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Measuring Secretion of Capsidiol in Leaf Tissues of Nicotiana benthamiana

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The Plant Cell
May 2016



Plant species produce a wide variety of antimicrobial metabolites to protect themselves against potential pathogens in natural environments. Phytoalexins are low molecular weight compounds produced by plants in response to attempted attacks of pathogens. Accumulation of phytoalexins in attacked plant tissues can inhibit the growth of penetrating pathogens. Thus phytoalexins play a major role in post-invasion defense against pathogens. Major phytoalexins produced by Solanaceous plants are sesquiterpenoids such as capsidiol produced by Nicotiana and Capsicum species, and rishitin produced by Solanum species, which are synthesized in the cytosol and secreted into the intercellular space of plant tissues. We previously reported that deficiency in capsidiol secretion causes enhanced susceptibility of Nicotiana benthamiana to potato late blight pathogen, Phytophthora infestans. Here, we describe a practical protocol to measure the secreted capsidiol in N. benthamiana.

Keywords: Capsidiol (辣椒醇), Nicotiana benthamiana (本氏烟), Phytoalexins (植物抗毒素), Secretion (分泌), Transporter (转运蛋白)


This protocol provides a quick and simple method to quantify the secretion of capsidiol as performed in Shibata et al. (2016). Because cyclohexane is commonly used as the solvent to wash off pollen coat layers but keeps pollen viable (Doughty et al., 1993), we developed this method to wash off secreted metabolites from the leaf surface to quantify extracellular capsidiol. The function of plasma-membrane localized transporters can be analyzed using this method, while also allowing it to be used in conjunction with other methods, such as biochemical transport analysis using plasma membrane vesicles (e.g., Sugiyama et al., 2007), in order to determine the substrate of the examined transporter.

Materials and Reagents

  1. Pipette tips (e.g., PIPETMAN Diamond Tips, Gilson)
  2. Needleless syringe (e.g., TerumoTM Tuberculin Syringes 1 ml, Terumo Medical, catalog number: SS-01T )
  3. 50 ml tubes (e.g., 50 ml Falcon centrifuge tube)
  4. 200 ml round-bottom flask
  5. 2 ml tubes (e.g., microcentrifuge tube, 2 ml with lid, BRAND, catalog number: 780550 )
  6. Leaves of 4-5 weeks old wild-type or gene-silenced Nicotiana benthamiana
    1. For Virus-induced gene silencing (VIGS) of N. benthamiana, see Zhang and Liu, (2014).
    2. Similar method can be applied for other plant species (e.g., see Khare et al., 2017).
  7. INF1 elicitor produced by P. infestans or in E. coli
    1. For the method of INF1 preparation, see Takemoto et al. (2005) or Shibata et al. (2010).
    2. Other elicitors may be used as well. 
  8. Capsidiol
    Note: Capsidiol is not commercially available. For the method of capsidiol purification, see Matsukawa et al. (2013).
  9. 99.5% Cyclohexane (Wako Pure Chemical Industries, catalog number: 034-05006 )
  10. 99.5% Cyclohexane/Ethyl acetate (1:1, v/v) (Ethyl acetate, Wako Pure Chemical Industries, catalog number: 051-00351 )
  11. 99.5% acetonitrile (Wako Pure Chemical Industries, catalog number: 019-08631 )
  12. Liquid nitrogen
  13. Methanol (Wako Pure Chemical Industries, catalog number: 131-01826 )


  1. Pipettes (e.g., PIPETMAN P, Gilson)
  2. Electronic scale (e.g., Mettler-Toledo International, model: AG245 Analytical balance)
  3. Freezer
  4. Rotary evaporator with water bath (e.g., Yamato Scientific, model: RE-301-BW )
  5. Centrifugal concentrator (e.g., TOMY DIGITAL BIOLOGY, model: CC-105 with Vacuum Pump)
  6. Microtube mixer (e.g., TAITEC, model: E-36 )
  7. Sonicator Bath (e.g., SND, model: US-101 )
  8. Mortar and pestle
  9. Microcentrifuge (e.g., KUBOTA, model: 3740 )
  10. High-performance liquid chromatography (HPLC) system (e.g., Prominence Modular HPLC system, Shimadzu, Japan)
  11. ODS column (e.g., Nomura Chemical, model: Develosil ODS-UG-3 )


  1. Treatment of elicitor and preparation of extracellular metabolites of N. benthamiana leaves
    1. Treat leaves of Nicotiana benthamiana with 150 nM INF1 elicitor solution by injecting the solution into the intracellular spaces of the abaxial sides of the leaves using a needleless syringe (Figure 1A). Apply gentle pressure during infiltration, in order to avoid damaging the leaf tissue. Inject the solution at 5-10 locations to ensure sufficient coverage of the leaf. Incubate for 9 h at 23 °C under light.
    2. Cut off treated leaves from the plant (Figure 1B) and measure the weight of each leaf using an electronic scale (generally, approx. 1 g for leaf of middle-sized).
      Note: Process Steps A2 to A4 one leaf at a time.
    3. Put each leaf into a 50 ml tube (Figure 1C). Avoid rupture of the central vein.
    4. Washing step: Add approx. 50 ml cyclohexane into each tube. Shake gently for approx. 30 sec at RT (Figure 1D) and then pour the cyclohexane into a 200 ml round-bottom flask (or a fresh 50 ml tube for storage in the freezer) to collect cyclohexane-soluble metabolites on the leaf surface. Freeze and keep the leaf tissue in the same 50 ml tube to extract intracellular (intra-tissue) metabolites later (see steps in Procedure B).

      Figure 1. Treatment of elicitor and preparation of extracellular metabolites of N. benthamiana leaves. A. Treatment of elicitor by injecting the solution into the intracellular spaces of the leaves using a needleless syringe. B. Cut off treated leaves from the plant. C. Put each leaf into a 50 ml tube. D. Add approx. 50 ml cyclohexane and shake gently for approx. 30 sec.
      Note: Time for shaking can be longer (e.g., a couple of minutes) but it is important to fix the time for all samples. Test leakage of chlorophyll into cyclohexane to make sure that there was no major damage to leaf cells by cyclohexane. See Shibata et al., 2016 for details.

    5. Evaporate the cyclohexane obtained in Step A4 using a rotary evaporator (at approx. 40 °C in a water bath).
    6. To collect the metabolites dried at the bottom of the round-bottom flask, wash the bottom twice with 1 ml of cyclohexane/ethyl acetate (1:1, v/v) by pipetting and collect the solution (total 2 ml) into a 2 ml tube.
      Note: Let the flask cool down to room temperature before adding solvent.
    7. Evaporate the solvent in the 2 ml tube using a centrifugal concentrator (This step usually takes about 1 h).
    8. Re-dissolve the precipitate in 100 µl acetonitrile, mix thoroughly and add 50 µl ddH2O.
    9. Shake the 2 ml tubes well using a microtube mixer for 5 min.
    10. Sonicate the samples by floating the tubes in a sonicator bath for 5 min.
    11. Centrifuge at 16,000 x g for 1 min at room temperature and collect the supernatant.
      Note: Carefully collect 120 µl supernatant to avoid contamination with precipitates.
    12. Analyze the metabolite by HPLC. See Procedure C.

  2. Preparation of intracellular (intra-tissue) metabolites of N. benthamiana leaves
    1. Grind leaves of N. benthamiana from Step A4 in liquid nitrogen with a mortar and pestle, and suspend in 50% methanol (1 ml/300 mg leaf tissue).
    2. Centrifuge at 16,000 x g for 1 min at room temperature and collect the supernatant.
    3. Add the same volume of cyclohexane/ethyl acetate (1:1, vol/vol) and vortex.
    4. Centrifuge at 3,000 x g for 5 min at room temperature, and collect the upper phase (ethyl acetate layer).
    5. Evaporate solvent using a centrifugal concentrator. (This step usually takes ~1 h).
    6. Re-dissolve precipitate in 100 µl acetonitrile and add 50 μl ddH2O.
    7. Shake 2 ml well using microtube mixer for 5 min.
    8. Sonicate samples by floating tubes in a sonicator bath for 5 min.
    9. Centrifuge at 16,000 x g for 1 min at room temperature and collect the supernatant.
      Note: Carefully collect 120 µl supernatant to avoid contamination with precipitate.
    10. Analyze the metabolite by HPLC. See Procedure C.

  3. High-performance liquid chromatography (HPLC) analysis
    Samples from Procedure A and B are subjected to HPLC analysis under the following conditions.
    Note: Other conditions may be used though this will influence the elution times of the products.
    Column: ODS column (e.g., Develosil ODS-UG-3, Nomura Chemical, Japan).
    Solvent: 50% acetonitrile (0 to 3 min), linear increase from 50% to 80% acetonitrile (3 to 13 min), and from 80% to 100% acetonitrile (13 to 14 min).
    Injection volume: 5 µl.
    Flow rate: 1.0 ml/min.
    Detection at 205 nm.
    Capsidiol and capsidiol 3-acetate elute at 4.6 and 12.8 min, respectively (see Figure 2). Use peak areas for quantitative analysis.
    Note: For the method of capsidiol 3-acetate purification, see Matsukawa et al. (2013).

    Figure 2. Elution profile of extracellular metabolites of TRV-infected or Nb-ABCG1/2-silenced N. benthamiana. Leaves of TRV-infected (A) or NbABCG1/2-silenced plants (B) were treated with 150 nM INF1. Elution profiles of washing fluids were analyzed by HPLC (detection at 205 nm).

Data analysis

  1. Test purified capsidiol first for HPLC analysis to determine the score of the peak area per amount of capsidiol, and elution time of capsidiol under your experimental condition.
  2. Calculate the ratio of secreted/intracellular capsidiol by comparing the amount of capsidiol detected in samples prepared by Procedure A and B from the same leaf.
  3. To compare the secretion of capsidiol between wild-type and transport deficient gene-silenced N. benthamiana, compare the elution profiles of extracellular metabolites between N. benthamiana lines. See example shown in Figure 1 for gene-silencing of probable capsidiol transporters NbABCG1/2. Peaks of the other metabolites can be used as internal standard to assure consistent extraction (washing) efficiency.


  1. The ratio of secreted/intracellular capsidiol of control N. benthamiana leaves treated with 150 nM INF1 for 9 h is around 1.5-2.5 under our experimental conditions. The ratio will be affected by the activity of the elicitor (lot-to-lot difference), the time point of sampling and the age of the leaf, etc. It is therefore important to fix your experimental condition.
  2. Avoid using elicitors which induce cell death of leaf tissue, since cell death can cause leakage of intracellular metabolites.


We thank Prof. Makoto Ojika (Nagoya University, Japan) for technical advice. The work was supported by a Grant-in-Aid for Scientific Research (B) (26292024 and 17H03771) from the Japan Society for the Promotion of Science. No potential conflicts of interest were disclosed.


  1. Doughty, J., Hedderson, F., McCubbin, A., and Dickinson, H. (1993). Interaction between a coating-borne peptide of the Brassica pollen grain and stigmatic S (self-incompatibility)-locus-specific glycoproteins. Proc Natl Acad Sci U S A 90(2): 467- 471.
  2. Khare, D., Choi, H., Huh, S. U., Bassin, B., Kim, J., Martinoia, E., Sohn, K. H., Paek, K. H. and Lee, Y. (2017). Arabidopsis ABCG34 contributes to defense against necrotrophic pathogens by mediating the secretion of camalexin. Proc Natl Acad Sci U S A 114(28): E5712-E5720.
  3. Matsukawa, M., Shibata, Y., Ohtsu, M., Mizutani, A., Mori, H., Wang, P., Ojika, M., Kawakita, K. and Takemoto, D. (2013). Nicotiana benthamiana calreticulin 3a is required for the ethylene-mediated production of phytoalexins and disease resistance against oomycete pathogen Phytophthora infestans. Mol Plant Microbe Interact 26(8): 880-892.
  4. Shibata, Y., Kawakita, K. and Takemoto, D. (2010). Age-related resistance of Nicotiana benthamiana against hemibiotrophic pathogen Phytophthora infestans requires both ethylene- and salicylic acid-mediated signaling pathways. Mol Plant Microbe Interact 23(9): 1130-1142.
  5. Shibata, Y., Ojika, M., Sugiyama, A., Yazaki, K., Jones, D. A., Kawakita, K. and Takemoto, D. (2016). The full-size ABCG transporters Nb-ABCG1 and Nb-ABCG2 function in pre- and postinvasion defense against Phytophthora infestans in Nicotiana benthamiana. Plant Cell 28(5): 1163-1181.
  6. Sugiyama, A., Shitan, N. and Yazaki K. (2007). Involvement of a soybean ATP-binding cassette-type transporter in the secretion of genistein, a signal flavonoid in legume-Rhizobium symbiosis. Plant Physiol 144(4): 2000-2008.
  7. Takemoto, D., Hardham, A. R. and Jones, D. A. (2005). Differences in cell death induction by Phytophthora elicitins are determined by signal components downstream of MAP kinase kinase in different species of Nicotiana and cultivars of Brassica rapa and Raphanus sativus. Plant Physiol 138(3): 1491-1504.
  8. Zhang, H. and Liu, Y. (2014). VIGS assays. Bio-protocol 4(5): e1057.


植物物种产生多种抗微生物代谢物,以保护自身免受自然环境中潜在的病原体的侵害。植物毒素是由植物响应于病原体的企图攻击而产生的低分子量化合物。植物抗毒素在受攻击的植物组织中的积累可以抑制穿透性病原体的生长。因此,植物抗毒素在入侵后对抗病原体的防御中起主要作用。茄科植物产生的主要植物抗毒素是倍半萜类化合物,如由 Nicotiana 和 Capsicum 物种产生的衣壳菌素,以及由 Solanum 物种产生的rishitin,其在胞质溶胶分泌到植物组织的细胞间隙中。我们以前曾报道,衣壳菌素分泌不足会导致本塞姆氏烟草对马铃薯晚疫病菌致病疫霉的敏感性增加。在这里,我们描述了一种测量 N中分泌的衣壳二醇的实用方案。本塞姆氏

【背景】该方案提供了一种快速简便的方法来量化衣壳菌醇的分泌,如Shibata 等(2016)所述。 因为环己烷通常用作洗去花粉层的溶剂,但能保持花粉活力(Doughty et al。,1993),我们开发了这种方法从叶子表面洗去分泌的代谢物来量化细胞外capsidiol。 可以使用该方法分析质膜局部转运蛋白的功能,同时还允许其与其他方法结合使用,例如使用质膜囊泡的生化转运分析(例如,Sugiyama 等人,,2007),以确定所检查的转运蛋白的底物。

关键字:辣椒醇, 本氏烟, 植物抗毒素, 分泌, 转运蛋白


  1. 移液器吸头(例如,PIPETMAN Diamond Tips,Gilson)
  2. 无针注射器(例如,Terumo TM Tuberculin Syringes 1 ml,Terumo Medical,目录号:SS-01T)
  3. 50毫升管(例如,50毫升Falcon离心管)
  4. 200毫升圆底烧瓶
  5. 2毫升管(例如,微量离心管,2毫升带盖,品牌,目录号:780550)
  6. 4-5周龄野生型或基因沉默的 Nicotiana benthamiana
    1. 对于本塞姆氏烟草的病毒诱导的基因沉默(VIGS),见Zhang和Liu,(2014)。
    2. 类似的方法可以应用于其他植物物种(例如,见Khare等,2017)。
  7. INF1激发子由 P产生。大批出没或 E。大肠杆菌
    1. 关于INF1制备的方法,参见Takemoto等。 (2005)或Shibata等。 (2010)。
    2. 也可以使用其他引发器。 
  8. Capsidiol
    注意:Capsidiol不可商购。关于衣壳醇纯化的方法,参见Matsukawa等人。 (2013)。
  9. 99.5%环己烷(Wako Pure Chemical Industries,目录号:034-05006)
  10. 99.5%环己烷/乙酸乙酯(1:1,v / v)(乙酸乙酯,Wako Pure Chemical Industries,目录号:051-00351)
  11. 99.5%乙腈(Wako Pure Chemical Industries,目录号:019-08631)
  12. 液氮
  13. 甲醇(Wako Pure Chemical Industries,目录号:131-01826)


  1. 移液器(例如,PIPETMAN P,Gilson)
  2. 电子秤(例如,Mettler-Toledo International,型号:AG245分析天平)
  3. 冰柜
  4. 带水浴的旋转蒸发器(例如,Yamato Scientific,型号:RE-301-BW)
  5. 离心浓缩器(例如,TOMY DIGITAL BIOLOGY,型号:CC-105带真空泵)
  6. 微管混合器(例如,TAITEC,型号:E-36)
  7. Sonicator Bath(例如,SND,型号:US-101)
  8. 砂浆和杵
  9. 微量离心机(例如,KUBOTA,型号:3740)
  10. 高效液相色谱(HPLC)系统(例如,Prominence Modular HPLC system,Shimadzu,Japan)
  11. ODS专栏(例如,Nomura Chemical,型号:Develosil ODS-UG-3)


  1. 诱导子的处理和 N的细胞外代谢产物的制备。本生的叶子
    1. 通过使用无针注射器将溶液注射到叶的远轴侧的细胞内空间中,用150nM INF1激发剂溶液处理本塞姆氏烟草叶(图1A)。在渗透过程中施加轻微的压力,以避免损坏叶组织。在5-10个位置注入溶液以确保叶子的充分覆盖。在光照下在23°C孵育9小时。
    2. 切断植物的处理过的叶子(图1B)并使用电子秤测量每片叶子的重量(一般来说,中等大小的叶片约为1克)。
    3. 将每片叶子放入50ml管中(图1C)。避免中央静脉破裂。
    4. 洗涤步骤:加入约。每管50ml环己烷。轻轻摇晃约在室温下30秒(图1D),然后将环己烷倒入200ml圆底烧瓶(或新鲜的50ml管中以储存在冰箱中)中以收集叶表面上的环己烷可溶性代谢物。冷冻并将叶组织保持在相同的50 ml管中,以便稍后提取细胞内(组织内)代谢物(参见程序B中的步骤)。

      图1.诱导子的处理和 N的细胞外代谢物的制备。通过无针注射器将溶液注入叶子的细胞内空间来处理诱导子。 A. B.从植物上切下处理过的叶子。 C.将每片叶子放入50ml管中。 D.添加约。 50毫升环己烷,轻轻摇动约。 30秒。

    5. 使用旋转蒸发器(在水浴中在约40℃下)蒸发步骤A4中获得的环己烷。
    6. 为了收集在圆底烧瓶底部干燥的代谢物,用移液管用1ml环己烷/乙酸乙酯(1:1,v / v)洗涤底部两次,并将溶液(总共2ml)收集到2中。毫升管。
    7. 使用离心浓缩器蒸发2 ml管中的溶剂(此步骤通常需要约1小时)。
    8. 将沉淀重新溶解在100μl乙腈中,充分混合并加入50μlddH 2 O。
    9. 使用微管混合器将2ml管充分摇动5分钟。
    10. 通过将管在超声波浴中漂浮5分钟来超声处理样品。
    11. 在室温下以16,000 x g 离心1分钟并收集上清液。
    12. 通过HPLC分析代谢物。见程序C.

  2. 制备 N的细胞内(组织内)代谢物。本生的叶子
    1. 研磨 N的叶子。来自步骤A4的本塞姆氏菌在液氮中用研钵和研杵,并悬浮在50%甲醇(1ml / 300mg叶组织)中。
    2. 在室温下以16,000 x g 离心1分钟并收集上清液。
    3. 加入相同体积的环己烷/乙酸乙酯(1:1,体积/体积)并涡旋。
    4. 在室温下以3,000 x g 离心5分钟,并收集上层相(乙酸乙酯层)。
    5. 使用离心浓缩器蒸发溶剂。 (这一步通常需要约1小时)。
    6. 将沉淀重新溶解在100μl乙腈中并加入50μlddH 2 。
    7. 用微管混合器摇匀2毫升,5分钟。
    8. 通过在超声波浴中漂浮管5分钟来超声处理样品。
    9. 在室温下以16,000 x g 离心1分钟并收集上清液。
    10. 通过HPLC分析代谢物。见程序C.

  3. 高效液相色谱(HPLC)分析
    专栏:ODS专栏(例如,Develosil ODS-UG-3,Nomura Chemical,日本)。
    Capsidiol和Capsidiol 3-acetate分别在4.6和12.8分钟洗脱(参见图2)。使用峰面积进行定量分析。
    注意:关于衣壳菌-3-乙酸酯纯化的方法,请参见Matsukawa等。 (2013)。

    图2. TRV感染或Nb- ABCG1 / 2 - 沉默 N的细胞外代谢物的洗脱曲线。用150nM INF1处理TRV感染的(A)或 NbABCG1 / 2 - 沉默的植物(B)的叶子。通过HPLC分析洗涤液的洗脱曲线(在205nm处检测)。


  1. 首先测试纯化的衣壳二醇用于HPLC分析,以确定每个量的衣壳醇的峰面积评分,以及在实验条件下衣壳二醇的洗脱时间。
  2. 通过比较由来自相同叶片的方法A和B制备的样品中检测到的衣壳二醇的量来计算分泌的/细胞内衣壳二醇的比例。
  3. 比较野生型和转运缺陷基因沉默的 N之间衣壳二醇的分泌。 benthamiana ,比较 N之间细胞外代谢物的洗脱曲线。本哈台那条线。参见图1中所示的实例,用于可能的衣壳体转运蛋白NbABCG1 / 2的基因沉默。其他代谢物的峰可用作内标,以确保一致的提取(洗涤)效率。


  1. 对照 N的分泌/细胞内衣壳二醇的比例。在我们的实验条件下,用150nM INF1处理9小时的本塞姆氏叶在1.5-2.5左右。该比例将受到诱导子活动(批次间差异),采样时间点和叶片年龄等因素的影响。因此,确定实验条件非常重要。
  2. 避免使用诱导叶片组织细胞死亡的诱导子,因为细胞死亡会导致细胞内代谢物的泄漏。


我们感谢Makoto Ojika教授(日本名古屋大学)提供技术建议。这项工作得到了日本科学促进会的科学研究资助(B)(26292024和17H03771)的支持。没有披露潜在的利益冲突。


  1. Doughty,J.,Hedderson,F.,McCubbin,A。和Dickinson,H。(1993)。 Brassica 花粉粒的涂层肽与柱头之间的相互作用S(自交不亲和) - 特异性糖蛋白。 Proc Natl Acad Sci USA 90(2):467-471。
  2. Khare,D.,Choi,H.,Huh,S.U.,Bassin,B.,Kim,J.,Martinoia,E.,Sohn,K.H.,Paek,K.H。和Lee,Y。(2017)。 拟南芥ABCG34通过调节camalexin的分泌,有助于防御坏死性病原体。 Proc Natl Acad Sci USA 114(28):E5712-E5720。
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引用:Kuroyanagi, T., Camagna, M. and Takemoto, D. (2018). Measuring Secretion of Capsidiol in Leaf Tissues of Nicotiana benthamiana. Bio-protocol 8(15): e2954. DOI: 10.21769/BioProtoc.2954.