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Growth Assay for the Stem Parasitic Plants of the Genus Cuscuta
菟丝子属茎寄生植物的生长测定

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Abstract

Cuscuta spp. are widespread obligate holoparasitic plants with a broad host spectrum. Rootless Cuscuta penetrates host stems with so called haustoria to form a direct connection to the host vascular tissue (Dawson et al., 1994; Lanini and Kogan, 2005; Kaiser et al., 2015). This connection allows a steady uptake of water, assimilates and essential nutrients from the host plant and therefore enables Cuscuta growth and proliferation. To quantify the parasites’ ability to grow on potential host plants one can use the quantitative growth assay (Hegenauer et al., 2016) described herein, which exclusively utilizes fresh weight measurement as readout.

Keywords: Cuscuta reflexa(大花菟丝子), Dodder(菟丝子), Growth assay(生长测定), Haustoria(吸器), Holoparasitic plant(全寄生植物)

Background

In research fields of plant-pathogen resistance, either in basic research or in economic plant breeding, it is unavoidable to have an assay to quantify resistance against pathogen infection. To quantify the resistance/susceptibility of different plants against Cuscuta infections the simplest way is to measure the gain of biomass of Cuscuta growing on a plant of interest. This is a reliable method since Cuscuta is a holoparasite and its gain of biomass is completely depending on its ability to successfully infect another plant. Thus, unsuccessful infection of a plant leads to a decrease in biomass and subsequently the death of the parasite Cuscuta.

Materials and Reagents

  1. Gloves and lab suit (Cuscuta sap causes stains on skin and clothes)
  2. Mature Cuscuta (e.g., Cuscuta reflexa; see Note 1 for cultivation)
  3. Putative host plants

Equipment

  1. Weighing machine/balance (mass range between 0.01-100 g)
  2. Wooden planting rods (bamboo; diameter appropriate to the particular host plants stem diameter), available in gardening shops
  3. Scissor to cut Cuscuta shoots

Procedure

The whole experiment is performed under optimal conditions for the host plant. The optimal host plant conditions are provided by the seed supplier and can’t be generalized.

  1. Cut Cuscuta shoots (length ~15 cm) including the shoot tip from a mature plant and wind it around upright wooden sticks; be aware of winding direction (counter clockwise from bottom to top; Figure 1A) and integrity of the tip (see also Note 2). Keep the stick with the Cuscuta shoot in a vertical position for one day (e.g., by sticking it into Styrofoam, soil or sand). The Cuscuta shoot should hold by itself on the planting rod without contact to anything else (Figure 1A).


    Figure 1. Starting the Cuscuta infection. A. Cuscuta reflexa shoot enwinding a wooden stick. Winding direction is counter clockwise from bottom to top. Red frame indicates the area where haustoria will most likely form. B. Cuscuta reflexa shoot wound around N. benthamiana plant after preconditioning around the wooden stick (A).

  2. After one day (at the beginning of prehaustoria formation), when the shoot has been carefully uncoiled from the stick, weigh the shoots and transfer them to their host plants considering the winding direction (see also Note 3). The haustoria will form close to the shoot tip (Figure 1, red box) so make sure to wind this part around the host plant stems thoroughly. Remaining of the Cuscuta shoot will provide a source of nutrients and water until the haustoria connect with the host’s vascular system. Therefore, an equal length (e.g., 15 ± 1 cm) as well as an approximately same weight (e.g., 0.5-0.7 g) of the parasite shoots is relevant for reproducible results.
  3. After transfer (Figure 1B, see also Note 4), let the Cuscuta spp. shoots grow for the same time period (14-21 days). After that time of growth, remove individual shoots from the host plant and measure the fresh weight immediately for each individual.

Data analysis

Cuscuta’s ability and speed to accumulate biomass depends on the number of haustoria to acquire nutrients, and thus, on the time frame a single shoot needs to establish a successful haustorial connection to the host’s vascular tissue. For this reason, the variance of the Cuscuta growth can be occasionally high. A big number (> 10) of replicates and multiple repeats are recommended. The outcome of this experiment is the gain of biomass (in g) for every single Cuscuta shoot for the distinct timeframe. So resistance against Cuscuta (or susceptibility for the infection) can be quantified and compared by its ability to gain biomass on two distinct sets of host plants. A correction factor is not necessary if there is no great variance in the initial weight, never the less if this is the case it could be helpful to show the result as mass change (ΔFw = final shoot weight - initial shoot weight) during the time frame. This depends on the particular experiments experimenters will perform to answer their research questions.
The results can be presented as the mean of the Cuscuta shoot biomass 21 dpi (days post infection) of n replicates with standard deviation comparing two types of hostplants.
For the reduction of outlier effects, ranked data analysis and nonparametric tests like Mann-Whitney U-test can be used.
See also Note 5.

Notes

  1. Cultivation of Cuscuta: Cuscuta can be cultivated on many plants. In our Lab we use Coleus (Solenostemon scutellarioides) because it’s a robust plant and its red color gives a good contrast to spot Cuscuta shoots. You can easily cultivate Cuscuta when you place 6 to 10 host plants in close proximity so that Cuscuta can overgrow all of them. It’s important to concern that after Cuscuta overgrows the host plant it cannot be potted anymore, so make sure to provide enough nutrients for the host plant. Cuscuta should be cultivated under long day conditions with 17 h daylight. Cuscuta seeds and instructions how to start the culture can be requested in University botanical gardens or other research groups.
  2. The pre winding of Cuscuta around a wooden planting rod facilitates the later infection of the host plant due to an efficient prehaustoria formation, a clearly visible swelling of the shoot at the shoot-stick contact sites.
  3. It is important not to harm the Cuscuta shoot during the initial weight measurement and transfer to the host plant.
  4. In the first days of infection the Cuscuta shoot may search in the surrounding area of the host plant for other hosts despite infecting the plant it is sitting on. Therefore, the experimenter has to softly redirect the shoot back to its host.
  5. Blinded set up of the experiment: Comparison of e.g., N. benthamiana wild type plants with resistant transgenic CuRe1-expressing plants was performed as blind experiment in Hegenauer et al. (2016). One person randomly assigned numbers to each tested plant and a second person performed the infection with Cuscuta reflexa shoots.
  6. A successful infection of Coleus by Cuscuta reflexa is shown in Figure 2. At the side of haustorial infection Cuscuta is thickened (red frame). After successful infection Cuscuta starts growing and branching (blue arrow).


    Figure 2. Coleus successfully infected by Cuscuta reflexa. Red frame: sides of haustorial development. Blue arrow: branching of Cuscuta

Acknowledgments

The work was funded by DFG-grant (AL1426/1-2).

References

  1. Dawson, J. H., Musselman, L. J., Wolswinkel, J. P. and Dörr, I. (1994). Biology and control of Cuscuta. Weed Sci 6: 265-317.
  2. Hegenauer, V., Furst, U., Kaiser, B., Smoker, M., Zipfel, C., Felix, G., Stahl, M. and Albert, M. (2016). Detection of the plant parasite Cuscuta reflexa by a tomato cell surface receptor. Science 353(6298): 478-481.
  3. Kaiser, B., Vogg, G., Furst, U. B. and Albert, M. (2015). Parasitic plants of the genus Cuscuta and their interaction with susceptible and resistant host plants. Front Plant Sci 6: 45.
  4. Lanini, W. T. and Kogan, M. (2005). Biology and management of Cuscuta in crops. Cienc Investig Agrar 32: 165-179.

简介

镰刀菌属 spp。是具有广泛宿主谱的普遍的专性花岗质植物。无根C uta uta uta> with rates rates rates rates oria oria oria oria em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em em >等,,2015)。这种连接使得能够稳定地摄取来自宿主植物的水,同化物和必需的营养物质,从而使得猕猴桃生长和增殖。为了量化寄生虫在潜在宿主植物上生长的能力,可以使用本文所述的定量生长测定法(Hegenauer等,2016),其仅将新鲜体重测量用作读出。

在植物病原体抗性的研究领域,无论是在基础研究还是在经济植物育种中,都不可避免地有一种能够定量抵抗病原体感染的药物。为了量化不同植物抵抗镰刀菌感染的抗性/易感性,最简单的方法是测量在感兴趣的植物上生长的镰刀菌生物量的增益。这是一种可靠的方法,因为Cuscuta 是一种holoparasite,其生物量的增加完全取决于其成功感染另一种植物的能力。因此,植物的不成功的感染导致生物量的降低,并且随后导致蛔虫寄生虫的死亡。

关键字:大花菟丝子, 菟丝子, 生长测定, 吸器, 全寄生植物

材料和试剂

  1. 手套和实验室套装(C uta uta uta sap sap sap sap on on on on on on)))))
  2. 成熟的Cuscuta (例如,Cuscuta reflexa ;参见注1)培养)
  3. 推定寄主植物

设备

  1. 称重机/天平(质量范围在0.01-100 g之间)
  2. 木制种植棒(竹子;直径适合特定寄主植物茎直径),可在园艺店铺购买
  3. 裁剪剪刀C uta ots ots ots ots

程序

整个实验在宿主植物的最佳条件下进行。最佳宿主植物条件由种子供应商提供,不能推广。

  1. 将Cuscuta 切开(长度〜15厘米),包括成熟植物的枝梢,并将其缠绕在直立的木棍上;请注意卷绕方向(从底部到顶部逆时针旋转;图1A)和尖端的完整性(另见注2)。将Çusc uta the the the the shoot shoot shoot shoot shoot for for for for for for for for by by))))))))))))。)。。。。。。。。。。。。。 C uta> shoot shoot shoot shoot by>>>>>> rod>>>。。。。。。。。。。。。。。。。。。。。。。。

    图1.启动咳嗽 感染 A.咳嗽反射射击木棍。卷绕方向从下到上是逆时针方向。红框表示最有可能形成的区域。 C.镰刀菌反射在N周围伤口。本木本植物在木条(A)附近预处理后。

  2. 一天之后(在排气开始形成时),当从棒上小心地放出拍摄时,称重拍摄并将其转移到主机上,考虑到卷绕方向(另见注3)。饵料会形成接近枝梢(图1,红色盒子),因此请确保将本部分彻底卷绕在宿主植物茎上。 C uta uta uta shoot shoot shoot shoot shoot。。。。。。。。。。。。。。。。。。。。。。。。。。。。因此,相同的长度(例如,15±1cm)以及大约相同的重量(例如,0.5-0.7g)的寄生虫芽是相关的可重复的结果。
  3. 转移后(图1B,另见注4),让我们的Cuscuta spp。芽生长在同一时期(14-21天)。在生长之后,从宿主植物中除去单个芽,并立即测量每个个体的鲜重。

数据分析

Cuscuta 的能力和积累生物量的速度取决于获取营养物质的数量,因此,在一段时间内,单次拍摄需要建立与宿主血管组织的成功连接。因此,"蔡司"增长的差异偶尔会很高。建议使用大量(> 10)重复和重复。该实验的结果是对于每个单独的Cuscuta 拍摄在不同时间段的生物量(g)的增益。因此,可以通过其在两个不同组的宿主植物上获得生物量的能力来量化并对其进行比较以抵抗镰孢霉素(或感染的易感性)。如果初始重量没有很大变化,则不需要校正因子,如果在时间内显示结果作为质量变化(ΔFw=最终枝条重量 - 初始枝条重量)可能有帮助的情况,则更少)帧。这取决于实验者将会执行的具体实验来回答他们的研究问题 结果可以以两种类型的宿主植物的标准偏差作为n重复的猕猴桃苗生物量21dpi(感染后天数)的平均值来表示。
为了减少异常值效应,可以使用诸如Mann-Whitney -test之类的分级数据分析和非参数测试。
另见注5。

笔记

  1. 可以在许多植物上栽培猕猴桃: Cuscuta 的种植。在我们的实验室里,我们使用了Coleus(Solenostemon scutellarioides),因为它是一个强壮的植物,它的红色与斑点Cuscuta 射击形成良好的对比。当您靠近6到10个寄主植物时,您可以轻松培养"蔡司"(Cuscuta),以便茧丝虫可以将其全部覆盖。重要的是要考虑到,在Cuscuta 过度繁殖宿主植物后,不能再盆栽,因此请确保为宿主植物提供足够的营养。镰刀菌应在日照条件下培育17小时。可以在大学植物园或其他研究小组中要求蔡司的种子和说明如何开始培养。
  2. 围绕木制种植棒预先蜿蜒的Cuscuta有利于宿主植物的后期感染,这是由于有效的排尿前体形成,在枝条接触部位的枝条清晰可见的肿胀。 >
  3. 在初始体重测量和转移到主机厂期间,不要伤害蔡斯库射击是很重要的。
  4. 在感染的第一天,尽管感染了它所在的植物,但是Cuscuta 拍摄可以在宿主植物的周围区域搜索其他宿主。因此,实验者必须轻轻地将拍摄重定向到其主机。
  5. 实验的盲目设置:比较例如, N。在Hegenauer等人的盲目实验中进行了具有抗性转基因CuRe1表达植物的本地植物野生型植物。 (2016)。一个人随机分配数字给每个被测试的植物,第二个人使用镰刀菌反射进行感染。
  6. 通过咳嗽反射成功感染Coleus如图2所示。在抽搐感染的一侧,Cuscuta 增厚(红框)。感染成功后,Cuscuta 开始生长和分支(蓝色箭头)。


    图2.由蔡斯反射成功感染的科莱斯 。红框:发育的两侧。蓝色箭头:蔡司的分支。 

致谢

这项工作由DFG-grant(AL1426/1-2)资助。

参考文献

  1. Dawson,JH,Musselman,LJ,Wolswinkel,JP和Dörr,I。(1994)。< a class ="ke-insertfile"href ="http://www.cabi.org/isc/abstract/19942309905"目标="_ blank">蔡司的生物和控制 Weed Sci 6:265-317。
  2. Hegenauer,V.,Furst,U.,Kaiser,B.,Smoker,M.,Zipfel,C.,Felix,G.,Stahl,M。和Albert,M。(2016)。通过番茄细胞表面检测植物寄生虫镰孢反射受体。 科学 353(6298):478-481。
  3. Kaiser,B.,Vogg,G.,Furst,UB和Albert,M。(2015)。  Cuscuta属的寄生植物及其与敏感和抗性宿主植物的相互作用。前植物科学 6:45 。
  4. Lanini,WT和Kogan,M.(2005)。生物与管理在Cuscuta 中的作物。 Cienc Investig Agrar 32:165-179。
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引用:Hegenauer, V., Welz, M., Körner, M. and Albert, M. (2017). Growth Assay for the Stem Parasitic Plants of the Genus Cuscuta. Bio-protocol 7(8): e2243. DOI: 10.21769/BioProtoc.2243.
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