们开发了该协议。我们的孢子接种方法可以轻松实现均匀接种，并且简单且可扩展。因此，任何处理白粉病的实验室都可以轻松使用这种方法。研究人员还可以在纸板箱上安装网眼，并将其用于孢子接种。如何去除孢子并将其刷过网状物是一个关键步骤。因此，在视频中演示这一点可能会非常有帮助。对于此协议，请获取一卷50微米尼龙膜网或48微米不锈钢网。将大型中密度胶合板切割成 24.5X10 英寸和 12X10 英寸的碎片，用于制作接种盒。要制作带有网眼的可拆卸顶盖，请将长木方形销钉切成两组 26 英寸和 12.75 英寸。将四块粘合并钉在盒子周围。拿起亚克力板，使用八个角夹将所有亚克力板组装到一个盒子里。在盒子的亚克力板正面切一个 7X2 英寸的开窗。为了制备新鲜的白粉病孢子作为接种物，请在22摄氏度和65%相对湿度的生长室中种植干净的拟南芥免疫受损的pad4-1突变植物六到八周。种植植物以确定标准公寓的感染表型。接种一株或半株PAP4-1植物，以形成足够的新鲜白粉病孢子。当有来自受感染植物的新鲜孢子时，将一整株植物移入接种室内的接种箱中进行感染测试。切下15至20片严重感染的拟南芥叶子，如果有冷凝水，则让它们干燥。用一对长镊子用一只手倒置握住一到两片叶子。然后将双手穿过腔室的开口，用一把剪刀轻轻敲击镊子的手臂，同时慢慢地将叶子移动到安装在接种盒上的网上方。用细扇式搅拌机刷子将孢子从不同方向刷过整个网状表面，以最大限度地提高孢子在植物上的均匀分布。用刷子轻轻拍打网眼几次，以甩掉卡在网眼中的孢子。孢子沉淀后，从接种箱中移出装有接种植物的平板。用塑料圆顶盖住平面，并将其放置在22摄氏度和65%相对湿度的生长室中。疾病表型可在接种后 5 天或 8 至 12 天进行。通过移除盒子的顶部和底部，用胶带粘住角落以使其牢固，并在顶部用胶水安装一个 50 微米的网格，将纸板箱转换为接种盒。将包含植物的平板移动到接种室内，并将较小的接种盒放在平板顶部以覆盖植物。然后移开并刷拭孢子。制作一个比培养皿稍大的迷你接种盒。将盘子存放在四摄氏度的冰箱中，直到使用。从叶柄的基部切下一到两片完全展开的叶子，用于每株要测试的植物。将分离叶的叶柄以30度角插入琼脂培养基中。在接种室内接种叶子，并将带盖的板移动到生长室中。为了评估感染表型，在接种后四到五天将叶子转移到新鲜无菌MS琼脂平板上。切下叶柄的基部，然后将叶子插入新鲜琼脂培养基中。在接种后 8 或 9 天评估疾病表型。显微分析显示孢子均匀分布在平板底部的显微载玻片上。光照接种后分布在6个不同位置的孢子密度计算结果无显著差异。在用孢子大量接种后也取得了类似的结果。在大量接种后12 d检查不同植物基因型的接种均匀性。与野生型植物哥伦比亚0相比，pad4-1植物表现出更强的疾病易感性。在易感植物上接种后约10天产生的足够新鲜孢子很重要，因为新鲜孢子更容易去除，更有可能建立定植。白粉病仅定植于植物表皮细胞。通过剥离表皮或激光显微解剖来分离白粉病感染的细胞，将使宿主反应的更有针对性的转录组学和蛋白质组学研究成为可能。该方法将帮助研究人员更有效地确定白粉病感染表型，因此应有助于更有效地研究植物白粉病相互作用的功能。

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An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

的RNA分离方法的改进火炬松（ P。火炬松 L）和其他针叶树种

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

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Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

冰帽：为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Construction of an Affordable and Easy-to-Build Zebrafish Facility

一个经济实惠，易于构建斑马鱼设施建设

In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it ...

An Easy Method for Plant Polysome Profiling

植物多聚核糖体剖面的简单方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

三维球体制作一个高效，灵活细胞聚集方法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...