1。白粉虱殖民地维护<ol><li> 环境条件：白粉虱的殖民地应保持受控生长室。相对湿度，温度，光周期，光强度的控制是必不可少的，以获得最佳的集落生长（ 图1）。温度28℃，相对湿度30-50％，和一个14小时的光照将产生一个殖民地，从卵到成虫羽化18天（这个时间随植物宿主）发展。阻止昆虫和植物病原真菌的生长，相对湿度应保持在70％以下。必须减少化肥和浇水，阻止真菌病原体和积盐。应该是相当高的光强度为殖民地。 VHO荧光灯灯泡产生足够数量的树冠高度约800-1000英尺烛光是足够的大多数植物（ 即棉花，菜豆，利马豆，番茄）。清洁是必不可少的烟粉虱的殖民地，以保持最佳的饲养条件。 </li><li> 殖民地笼应保持对植物粉虱在笼子里，而不是在生长室。笼可以通过各种材料构成，但必须允许通风，易于访问能力，以防止烟粉虱逸出或浸润，并有足够的尺寸，以保持足够的植物产生所需粉虱人口。与的纱加铝筛选（强度）以及防粉虱筛选（见表特定设备及用品），已取得了良好的效果。 </li><li> 殖民地植物制剂：种植物的选择是一个重要的考虑因素时，开始粉虱殖民地。工厂应能够支持高虫口没有倒塌。选择为一个nonviruliferous集落的植物应该是一个非寄主的病毒或病毒的目的是要被发送的菌落中，以避免污染，如果维持比鲁利的生苗殖民地，它必须是该病毒的宿主。植物应较快地增长，以支持在18天内被引入到殖民地，但出现了新的成人不会这么快就长大了四个星期内笼。矮人，灌木，或庭院型植物品种的建议，因为他们往往​​会产生一个类似的叶面积高的品种，但无茎伸长。 </li><li>这是非常重要的殖民地后植物在温室粉虱和其他昆虫在笼子里排除。使用植物，粉虱，蓟马，螨出没，他们进入前殖民地可以导致菌落崩溃。被感染的植物也有可能被感染虫媒病毒会干扰病毒传播研究。 </li></ol> 2。白粉虱殖民地建立<ol><li>开始第一次使用清洁粉虱 - 植物病毒，其他昆虫和昆虫病原体的一个殖民地。这些可以得到从合作者或从外地。如果实地收集，粉虱应至少8周饲养的植物病毒的非寄主植物上，植物症状的情况下检查，以确保它们是植物病毒。 </li><li>第1周：第一笼植物引进清洁粉虱。粉虱可以引入吸已知的数字，或从另一个源植物上的殖民地的需求取决于粉虱轻轻晃动。在第1周，粉虱产卵植物叶片表面的底部。用于产卵，产卵的平均数量由女性粉虱寄主植物有问题的成年人数的基础上每出现一个近似的人口规模可以预见。 </li><li>第2周：开始另一个笼子，通过引入新的寄主植物的烟粉虱产卵。第1周开始在笼中的植物，将有蛋，和一些未成熟粉虱覆盖的叶子（ 图2）的底面。一些1周期间推出的成人粉虱仍然活着。 </li><li>第3周：开始第三个笼子，通过引入新的植物的烟粉虱产卵。在第1周开始在笼子里的植物将有不成熟以及许多新兴的新成人粉虱在成人粉虱人口应该有一个非常明显的增加。第2周开始在笼中的植物，将有多个卵和未成熟粉虱覆盖的叶子的底面。 </li><li>第4周：开始的第四个笼子，通过引入新的植物的烟粉虱产卵。第1周开始，在笼子里的植物将有大约一个星期后出现的许多成人粉虱。在笼子里的植物，在第2周开始，将有许多新的成人粉虱新兴市场和粉虱人口应该有一个非常明显的增加。第3周开始在笼中的植物，将有覆盖的叶子的底面许多鸡蛋和未成熟的粉虱。 </li><li>每周前4后凌晨KS：每个星期，开始一个新的笼子通过引入成人粉虱从第四个最古老笼（笼1周开始），到新的植物，在一个新的，干净的笼子。老笼应该除掉，并废弃工厂。这些粉虱是约1周后出现。在带毒殖民地的情况下，这些粉虱传播病毒到新的植物，以及产卵粉虱的下一代。 </li><li>计划传输实验使用粉虱将会出现在第三周的笼子。如果需要更多粉虱，植物数量可以增加，可以添加更多的粉虱周1笼增加粉虱人口。 </li></ol> 3。测试植物接种方法<ol><li>为了保证高传输速率：1）必须处理粉虱尽量轻柔，以防止损坏的昆虫，这将降低传输速率; 2）必须有足够的叶面积粉虱企业所得税她的探头或饲料。采集时间的增加或增加收购寄主植物的数量可能会增加低利率的传染性测试植物因收购或接种访问期间拥挤。 </li><li>以下是收集粉虱。粉虱实验目的所需的精确的数字采集和运动的程序。要移动大量的烟粉虱，例如抗性筛选是必要的，它是只需要轻轻摇晃的粉虱出没的植物在试验植物或病毒感染的收购主机（以及后来的测试厂）。是撼动过许多地方的植物，以减少聚集效应。 </li><li>新出现的成人粉虱（1-3天出苗后）非常活跃，经常喂因此往往给予最高传输速率4。大龄粉虱仍然发送，但在较低的频率。多粉虱每个测试厂（解放军15-40元NT）应该用于高速率的传输，因为每株1粉虱的比例往往导致不可接受的低传输速率3,4。需要取决于病毒和物种的采集和测试工厂。 </li><li>组装吸入装置和收集瓶。 （ 图3） </li><li>用一只手按住一个黄色的塑料卡，里面包含要收集的烟粉虱的殖民地笼。轻轻拍打植物，鼓励成年粉虱飞。粉虱将被吸引到了一张黄牌，并从工厂飞到卡在那里他们可以收集使用吸引器和一个非常温和的气息。不要吸烟粉虱取食植物。他们的钢丝为嵌入在植物，同时投喂所以拉他们关闭工厂将打破他们的口针，使他们无法获取或传播病毒（ 图4）。 </li><li>收集约20只成年粉虱成一个单一的科尔CTION小瓶昆虫反复的愿望（ 图5），以尽量减少物理伤害。 </li><li>要改变收集瓶，轻轻拍打小瓶粉虱在坚硬的表面和瓶盖封口膜，而迷失方向的自攻螺丝。 </li><li>将一个新的收集瓶中吸气，重复，直到收集所需粉虱数量。粉虱可以保持几个小时，在室温下将收集瓶。 </li><li> 收购粉虱被放置在一个感染病毒的植物，并允许48-72小时，在受感染的植物为食。收购期间长于72小时，一般不增加传输速率。对于那些在非持久性或半持久性的方式，1小时，几个小时，分别发送的病毒，是足够的采集访问期间（ 表1）。最好的结果是当粉虱免费访问植物在笼子里，限制到一个单一的植物或昆虫多个工厂。然而，一些实验需要采集在这种情况下，剪辑笼可用于从特定的叶片。使用剪辑笼当它是重要的叶子的底面以容纳粉虱取食选择。此外，拥挤在剪辑笼烟粉虱，可以降低传输速率 - 可接受的传输速率已经达到女10粉虱每片段笼直径2.5厘米。 </li><li> 接种准备一个笼子适当接种的大小，将试验植物在里面。如果只有一个工厂要接种考虑一个单株笼。如果考虑一个小的PVC帧/欧根纱袋笼或铝笼（ 图6），一个以上的工厂要接种。用于接种的笼子的大小应稍大一些的大小（s）被接种的植物。粉虱时获得更高的传输速率保持着密切植物冠层，并没有给出一个很大的自由空间。 </li><li>采集主机粉虱的集合取决于实验的需要。如果需要小的数字，吸烟粉虱如上所述。将收集瓶粉虱内笼附近的植物和取下盖子（ 图7）。打开的小瓶和释放粉虱或反转，轻轻拍打的小瓶释放粉虱。如果需要大量简单的移动采集植物与粉虱成的笼子里，测试厂位于轻轻摇晃关闭收购粉虱寄主植物。对于这两种类型的介绍，一定要分发整个植物的烟粉虱，，减少聚集和确保一个统一的接种。 </li><li>允许粉虱探测或饲料为适当的时间后，再次依赖于病毒的寄主植物（ 表1）在一定程度上。 </li><li>粉虱检查至少一次，以确保期间接种访问期间，粉虱探测或打开笼子，每个工厂翻身一些树叶喂养。 </li><li>对于较长的接种访问期间，轻轻扰乱植物（使用竹签或同等学历），刷顶部的植物，，再分配对植物和鼓励粉虱。烟粉虱的再分配有助于确保更高的传输速率打击总粉虱的自然倾向。 </li><li> 终止接种访问期间结束杀死粉虱批准的化学品。应用两种杀虫剂，一前一后：接触杀虫剂，迅速 ​​终止成年粉虱和内吸性杀虫剂，终止任何粉虱的发展在接下来的几个星期，那些错过了接触杀虫剂（ 图8）。一接触杀虫剂杀虫肥皂和全身的杀虫剂，如吡虫啉，吡蚜酮等已被用于具有良好的成功。 </li></ol>

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没有利益冲突的声明。

这里描述的方法被开发过了一段二十年是基于许多粉虱传播，行为和生物学的研究提供的基本信息。由于有很多出版物，这份手稿是指主要的评论，并选定几个特定出版物说明用于开发这项技术的数据类型。这是非常重要的，在此过程中，烟粉虱轻轻地处理自腿和天线打破可以导致异常行为和降低传输率。真空和其他机械抽吸装置收集从未产生了良好的效果。成功的另一个要考虑的是，用于获得100％的传输试验植物的烟粉虱的数量，必须确定每个组合的植物宿主和病毒。这个最佳比例只需要建立一次。已获得了最好的结果，使用年轻成人粉虱（过去1-3天出现），因为他们克香港专业教育学院的最高传输速率。老年人可以使用，但将需要大量粉虱，以弥补其传播率减少。女性被称为传输率高于男性，因为他们养活更多的往往比男性。然而，它通常是不值得的时间用于传输两性分开。此过程可以在许多方面进行修改，以满足研究者的资源和需要。 多种病毒工作时，最好是保持病毒的非寄主植物上烟粉虱饲养的单个菌落。这些粉虱可以收集病毒感染的植物放在收购和收购后，可以移动粉虱再次测试厂。用于殖民地的植物生长在大棚在笼子里，以防止引进粉虱可能与其他病毒感染它们。这些植物的螨类，蓟马，和其他植物病虫害的侵扰，可以妥协的health的殖民地，并导致它的崩溃。最后的殖民地植物浇水护理有许多工作要做。这些植物喂养这么多的烟粉虱和低于正常光照条件下的极端压力。如果植物甚至overwatered一次，根腐菌在无土混合泥炭介绍可以成为一个问题。预处理中的植物用杀真菌剂淋可以消除的一些问题。 虽然烟粉虱传播植物病毒可能会非常耗时，需要宝贵的资源（如生长室），它是必不可少的一些病毒的传播，我们没有其他的传播途径。抗病毒的筛选设备，它也是一种有价值的手段，因为它使用相同类型的传输将有望在该领域的植物抵抗。更多的人工传输手段的使用并不总是产生最好的结果种质评价维鲁抗S-。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the Item</td>
			<td>Company</td>
			<td>Catalogue No.</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Whitefly proof screening</td>
			<td>Klayman Meteor Ltd. Petah Tikva 49130, Israel</td>
			<td>---</td>
			<td>BioNet 50 mesh screen Antivirus Insect Screen</td>
		</tr>
		<tr>
			<td>Whitefly proof screening</td>
			<td>Hummert International (www.hummert.com)</td>
			<td>27-4050-1 - 27-4062-1</td>
			<td>BioNet 50 mesh screen Antivirus Insect Screen</td>
		</tr>
		<tr>
			<td>Whitefly Collection Devices</td>
			<td>BioQuip</td>
			<td>1135A</td>
			<td>Can also be made using eye droppers, flexible plastic tubing, plastic vacuum adaptors, and cheesecloth</td>
		</tr>
		<tr>
			<td>Collection vials</td>
			<td>BioQuip</td>
			<td>8909</td>
			<td>Vials used depend on which aspirator is used.</td>
		</tr>
		<tr>
			<td>Yellow plastic cards</td>
			<td>any company</td>
			<td>---</td>
			<td>Any bright yellow plastic card will work, can be purchased in many places.</td>
		</tr>
		<tr>
			<td>Whitefly Cages</td>
			<td>---</td>
			<td>---</td>
			<td>These are not commercially available and must be constructed. 60 cm x 60 cm x 60 cm cages work well for us. Our preference is for those constructed of aluminum window screening, nylon organdy, vinyl plastic, with a structure created using aluminum window frames. Doors are attached using piano hinges and sealed with weather stripping; tops of the cages are vinyl plastic. Cages can also be made of 1) plexiglass glued together on the edges with ventilation provided by screened openings cut on the side pieces; 2) large bags of nylon organdy, organza, or 50 mesh whitefly screening with internal supports provided by PVC pipes and fittings cut to desired lengths and assembled inside the bags, 3) wood frames and nylon organdy, organza, or 50 mesh whitefly screening.</td>
		</tr>
	</tbody>
</table>

transmitting plant viruses using whiteflies

粉虱， 半翅目：粉虱，烟粉虱 ，一个复杂的形态indistinquishable的5种，是许多植物病毒载体。这些粉虱传播植物的病毒（Carlavirus菜豆金色花叶病毒，Crinivirus，Ipomovirus，Torradovirus）的几个属新兴经济重要的粮食和纤维作物（审阅9,10,16）的重要病原体，包括几百种。这些病毒不复制，但在他们的矢量移动容易从工厂到工厂通过各种手段（2,6,7,9,10,11,17审阅）成人粉虱。对于大多数粉虱取食这些病毒收购和接种需要，而对于其他人只需要探测。许多这些病毒是不能或不能容易地以其他方式传送。因此，维护病毒培养，生物和分子鉴定（鉴定寄主范围和症状）3,132,12，生态，需要被发送到该病毒实验主机使用粉虱向量。此外，开发新的管理方式，如评估新的化学品14或15日 ，新的文化方法1,4,19，或抗病品种的选择和发展的7,8,18，需要使用粉虱病毒的传播。粉虱传播植物病毒的抗病品种的选择和发展育种计划的使用尤其具有挑战性。有效电阻的选择和筛选采用大量的植物，有必要为100％的接种的植物，以便找到具有抗性基因的少数基因型。这些研究使用大量带毒粉虱，通常每年数次。 在这里可以产生数百或数千白粉虱维修成人粉虱的植物，每星期，一年四季，没有其他植物病毒的污染。必须引入粉虱菌落每星期免费粉虱和病毒的植物。粉虱文化必须保持烟粉虱的病原体，寄生虫，寄​​生蜂，可以降低烟粉虱种群和/或减少病毒的传输效率。描述的方式产生的菌落，可以快速缩放以增加或减少人口数量根据需要，可以进行调整，以容纳进给的喜好的粉虱基于病毒的植物宿主中。 有两种基本类型的的粉虱菌落，可以保持：nonviruliferous和带毒粉虱集落。该nonviruliferous集落是由饲养无病毒植物的烟粉虱，粉虱，可用于发送来自不同文化的病毒可以每周可用的。带毒粉虱殖民地，由病毒感染的植物饲养粉虱，允许每周可用性粉虱爱滋病病毒感染者，从而省略病毒传播过程中的一个步骤。

这些粉虱细胞集落的建立和维护，以及操纵传输植物病毒的方法已经成功地使用在许多的研究4,11,12,14，以及许多其它的没有引用。使用这些方法坚持传播的病毒，我们经常获得所需100％的传输率选择阻力，干扰传输能力（和评估的抗性诱导化合物）13,14,17,18筛选杀虫剂（ 图9）。这里描述的程序，并已被改编到许多地方在公共和私人研究设施。 <table border="1"><tbody><tr><td> 传输类型 </td><td> 收购访问期间（小时） </td><td> 接种访问期间（小时） </td><td> 楼盘。 </td></tr><tr><td>非永久性</td><td> 1 </tD&gt; <td> 2-24 </td><td> 10 </td></tr><tr><td>半持久性</td><td> 6-24 </td><td> 8-24 </td><td> 10，16 </td></tr><tr><td>一贯</td><td> 48-72 </td><td> 48-72 </td><td> 3，6，8，9 </td></tr></tbody></table> 表1的估计时间，可用于病毒的传播，生产率高，与不同类型的关系，他们的载体烟粉虱复合种时间估计的基础上公布的最低所需的时间为80〜100％的传输，并在某些情况下，改性占潜伏期和粉虱种群，病毒和寄主植物之间的差异。 <img alt="图1" src="/files/ftp_upload/4332/4332fig1.jpg" /> 图1。带毒粉虱殖民地的一个例子，这个增长空间包含笼感染与双生病毒和INFE的植物STED带毒粉虱。 <img alt="图2" src="/files/ftp_upload/4332/4332fig2.jpg" /> 图2。左：幼体阶段，白粉虱， 烟粉虱 MEAM分支意义上的巴罗6约1周后出现右：成人白粉虱，许多植物病毒的有效载体。 <img alt="图3" fo:content-width="5.5in" fo:src="/files/ftp_upload/4332/4332fig3highres.jpg" src="/files/ftp_upload/4332/4332fig3.jpg" /> 图3。采集装置的类型，可用于收集植物病毒传播的烟粉虱。 <img alt="图4" src="/files/ftp_upload/4332/4332fig4.jpg" /> 图4。左：黄色的塑料卡用于显示粉虱准备收集到收集粉虱成人，<html>右：收集粉虱从一个黄色的塑料卡。粉虱可以看到卡上的小白色/灰色斑点。 <img alt="图5" src="/files/ftp_upload/4332/4332fig5.jpg" /> 图5。粉虱所收集的愿望（20小瓶）从殖民地的准备无论是收购或接种。 <img alt="图6" fo:content-width="5.5in" fo:src="/files/ftp_upload/4332/4332fig6highres.jpg" src="/files/ftp_upload/4332/4332fig6.jpg" /> 图6。上左：单厂笼用于收购或接种要么。右上：较大的PVC管和透明硬纱制成的笼子。较低：铝/屏幕和蝉翼纱的笼在温室白粉虱的殖民地用于含有植物。 32fig7.jpg“/&gt; 图7。粉虱被转移到一个单一的植物接种（或收购）。 <img alt="图8" src="/files/ftp_upload/4332/4332fig8.jpg" /> 图8。内吸性杀虫剂淋终止粉虱接种访问期间除了。 <img alt="图9" src="/files/ftp_upload/4332/4332fig9.jpg" /> 图9所示。场厂正在评估所有的植物抗番茄黄化曲叶病毒 。接种5周龄苗，然后移植到现场进行评估。在前景中的植物是抗性种质线，在后台很容易的植物种质系。

Watch this Scientific Journal Video about Transmitting Plant Viruses Using Whiteflies at JoVE.com

Transmitting Plant Viruses Using Whiteflies

提高我们的理解和我们的能力来管理许多昆虫传播植物病毒需要使用的向量。昆虫传播植物病毒是一种营养层相互作用，因此需要操纵的昆虫病毒和植物。向量必须大量饲养和操作，在这样一种方式，以保证的高速率传输到试验植物。的基本饲养和操纵粉虱，<em&gt; Bemisa粉虱</em&gt;（<em&gt;半翅目：粉虱</em&gt;），属于一些新兴的植物病毒属的植物病毒，较大量的经济意义的病毒载体的方法。

发射使用粉虱的植物病毒

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several ...

transmitting-plant-viruses-using-whiteflies

Research

JoVE Journal

Biology

28.7K 次观看.  University of Florida. 提高我们的理解和我们的能力来管理许多昆虫传播植物病毒需要使用的向量。昆虫传播植物病毒是一种营养层相互作用，因此需要操纵的昆虫病毒和植物。向量必须大量饲养和操作，在这样一种方式，以保证的高速率传输到试验植物。的基本饲养和操纵粉虱， Bemisa粉虱（半翅目：粉虱），属于一些新兴的植物病毒属的植物病毒，较大量?...

视频: Transmitting Plant Viruses Using Whiteflies

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

测试使用微量注射蚜虫病毒传播的生理障碍

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

科研

生物学

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

使用人工饲料实验测试蚜虫的尼古丁公差

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

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

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression 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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

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

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

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 ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

使用转基因钙处理器 Aequorin 前向基因屏幕，以识别钙信号中的新目标

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...

High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

The protocol introduces a high-throughput method for measuring the relaxation of non-photochemical quenching by pulse amplitude modulated chlorophyll fluorometry. The method is applied to field-grown Glycine max and can be adapted to other species to screen for genetic diversity or breeding populations.

使用脉冲幅度调制叶绿素荧光法对作物中的非光化学猝灭进行高通量分析

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

使用原子力显微镜表征活植物器官中原代细胞壁的机械性能

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...