Name: a rapid and efficient method for assessing pathogenicity of ustilago maydis on maize and teosinte lines
Uploaded: 2014-01-03T18:00:00
Description: Watch this Scientific Journal Video about A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines at JoVE.com

我们感谢埃米尔·伊马诺维奇博士提供的实验室和温室援助。我们还感谢雪莉·弗林特-加西亚博士提供玉米x特奥辛特内向线。

1. 植物材料的增长
<ol><li>选择植物线进行接种和筛选。本作品使用了两条玉米线、五条特奥辛特线和四十条具有非特异性抗性玉米x特奥辛特线（表1）。</li>
	<li>植物种子用于实验（美国梅迪斯 注射）和控制（注水）针注射接种实验。为每个工厂生产线这样做。</li>
	<li>在小平底中为每株植物线种植四个种子（复制品），用手指将种子推入土壤约1/2英寸，并轻轻覆盖土?...

<ol>
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S., Doehlemann, G., Fernandes, J., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+tumors+caused+by+Ustilago+maydis+require+organ-specific+genes+in+host+and+pathogen.">Maize tumors caused by Ustilago maydis require organ-specific genes in host and pathogen.</a> Sci.. 328, 89-92 (2010).</li><li>Kamper, 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=Insights+from+the+genome+of+the+biotrophic+fungal+plant+pathogen+Ustilago+maydis.">Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.</a> Nature. 444, 97-101 (2006).</li><li>Allen, A., Kaur, J., Gold, S., Shah, D., Smith, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+maize+plants+expressing+the+Totivirus+antifungal+protein,+KP4,+are+highly+resistant+to+corn+smut.">Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut.</a> Plant Biotechnol. J. 8, 857-864 (2011).</li><li>Gold, S. E., Brogdon, S. M., Mayorga, M. E., Kronstad, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ustilago+maydis+regulatory+subunit+of+a+cAMP-Dependent+protein+kinase+is+required+for+gall+formation+in+maize.">The Ustilago maydis regulatory subunit of a cAMP-Dependent protein kinase is required for gall formation in maize.</a> , (1997).</li><li>Gold, S. E., Kronstad, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+two+chitin+syn-+thase+genes+in+the+phytopathogenic+fungus+Ustilago+maydis.">Disruption of two chitin syn- thase genes in the phytopathogenic fungus Ustilago maydis.</a> Mol. Microbiol. 11, 897-902 (1994).</li><li>Brefort, T., Doehlemann, G., Mendoza-Mendoza, A., Reissmann, S., Djamei, A., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ustilago+maydis+as+a+Pathogen.">Ustilago maydis as a Pathogen.</a> Annu. Rev. 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在这项研究中，用于将一株 美国麦地的 针头注射接种方法成功输送到700株玉米和青金植物的茎中。此外，还使用修订后的抗病等级表来筛选植物和检测病原体发展。由于使用这两种方法，在700种玉米和特奥辛特植物中发现了抗 美国梅迪斯 的植物线，这些植物现在可以在育种计划中结合和测试，以提高抗病性。
与大多数接种方法一样，从同一系植物中复制相同?...

植物真菌病是农业面临的最突出的威胁之一。由于世界人口的粮食需求，开发抗病能力提高的作物的需求正在增加。植物病原体自然感染田间作物，引起疾病，对作物产量产生负面影响。结果表明，识别和利用抗性植物可以提高抗性，减少产量损失。许多植物品种，包括玉米、小麦、水稻和高梁，都通过给植物接种植物病原体和选择抗药性线7来识别抗性品种。因此，开发和使用有效的接种方法将使许多植物能够接种疫苗和筛选耐药性。各种接种方法已经使用，包括浸接种，管道病原体细胞悬浮培养到植物的旋转，针注射接种8-11。每种方法，病原体必须可靠地引入植物叶之间，病原体进入植物通过形成的复原体，以确保病原体的发展和植物感染12，13。
浸渍接种方法包括将植物幼苗浸入病原体细胞悬浮培养中，而管状方法要求将病原体细胞悬浮培养进入植物幼苗的旋转。但是，这两种方法都存在问题。首先，这两种方法都取决于病原体从叶面自然运动到植物组织，这是高度可变的。大多数病原体通过植物叶表面的口腔开口或伤口自然进入植物。然而，病原体通过叶面上的气雾和/或伤口穿透植物叶表面的能力存在显著变化。因此，无法用任何一种接种方法控制病原体渗透，这可能导致数据不一致。其次，在筛选大量植物时，将幼苗浸入病原体细胞悬浮培养物中可能非常耗时，并可能限制可筛选的植物数量。相反，此处描述的针头注射接种协议在植物叶之间提供病原体细胞悬浮培养，促进14型阿普索的形成。然后，病原体利用新开发的阿普西里亚进入植物，消除病原体渗透问题。此外，针头注射接种协议为已经接种了 美国梅迪斯 的玉米和青金植物提供了一系列表型，并表现出良好的感染。表型可以用作标记，以确定病原体细胞悬浮培养的最佳浓度，从而在不同的实验中和之间产生一致的植物表型。
植物接种后，与病原体细胞悬浮培养物，植物通常被筛选，以检测耐药或易感表型8-11，15。虽然疾病评级尺度被广泛用于筛选和分类植物表型，但等级等级因所分析的病原体而异。因此，一个疾病等级表协议建立为美国可能迪斯和玉米相互作用可用于类似的真菌病原体16。
本系列协议详细介绍了针头注射接种与 美国可能迪斯 细胞悬浮培养和抗病反应筛查玉米，硫酸盐，玉米x特异丙辛特内引线。目前的协议不限于针注射接种 美国可能迪斯 到玉米植物，但可用于相对任何真菌病原体和植物物种。因此，将这两种方法的细节包括在同一协议中将使研究人员能够直接利用协议进行接种和筛选，或操纵原始协议，以更好地适应病原体和植物物种的利益。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Seed for plants</td>
			<td></td>
			<td></td>
			<td>Collected from original crosses</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Conviron</td>
			<td>PGR14 REACH-IN</td>
			<td></td>
		</tr>
		<tr>
			<td>Planting flats</td>
			<td>Hummert International</td>
			<td>14-3385-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Soil (3 parts pine bark; 1 part peat moss with perlite)</td>
			<td>Hummert International</td>
			<td>10-1059-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Lab Conoco</td>
			<td>70875372</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol stock of pathogen (U. maydis) or fungal pathogen of interest</td>
			<td></td>
			<td></td>
			<td>Stocks were grown from original culture</td>
		</tr>
		<tr>
			<td>Sterile loop</td>
			<td>Fisher Scientific</td>
			<td>S17356A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato dextrose agar (PDA) plates</td>
			<td>Fisher Scientific</td>
			<td>R454311</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator set to 30 &#176;C</td>
			<td>Fisher Scientific</td>
			<td>11-690-650F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile toothpicks</td>
			<td>Walmart</td>
			<td></td>
			<td>Purchased from Walmart and sterilized by autoclave</td>
		</tr>
		<tr>
			<td>Potato dextrose broth (PDB)</td>
			<td>Fisher Scientific</td>
			<td>ICN1008617</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator-shaker set to 30 &#176;C</td>
			<td>New Brunswick</td>
			<td>14-278-179</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Fisher Scientific</td>
			<td>4001000</td>
			<td></td>
		</tr>
		<tr>
			<td>U. maydis&#160;cell suspension culture (1 x 106 cells/ml)</td>
			<td></td>
			<td></td>
			<td>Grown from glycerol stock as described in the methods</td>
		</tr>
		<tr>
			<td>3 ml Syringes</td>
			<td>Becton Dickinson</td>
			<td>309606</td>
			<td></td>
		</tr>
		<tr>
			<td>.457 mm x 1.3 cm Hypodermic needles</td>
			<td>Kendall Brands</td>
			<td>8881250321</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and efficient method for assessing pathogenicity of ustilago maydis on maize and teosinte lines

玉米是全世界主要的谷物作物。然而，易感性生物营养病原体是提高生产力的主要制约因素。美国麦迪斯是一种生物营养真菌病原体，也是玉米上玉米的因果因子。这种疾病导致美国每年损失约10亿美元。 然而，宿主电阻是管理玉米污迹的唯一实用方法。对各种生物营养病原体具有抗药性的作物作物，包括玉米、小麦和水稻的鉴定，每年可显著减少3-5个产量损失。因此，使用病原体接种方法，有效和可重复地将病原体输送到植物叶之间，将有助于快速识别抗美国玉米的玉米线。作为，向确定抗U.maydis的玉米线迈出的第一步，采用针注射接种法和抗性反应筛选法，为玉米、青金石和玉米x特奥辛特引进线接种，并选择抗性植物。
种植了由大约700株植物组成的玉米、青金石和玉米x特奥辛特自给系，接种了 一株美国梅迪斯，并进行了抗药性筛查。接种和筛选方法成功地确定了三条抗 美国梅迪斯的青金石线。这里介绍了玉米、青金石和玉米x青金石引种线的详细针头注射接种和抗性反应筛查方案。这项研究表明，针头注射接种是农业中一种宝贵的工具，可以有效地在植物叶之间输送 美国梅迪斯 ，并提供了对 美的 抗药性的植物线，现在可以在育种计划中结合和测试，以提高抗病性。

成功的针头注射接种可以通过可视化用美国梅迪斯（实验）接种的植物的表型来确定。大多数实验植物都容易感染美国梅迪斯病毒。易感植物表现出非常严重的疾病发展，表现为茎和基底胆形成与黑色三叶草孢子（图3D和3E，表2）。由于疾病的严重性，几种植物在接种后死亡。确定了三条抗美国梅迪斯的玉米x特奥辛特自入系。对于抗美...

Watch this Scientific Journal Video about A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines at JoVE.com

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

据介绍，使用针头注射法为玉米和青金植物注射生物营养病原体 Ustilago。 针头注射接种方法通过形成病原体，促进病原体进入植物的植物叶子之间的真菌病原体的可控输送。这种方法效率很高，使 美国梅迪斯的可重复接种成为可能。

评估 乌西拉戈 病原性的快速高效方法，可在玉米和特奥辛特线上使用

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

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Environment

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

科研

环境科学

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

自动系统（绿色饲料）的用于监视肠道的甲烷和二氧化碳从反刍动物排放

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

管理和检测蛋白标记的节肢动物的扩散研究

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

对于在野外条件下评估种子萌发，休眠和死亡率可靠的方法

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

评估环境和遗传因素对鹿茸和长寿命的鹿的身体生长的相对影响的议定书 》

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

使用 DMAv为环境应用 DMMTAv和 DMDTAv的准备: 合成、纯化和确认

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

夏威夷咖啡莓科学监测协议: 全球咖啡农田模型

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

玉米加工参数和营养品质分析的高通量、微尺度协议 (玉蜀黍)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

评估 乌西拉戈 病原性的快速高效方法，可在玉米和特奥辛特线上使用

评估乌西拉戈病原性的快速高效方法，可在玉米和特奥辛特线上使用