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EdU Based DNA Synthesis and Cell Proliferation Assay in Maize Infected by the Smut Fungus Ustilago maydis
在玉蜀黎黑粉菌感染玉米中进行基于EdU的DNA合成和细胞增殖实验   

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Abstract

The basidiomycetous smut fungus Ustilago maydis (U. maydis) infects all aerial parts of its host plant maize (Zea mays L.). Infection is seen in the form of prominent tumorous symptoms after the establishment of a biotrophic interaction with the host, usually around 5-6 days after infection. The fungus colonizes the various developmentally distinct aerial organs at different stages of development. Formation of tumors is coupled with the induction of host cell division. Activation of cell division can be understood as a measure of DNA synthesis which is triggered to induce rapid divisions in host cell. This developed protocol helps in tracking tumor induction in U. maydis by monitoring of DNA synthesis in planta. Infected leaves were treated with 5-ethynyl-2-deoxyuridine (EdU) at several stages of infection in the seedling leaves and labeled. EdU incorporation in the S phase cells, was visualized by attaching a fluorescent tag and non-dividing maize nuclei were stained with propidium iodide (PI). This protocol helped to understand the tumor development in U. maydis by confocal laser scanning microscopy (Kelliher and Walbot, 2011; Redkar et al., 2015)

Keywords: DNA Synthesis(DNA合成), EdU(edu), Quantification(量化), Maize(玉米), Ustilago maydis(玉米黑粉菌)

Materials and Reagents

  1. 1 ml Syringes (Henke Sass-Wolff, catalog number: 5100.200V0 )
  2. Needles (16 G, 40 mm) (Premier Healthcare & Hygiene, BD microlance, catalog number: 300637 )
    Note: A needle of different thickness can be used depending upon the maize variety.
  3. Falcon tubes (15 ml)
  4. Microcentrifuge tubes
  5. Click iT EdU imaging kit (Life Technologies, catalog number: 10337 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: 10337”.
    1. 5-ethynyl- 2’-deoxyuridine (EdU)
    2. Wheat germ Agglutinin-Alexa Fluor 488 (WGA AF 488)
    3. Dimethylsulfoxide (DMSO)
    4. Click iT EdU reaction buffer
    5. Copper sulphate (CuSO4)
    6. Click iT EdU buffer additive
    7. Hoechst 33342
  6. Phosphate buffer saline (PBS, pH 7.4) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  7. Permeabilization reagent [e.g. 0.5% Triton X-100 in PBS (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: R37602 )]
  8. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A8531 )
  9. 100% Ethanol
  10. Propidium iodide (PI) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P1304MP )
    Note: PI is toxic and may cause skin and respiratory irritation. Hence it is good to handle PI in the fume hood with the personal safety protection.
  11. Double distilled water
  12. 2% BSA in PBS (pH 7.4) (See Recipes)
  13. Propidium iodide stock solution (see Recipes)

Equipment

  1. EdU Labeling chambers
    Note: These plastic chambers are custom made to fit in the desired sample). An image showing the details of this chamber is shown below (Figure 1B). As an alternative to the labeling chambers 50 ml Falcon Tubes can also be used to labeling the leaves by dipping the infected leaf in 10 µM EdU.
  2. Confocal microscope (e.g. Leica Microsystems, model: TCS-SP5 )
  3. Vacuum Pump
  4. Rocker

Software

  1. Leica Imaging Software (Leica)

Procedure

  1. Preparation of stock solutions
    1. Allow all the vials to warm to room temperature.
    2. Add 2 ml DMSO or an aqueous solution to EdU (Molar mass 252.23 g mol-1) to make a 10 mM EdU stock solution. This stock is generally stable for one year when stored at -20 °C.
    3. Prepare 10x Click-iT EdU reaction buffer by adding 3.6 ml of deionized water to Click iT EdU reaction buffer. Stored at 2-8 °C.
    4. Prepare Alexa Fluor azide solution by adding 70 μl DMSO to Alexa Fluor 488 Azide. Mix and store well at -20 °C.
    5. Prepare 10x Click-iT EdU buffer additive by adding 2 ml deionized water to Click iT EdU buffer additive and mix. Stored at -20 °C.

  2. Labeling cells with EdU and fixation
    Note: Ustilago maydis infections are done in the desired maize organ as described previously (Redkar and Doehlemann, 2016).

    For seedling samples
    1. The U. maydis seedling or tassel infections are carried out by the syringe inoculation method by injecting the inoculum into the desired maize organ (Redkar and Doehlemann, 2016).
    2. The EdU labeling is done at the time-point for which DNA synthesis is to be measured. In case of a U. maydis infected seedling the 4 days post infection (dpi) time-point was chosen. In the U. maydis infected seedling the third infected leaf where the first infection symptoms appear is used for the EdU labeling assay.
    3. This leaf is incubated for 5 h with 10 µM EdU in small chambers (details of which have been previously mentioned) designed for labeling physiologically active leaves, taking care that the 2 cm area below the infection mark which is actively colonized is well immersed (Figure 1A). The labeling is done in the temperature controlled greenhouse to ensure the active imbibition of EdU into the leaf cells.


      Figure 1. EdU labelling to detect DNA synthesis in U. maydis infected maize seedlings. A. Labeling of EdU in a maize seedling to detect S phase cells in infected leaves. B. An image of the EdU labeling chamber with the lid (Left), The lid of the chamber removed for insertion of the seedling sample (Right). C. Cell division events observed in a maize seedling infected by U. maydis wild-type strain SG200 at 4 dpi. (Left) EdU incorporation into a cell will result in equally labeled contiguous daughter cells after cell division as indicated by yellow arrows. (Right) The mitotic events are nicely preserved by this EdU method of labeling (scale bars = 25 µm).

    4. After 5 h of labeling, the 2 cm area below the syringe mark on the leaves is detached and fixed in 100% (v/v) ethanol. The material is collected in 15 ml Falcon tubes. This area of the maize leaf shows maximum colonization with U. maydis as it shows chlorosis to small tumorous symptoms at 4dpi.

    For tassel samples
    1. U. maydis tassel infections are performed by a syringe inoculation method by delivering 1 ml of sporidial inoculum as previously described (Walbot and Skibbe, 2010).
    2. For the tassel tissues, the EdU labeling assay is done at 3 dpi, to capture the mitotically active anthers before they switch to meiosis.
    3. At 3 dpi the immature infected tassel (anther size approx. 1-1.5 mm; Figure 2) is bathed with 1 ml of 20 µM EdU after delivery.


      Figure 2. Immature maize tassel at time point of EdU treatment

    4. The injected EdU enters through the small air spaces between the external organs of the spikelets and reaches anthers over the labeling time of 5 h.
    5. After the labeling procedure, the tassel is dissected out and around 150 anthers from different parts of the tassel were collected to ensure random sampling with equal probability of labeled anthers and fixed in 100% (v/v) ethanol 5 h after labeling.

  3. Permeabilization and staining of labeled EdU
    1. The EdU staining procedure is done as described by implementation of a modified protocol (Kelliher and Walbot, 2011). For seedling samples, approx. 1 cm pieces of the collected tissues which are fixed in ethanol are used. For tassel samples, around 100 individual anthers from different parts of the tassel are used. The labeling procedure is done in 2 ml microcentrifuge tubes for seedling and anther samples.
    2. The fixed samples in ethanol are washed once with fresh 100% (v/v) ethanol followed by two washes in PBS (pH 7.4) + 2% (w/v) BSA. More washes with the PBS buffer are helpful for complete removal of chlorophyll from the seedling samples.
    3. The samples are then transferred to permeabilization solution [PBS +1% (v/v) Triton X-100] at room temperature for 20 min with rocking for 50-60 rotations per minute (rpm)/min. This step can also be done on a regular shaker with the same rpm. Triton treatment results in active nuclear staining and is compatible with EdU co-staining. It is known to prevent cell shrinkage and quick penetration of the fixative to allow better preservation of mitotic stages (Kotogany et al., 2010). The image representation of a cell stained with EdU and showing the well preserved mitotic event is shown in Figure 1B.
    4. The samples are washed twice in PBS + 2% (w/v) BSA then directly incubated for 30 min at room temperature with EdU Click-IT cocktail for detection. E.g., for approx. 2.5 ml EdU cocktail the following components were added (Click iT EdU reaction buffer - 2.2 ml; CuSO4 -100 µl; Alexa Fluor azide - 6 µl; Reaction buffer additive - 250 µl). For the seedling samples, 500 µl of this cocktail solution was used in each transparent microcentrifuge tube where as for anthers this corresponded to 100 µl.
    5. The EdU reaction cocktail is also supplemented with 20 μg/ml Propidium iodide (PI). This is mainly to co-stain the fungal hyphae and the non-dividing host cell nuclei.
    6. This EdU reaction cocktail is then incubated at room temperature for 50 min and then vacuum infiltrated at 250 millibar 3 times for 5 min each along with a regular interval of 5 min with atmospheric pressure.
    7. After the infiltration, the samples are washed twice in PBS (pH 7.4) + 2% (w/v) BSA, transferred to new PBS (pH 7.4), and can be kept at 4 °C in the dark (up to several days) before imaging.

  4. Microscopy of the EdU labeled leaves and anthers
    All confocal analysis may be performed on a TCS SP5 confocal laser scanning microscope, or a comparable device. The laser channels used for confocal fluorescence analysis of excitation and detection wavelengths are summarized in Table 1.

    Tabel 1. Lasers used and the excitation and detection wavelength
    Detection
    Excitation wavelength
    Detection wavelength
    Pinhole used
    EdU coupled to WGA AF488
    488 nm
    490-540 nm
     ~ 90 µm
    Propidium iodide
    561 nm
    570-640 nm
     ~ 90 µm

    All the images taken by the Leica TCS SP5 Confocal microscope were processed and analyzed by the Leica Imaging Software.

Recipes

  1. Bovine serum albumin (BSA) (2%) in PBS (pH 7.4)
    Add 20 g of BSA to 1 L of PBS buffer (pH 7.4)
  2. Propidium iodide stock solution
    10 mg/ml in PBS (storage in the dark at -20 °C). This stock solution is directly added to the EdU reaction cocktail to include the co-stain in working concentration.

Acknowledgments

Our work was funded by the Max Planck Society, the Deutsche Forschungsgemeinschaft (DFG), the Deutscher Akademischer Austauschdienst (DAAD) and the Cluster of Excellence on Plant Science (CEPLAS). The protocol is adapted from Kelliher and Walbot (2011) and Redkar et al. (2015).

References

  1. Kelliher, T. and Walbot, V. (2011). Emergence and patterning of the five cell types of the Zea mays anther locule. Dev Biol 350(1): 32-49.
  2. Kotogany, E., Dudits, D., Horvath, G. V. and Ayaydin, F. (2010). A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine. Plant Methods 6(1): 5.
  3. Redkar, A. and Doehlemann, G. (2016). Ustilago maydis virulence assays in maize. Bio-protocol 6(6): e1760.
  4. Redkar, A., Hoser, R., Schilling, L., Zechmann, B., Krzymowska, M., Walbot, V. and Doehlemann, G. (2015). A secreted effector protein of Ustilago maydis guides maize leaf cells to form tumors. Plant Cell 27(4): 1332-1351.
  5. Walbot, V. and Skibbe, D. S. (2010). Maize host requirements for Ustilago maydis tumor induction. Sex Plant Reprod 23(1): 1-13.

简介

落叶青霉菌(Basidiomycetous smut fungus)Ustilago maydis( U。maydis )感染其宿主植物玉米的所有地上部分( Zea mays 感染在与宿主生物营养相互作用建立后通常在感染后约5-6天以突出的肿瘤症状的形式见到。真菌在不同发育阶段定殖各种发育不同的气生器官。肿瘤的形成与宿主细胞分裂的诱导相结合。细胞分裂的激活可以理解为DNA合成的量度,其被触发以诱导宿主细胞中的快速分裂。这种开发的协议有助于跟踪肿瘤的诱导。 maydis通过监测植物中的DNA合成来实现。 在幼苗叶中感染的几个阶段用5-乙炔基-2-脱氧尿苷(EdU)处理感染的叶并标记。通过连接荧光标签显现S期细胞中的EdU掺入,并用碘化丙啶(PI)染色未分裂的玉米核。这个协议有助于了解肿瘤的发展。 maydis 通过共聚焦激光扫描显微术(Kelliher和Walbot,2011; Redkar等人,2015)

关键字:DNA合成, edu, 量化, 玉米, 玉米黑粉菌

材料和试剂

  1. 1ml注射器(Henke Sass-Wolff,目录号:5100.200V0)
  2. 针(16G,40mm)(Premier Healthcare& Hygiene,BD microlance,目录号:300637)
    注意:根据玉米品种,可以使用不同厚度的针。
  3. Falcon管(15ml)
  4. 微量离心管
  5. 点击iT EdU成像试剂盒(Life Technologies,目录号:10337)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:10337" br />
    1. 5-ethynyl-2'-deoxyuridine(EdU)
    2. 小麦胚凝集素-Alexa Fluor 488(WGA AF 488)
    3. 二甲基亚砜(DMSO)
    4. 单击iT EdU反应缓冲液
    5. 硫酸铜(CuSO 4)
    6. 点击iT EdU缓冲添加剂
    7. Hoechst 33342
  6. 磷酸盐缓冲盐水(PBS,pH 7.4)(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  7. 渗透试剂[例如在PBS(自制)(Thermo Fisher Scientific,Molecular Probes TM ,目录号:R37602)中的0.5%Triton X-100]
  8. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A8531)
  9. 100%乙醇
  10. 碘化丙锭(PI)(Thermo Fisher Scientific,Molecular Probes ,目录号:P1304MP)
    注意:PI有毒,可能引起皮肤和呼吸道刺激。因此,在人身安全保护的情况下处理PI是很好的。
  11. 双蒸水
  12. 2%BSA的PBS(pH7.4)(参见配方)
  13. 碘化丙啶储备溶液(见配方)

设备

  1. EdU标签室
    注意:这些塑料室是定制的,以适合所需的样品)。显示该室的细节的图像如下所示(图1B)。作为标记室的替代方案,也可以使用50ml Falcon Tubes通过将受感染的叶浸入10μMEdU中来标记叶子。
  2. 共焦显微镜(例如,徕卡显微系统公司,型号:TCS-SP5)
  3. 真空泵
  4. 摇杆

软件

  1. Leica成像软件(Leica)

程序

  1. 储备溶液的制备
    1. 允许所有小瓶温热至室温。
    2. 加入2ml DMSO或 ?水溶液至EdU(摩尔质量252.23g.mol ),以制备10mM EdU储备溶液。该股一般稳定一年 储存于-20℃
    3. 准备10x Click-iT EdU反应缓冲液 加入3.6ml去离子水至iT EdU反应缓冲液。存储 ?在2-8℃
    4. 通过加入70μlDMSO至Alexa Fluor 488 Azide制备Alexa Fluor叠氮化物溶液。在-20°C下混合储存。
    5. 通过加入2ml去离子水制备10x Click-iT EdU缓冲液添加剂 水点击iT EdU缓冲液添加剂混匀。储存于-20°C。

  2. 用EdU和固定标记细胞
    注意:如前所述,Ustilago maydis感染在所需的玉米器官中进行(Redkar和Doehlemann,2016)。

    对苗化样品
    1. U。 maydis 幼苗或雄穗感染 注射器接种方法通过注射接种物到所需的 玉米器官(Redkar和Doehlemann,2016)
    2. EdU标签是 在要测量DNA合成的时间点进行。 例如 。 maydis 感染幼苗感染后4天(??dpi) ?选择时间点。在中。 maydis 感染幼苗第三 第一感染症状出现的感染叶用于 EdU标记测定。
    3. 将该叶与10μM温育5小时 EdU在小室(细节已经在前面提到) 设计用于标记生理活性叶子,小心 在积极定殖的感染标记以下的2cm区域是 (图1A)。标记在温度下进行 控制温室,以确保EdU的主动吸入 叶细胞

      图1。 A.在玉米幼苗中标记EdU 检测感染叶中的S期细胞。 B. EdU标记的图像 ?带盖的室(左),室的盖除去 插入幼苗样品(右)。 C.细胞分裂事件 在被U感染的玉米幼苗中观察到。 maydis 野生型菌株 SG200在4 dpi。 (左)EdU掺入细胞将导致 在细胞分裂后同样标记的连续的子细胞 由黄色箭头表示。 (右)有丝分裂事件是很好的 通过这种EdU标记法(比例尺=25μm)保存
    4. 标记5小时后,注射器标记下面的2厘米区域 叶分离并在100%(v/v)乙醇中固定。材料是 收集在15ml Falcon管中。这个区域的玉米叶显示 用最大定殖。 maydis ,因为它显示褪绿至小 肿瘤症状4dpi。

    对于流苏样品
    1. U。毛发感染通过注射器接种进行 方法通过递送1ml如前所述的孢子接种物 (Walbot和Skibbe,2010)。
    2. 对于穗状组织,EdU 标记测定在3dpi下进行,以捕获有丝分裂活性 花药在它们转向减数分裂之前。
    3. 在3 dpi的未成熟 感染的雄穗(花药大小约1-1.5mm;图2)用1 ?ml的交付后20μMEdU

      图2. EdU处理时间点的未成熟玉米穗状花序

    4. 注入的EdU通过之间的小空气空间进入 小穗的外部器官并在标记上到达花药 时间为5 h。
    5. 在标记程序后,流苏是 解剖出和约150个花药从流苏的不同部分 以确保随机抽样的概率相等 标记的花药,并在标记后5小时在100%(v/v)乙醇中固定。

  3. 标记的EdU的透化和染色
    1. EdU染色程序如实施a所述进行 (Kelliher和Walbot,2011)。对于幼苗样品, 约。将1cm收集的组织固定在乙醇中 使用。对于雄穗样品,约100个个体花药 使用流苏的不同部分。完成标记过程 在2ml微量离心管中用于苗和花药样品
    2. 将固定的样品在乙醇中用新鲜的100%(v/v) 乙醇,然后在PBS(pH 7.4)+ 2%(w/v)BSA中洗涤两次。更多 用PBS缓冲液洗涤有助于完全去除 来自幼苗样品的叶绿素
    3. 然后是样品 转移至透化溶液[PBS + 1%(v/v)Triton X-100] ?室温20分钟,摇动50-60转/分钟 ?(rpm)/min。该步骤也可以在具有该步骤的常规摇床上进行 rpm。 Triton处理导致活性核染色 与EdU共染色相容。已知防止细胞收缩 和快速渗透固定剂以允许更好的保存 有丝分裂阶段(Kotogany等人,2010)。 a的图像表示 细胞用EdU染色并显示良好保存的有丝分裂事件 如图1B所示。
    4. 样品在PBS + 2% (w/v)BSA,然后在室温下直接温育30分钟 用于检测的EdU Click-IT鸡尾酒。 2.5ml EdU 鸡尾酒中加入以下组分(点击iT EdU反应 缓冲液 - 2.2ml; CuSO 4-100μl; Alexa氟化叠氮化物 - 6μl;反应 缓冲添加剂 - 250μl)。对于幼苗样品500μl 混合溶液用于每个透明微量离心管中 其中花药这对应于100μl
    5. 爱德华 反应混合物还补充有20μg/ml碘化丙啶 (PI)。这主要是对真菌菌丝和不分裂的染色 宿主细胞核。
    6. 然后孵育该EdU反应混合物 在室温下50分钟,然后在250℃下真空渗透 毫巴3次,每次5分钟,定期间隔5分钟 与大气压力
    7. 渗透后,样品 在PBS(pH 7.4)+ 2%(w/v)BSA中洗涤两次,转移到新的PBS中 (pH 7.4),并且可以在4℃下在黑暗中保存(直到几天) 成像前。

  4. 显示EdU标记的叶和花药
    所有共聚焦分析可在TCS SP5共聚焦激光扫描显微镜或类似装置上进行。用于激发和检测波长的共焦荧光分析的激光通道总结在表1中
    Tabel 1.激光使用及激发和检测波长
    检测
    激发波长为
    检测波纹
    针孔使用
    EdU耦合到WGA AF488
    488 nm
    490-540nm
     ?90μm
    碘化丙啶
    561 nm
    570-640 nm
     ?90μm

    由Leica TCS SP5共聚焦显微镜拍摄的所有图像由Leica Imaging软件处理和分析。

食谱

  1. BSA(pH 7.4)中的牛血清白蛋白(BSA)(2%) 将20g BSA加入1L PBS缓冲液(pH 7.4)中
  2. 碘化丙啶储液
    10mg/ml在PBS中(在-20℃的黑暗中储存)。将该储备溶液直接加入到EdU反应混合物中,包括工作浓度的共染色。

致谢

我们的工作由马克斯普朗克学会,德意志交易所(DFG),德意志研究院(DAAD)和植物科学卓越集群(CEPLAS)资助。该协议改编自Kelliher和Walbot(2011)和Redkar等人(2015)。

参考文献

  1. Kelliher,T.和Walbot,V。(2011)。 五种细胞类型的玉蜀黍花药芽的出现和形成。 Dev Biol 350(1):32-49。
  2. Kotogany,E.,Dudits,D.,Horvath,G.V。和Ayaydin,F。(2010)。 用于检测植物细胞和组织中S期细胞周期进展的快速而稳定的测定法,使用乙炔基脱氧尿苷。植物方法 6(1):5.
  3. Redkar,A.和Doehlemann,G.(2015)。 Ustilago maydis 在玉米中的毒力测定。 /em> 6(6):e1760。
  4. Redkar,A.,Hoser,R.,Schilling,L.,Zechmann,B.,Krzymowska,M.,Walbot,V。和Doehlemann,G。 Ustilago maydis的分泌效应蛋白指导玉米叶细胞形成肿瘤。植物细胞 27(4):1332-1351。
  5. Walbot,V.和Skibbe,D.S.(2010)。 玉米宿主对 Ustilago maydis 肿瘤诱导的要求。 < em> Sex Plant Reprod 23(1):1-13。
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引用:Redkar, A. and Doehlemann, G. (2016). EdU Based DNA Synthesis and Cell Proliferation Assay in Maize Infected by the Smut Fungus Ustilago maydis. Bio-protocol 6(6): e1761. DOI: 10.21769/BioProtoc.1761.
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