搜索

Hyaloperonospora arabidopsidis (Downy Mildew) Infection Assay in Arabidopsis
拟南芥中活体营养型卵菌(霜霉病)的感染分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Hyaloperonospora arabidopsidis (Hpa; formerly Peronospora parasitica or Hyaloperonospora parasitica) is an oomycete downy mildew pathogen of the model plant Arabidopsis. The pathosystem between Arabidopsis and Hpa has been extensively used to study host/pathogen co-evolution (Coates and Beynon, 2010). As Hpa is an obligate biotrophic pathogen, its host is absolutely required for survival. Thus, Hpa must be maintained on susceptible Arabidopsis accessions and mutants. Growth of Hpa is evaluated in two ways; counting conidiospores (Asai et al., 2014) or counting sporangiophores after trypan blue staining (Holt et al., 2005). Here, we describe how to do inoculation with Hpa and how to evaluate Hpa growth on Arabidopsis.

Keywords: Downy mildew(霜霉病), Arabidopsis(拟南芥), Oomycete(卵菌), Hyaloperonospora arabidopsidis(活体营养型卵菌)

Materials and Reagents

  1. Materials and Reagents1. Conical tubes (50 ml) (BD Biosciences, Falcon®, catalog number: 352070 )
  2. Miracloth (Calbiochem®, catalog number: 475855 )
    Note: Currently, it is “EMD Millipore Corporation, catalog number: 475855”.
  3. Labeling Tape (Shamrock, catalog number: ST20 )
  4. Paper towels
  5. Arabidopsis lines and Hpa isolate. Ws-2 eds1-1 mutants [the accession previously reported as Ws-0 is in fact Ws-2 (Parker et al., 1996)] and Col-0 plants were used as susceptible and resistant accessions of Arabidopsis for Hpa isolate Emoy2, respectively.
  6. Ethanol (70%)
  7. Plastic tray (270 x 270 x 60 mm) with a transparent lid
  8. Sterile water
  9. Bright-LineTM Hemacytometer (Sigma-Aldrich, catalog number: Z359629 )
  10. Trypan blue (Sigma-Aldrich, catalog number: T6146 )
  11. Phenol (Wako Pure Chemical Industries, Siyaku, catalog number: 160-12725 )
  12. Chloral hydrate (Sigma-Aldrich, catalog number: C8383 )
  13. Glycerol (Wako Pure Chemical Industries, Siyaku, catalog number: 07500616 )
  14. Trypan blue solution (see Recipes)
  15. Chloral hydrate solution (see Recipes)

Equipment

  1. Scissors
  2. Biological safety cabinet (Labconco, model: Purifier Delta Series Class II Type A2 Cabinet )
  3. Airbrush Kit (Airtex, model: ASCF4 and KIDS105 )
  4. Growth chamber (NKsystem) (Nippon Medical & Chemical Instruments Co., catalog number: LPH410S )
  5. Weight (Sartorius, model: Quintix 224-1S )
  6. Water bath ( NE1-8 ) (Thermo Fisher Scientific, catalog number: 11459499)
  7. Labo shaker (BIO CRAFT, catalog number: BC730 )
  8. Stereomicroscope (Leica Microsystems, catalog number: M165FC )

Procedure

  1. Inoculation with Hpa
    1. Sterilize scissors and inside of the biological safety cabinet using 70% ethanol.
    2. Open a plastic tray with a transparent lid containing Arabidopsis plants that are densely covered by sporangiophores (Figure 1A) in biological safety cabinet to avoid diffusion of conidiospores.
      Note: To check contamination of different isolate(s) of Hpa, using both susceptible and resistant Arabidopsis accessions for propagation of Hpa is recommended (Figure 1A).
    3. Harvest aerial parts of the Arabidopsis plants in a 50 ml conical tube (Figure 1B).
      Note: Fill the tissues up to 20 ml marker on the tube (see Figure 1B). Avoid any soil contaminations because it may cause propagation of soil inhabiting pathogens.
    4. Put 15 ml sterile water in the 50 ml conical tube and shake it gently several times to obtain sporangia in water.
    5. Filter the obtained suspension using Miracloth (Figure 1C).
    6. Measure the concentration of conidiospores in the suspension using a hemacytometer (Figure 1D) and dilute with water to a concentration of 5 x 104 conidiospores per ml water.
      Note: To know how to count with hemacytometer, check the link (http://www.hemocytometer.org/).
    7. Saturate Arabidopsis plants with the suspension by spraying using Airbrush Kit.
      Note: Usually, 2-week-old plants and 5-day-old seedlings are used for evaluation of Hpa growth by counting conidiospores and counting sporangiophores, respectively. 2-week-old plants are used for propagation of Hpa. Arabidopsis plants are grown at 22 °C and 60% humidity under a 10 h photoperiod in environmentally controlled growth cabinets.
    8. Place the inoculated plants in a plastic tray with a transparent lid to maintain high humidity (90-100%) conditions (Figure 1E).
      Note: High humidity is very important for growth of Hpa. Thus, sealing off a plastic tray with a transparent lid using Labeling Tape is recommended.
    9. Incubate the Hpa-inoculated plants in a growth chamber at 16 °C under a 10 h photoperiod until the day of sampling.
      Note: Timing of sampling depends on the combinations of Hpa isolates and Arabidopsis accessions. Usually, from 4 to 7 days after the inoculation would be the proper timing.


      Figure 1. Procedure of inoculation with Hpa. A. Susceptible and resistant accessions of Arabidopsis 7 days after inoculation with Hpa. Ws-2 eds1-1 mutants (the accession previously reported as Ws-0 is in fact Ws-2; (Parker et al., 1996) and Col-0 plants were used as susceptible and resistant accessions of Arabidopsis for Hpa isolate Emoy2, respectively. Red boxes: Close-up shown in lower photographs. B. Arabidopsis plants harvested in 50 ml conical tube. C. The illustration of filtering the obtained suspension using Miracloth. D. An image of conidiospores on a hemacytometer. Red arrows indicate conidiospores. Scale bars = 200 μm. E. A plastic tray with a transparent lid containing Hpa-inoculated Arabidopsis plants.

  2. Evaluation of Hpa growth by counting conidiospores
    1. Harvest aerial parts of Hpa-inoculated plants in a 50 ml conical tube with sterile water.
      Note: The number of replicates and plants per replicate is dependent on the experimental design. Usually, we harvest 5 pools, each consisting of 3 plants in 1 ml water (total 15 plants) for each Hpa-inoculated Arabidopsis line.
    2. Vortex the conical tube.
    3. Count the number of conidiospores released in water using a hemacytometer (see Figure 1D).
    4. Pat the plants dry on paper towels and measure their fresh weight.
      Note: Growth of Hpa is represented as the number of conidiospores per ml water per gram fresh weight (conidiospores ml-1 g-1 FW). The intensity of disease symptom (the number of conidiospores) is variable, dependent on several conditions such as humidity, the intensity of light, timing for inoculation and age of host plants. The conditions can be controlled in each experiment by placing all the plant materials (e.g. WT and all the investigating mutants) in the same tray during infection.

  3. Evaluation of Hpa growth by counting sporangiophores after trypan blue staining
    Note: Protective wear should be applied at all steps because trypan blue solution and chloral hydrate are highly toxic chemicals.
    1. Harvest aerial parts of Hpa-inoculated seedlings in 50 ml conical tube and just cover the harvested seedlings with trypan blue solution.
      Note: At least 50 cotyledons for each of the investigating lines should be sampled.
    2. Boil for 1 min in water bath in fume hood.
    3. Incubate for 1 h at room temperature.
    4. Replace trypan blue solution with chloral hydrate solution and leave overnight on Labo shaker at room temperature.
    5. Replace chloral hydrate solution with 60% glycerol.
    6. Count the number of sporangiophores per cotyledon using a stereomicroscope.


      Figure 2. Typan blue-stained cotyledons. Resistant (left) and susceptible (right) accessions of Arabidopsis 7 days after inoculation with Hpa. Col-0 plants (left) and Ws-2 eds1-1 mutants (right) were used as resistant and susceptible accessions of Arabidopsis for Hpa isolate Emoy2, respectively. Yellow arrows indicate the hypersensitive response (HR), a programmed cell death that is commonly associated with plant disease resistance. Red arrows indicate sporangiophores.

Recipes

  1. Trypan blue solution
    10 ml lactic acid
    10 ml glycerol
    10 g phenol
    10 ml sterile water
    10 mg trypan blue
    The working solution is prepared by diluting the above solution with ethanol (1:1 v/v) and storing at room temperature.
  2. Chloral hydrate solution
    Add around 200 ml of sterile water and 500 g of chloral hydrate to a bottle and stir overnight in a fume hood.

Acknowledgments

Our work was supported by the Gatsby Foundation (http://www.gatsby.org.uk/), JSPS KAKENHI 15K18651 (S. A.) and 24228008 (K. S.) and RIKEN Special Postdoctoral Research Fellowship (S. A.). We thank Timothy Westlake for his help with the manuscript.

References

  1. Asai, S., Rallapalli, G., Piquerez, S. J., Caillaud, M. C., Furzer, O. J., Ishaque, N., Wirthmueller, L., Fabro, G., Shirasu, K. and Jones, J. D. (2014). Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid. PLoS Pathog 10(10): e1004443.
  2. Coates, M. E. and Beynon, J. L. (2010). Hyaloperonospora Arabidopsidis as a pathogen model. Annu Rev Phytopathol 48: 329-345.
  3. Holt, B. F., 3rd, Belkhadir, Y. and Dangl, J. L. (2005). Antagonistic control of disease resistance protein stability in the plant immune system. Science 309(5736): 929-932.
  4. Parker, J. E., Holub, E. B., Frost, L. N., Falk, A., Gunn, N. D. and Daniels, M. J. (1996). Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell 8(11): 2033-2046.

简介

( Hpa ;以前为寄生寄生霜霉菌或寄生寄生虫)是模式植物的卵菌霜霉病病原体 拟南芥。 拟南芥和 Hpa之间的细胞系统已广泛用于研究宿主/病原体共进化(Coates和Beynon,2010)。 作为Hpa 是一种专性的生物营养性病原体,其宿主是生存所必需的。 因此,必须在易感的拟南芥登录号和突变体上维持 Hpa 。 生长Hpa 以两种方式评估: (Asai等人,2014)或在台盼蓝染色后计数孢子生成(Holt等人,2005)。 在这里,我们描述了如何用 Hpa 进行接种以及如何在拟南芥上评估 Hpa

关键字:霜霉病, 拟南芥, 卵菌, 活体营养型卵菌

材料和试剂

  1. 材料和试剂1。锥形管(50ml)(BD Biosciences,Falcon ,目录号:352070)
  2. Miracloth(Calbiochem ?,目录号:475855)
    注意:目前,它是"默克密理博公司,目录号:475855"。
  3. 标签胶带(三叶草,目录号:ST20)
  4. 纸毛巾
  5. 行和 Hpa 隔离。 Ws-2 ems1-1突变体[先前报道为Ws-0的登录实际上是Ws-2(Parker等人,1996)]和Col-0植物分别用作Hpa 分离株Emoy2的拟南芥的易感和抗性登录。
  6. 乙醇(70%)
  7. 塑料托盘(270 x 270 x 60毫米),带有透明盖子
  8. 无菌水
  9. Bright-Line TM血细胞计数器(Sigma-Aldrich,目录号:Z359629)
  10. 台盼蓝(Sigma-Aldrich,目录号:T6146)
  11. 苯酚(Wako Pure Chemical Industries,Siyaku,目录号:160-12725)
  12. 水合氯醛(Sigma-Aldrich,目录号:C8383)
  13. 甘油(Wako Pure Chemical Industries,Siyaku,目录号:07500616)
  14. 台盼蓝溶液(见配方)
  15. 水合氯醛溶液(见配方)

设备

  1. 剪刀
  2. 生物安全柜(Labconco,型号:净化器Delta系列II类A2型)
  3. 喷枪套件(Airtex,型号:ASCF4和KIDS105)
  4. 生长室(NKsystem)(Nippon Medical& Chemical Instruments Co.,目录号:LPH410S)
  5. 重量(Sartorius,型号:Quintix 224-1S)
  6. 水浴(NE1-8)(Thermo Fisher Scientific,目录号:11459499)
  7. Labo摇动器(BIO CRAFT,目录号:BC730)
  8. 立体显微镜(Leica Microsystems,目录号:M165FC)

程序

  1. 接受 Hpa
    1. 使用70%乙醇消毒剪刀和生物安全柜内。
    2. 打开一个带有透明盖子的塑料托盘,其中含有被孢子囊磷脂密集覆盖的拟南芥植物(图1A) 生物安全柜,以避免分生孢子扩散 注意: ?检查Hpa的不同分离物的污染,使用两者 易感和抗性拟南芥品种繁殖的Hpa (图1A)。
    3. 在50ml锥形管中收获拟南芥植物的天花部分(图1B)。
      注意:在管上加入最多20ml的组织标记(参见图1B)。 避免任何土壤污染,因为它可能导致土壤的传播 居住的病原体。
    4. 将15毫升无菌水放入50毫升锥形管中,轻轻摇动几次,得到孢子囊水溶液。
    5. 使用Miracloth过滤所获得的悬浮液(图1C)
    6. 使用a测量悬浮液中分生孢子的浓度 ?血细胞计数器(图1D),并用水稀释至浓度为5 ?x 10 4 分生孢子/ml水 注意:要了解如何计数血球计,请检查链接( http://www.hemocytometer.org/ )。
    7. 使用喷枪套件通过喷雾使拟南芥植物悬浮。
      注意:通常使用2周龄的植物和5天龄的幼苗 通过计数分生孢子和计数评价Hpa生长 孢子囊菌。使用2周龄植物 Hpa的传播。拟南芥植物在22℃和60% 湿度在10小时光周期内环境控制生长 机柜。
    8. 将接种的植物放在一个塑料托盘中 透明盖保持高湿度(90-100%)条件(图 1E)。
      注意:高湿度对于Hpa的生长非常重要。从而, 使用标签带密封带有透明盖的塑料托盘 ?推荐。
    9. 在生长室中在16℃,10小时光照下孵育接种的Hpa 接种植物,直到采样日为止。
      注意:取样的时间取决于Hpa分离物的组合 和拟南芥(Arabidopsis)种质。通常,从4到7天后 接种将是适当的时机。


      图1.过程 接种Hpa 。 A.接种Hpa后7天的拟南芥的敏感和抗性登录 。 Ws-2 eds1-1 突变体 以前报告为Ws-0的登记事实上是Ws-2; (Parker等人, 1996)和Col-0植物用作敏感和抗性种质 ?的分离物Emoy2的 拟南芥。红色箱子:特写镜头 ?如下图所示。 B.在50ml中收获的拟南芥植物 锥形管。 C.过滤所得悬浮液的说明 使用Miracloth。 D.血细胞计数器上分生孢子的图像。红 箭头表示分生孢子。比例尺=200μm。 E.塑料托盘 其具有包含接种了拟南芥植物的 Hpa 接种的植物的透明盖。

  2. 通过计数分生孢子对hpa 生长的评价
    1. 用无菌水在50ml锥形管中收获接种的Hpa 接种植物的地上部分。
      注意:每个重复的复制数和植物数取决于 ?实验设计。通常,我们收获5个池,每个池 的3株植物在1ml水中(总共15株植物) 拟南芥系。
    2. 涡旋锥形管。
    3. 使用血细胞计数器计数在水中释放的分生孢子的量(见图1D)
    4. 用纸巾擦干植物,并测量其鲜重 注意:Hpa的生长表示为每孢子孢子数 ml水每克鲜重(分生孢子ml -1 /sup> FW)。的 疾病症状的强度(分生孢子数)是可变的, 取决于几个条件,如湿度,强度 光,接种时间和宿主植物的年龄。条件可以 ?在每个实验中通过放置所有植物材料来控制 (例如WT和所有研究的突变体) 感染。

  3. 通过在台盼蓝染色之后计数孢子生成来评价Hpa 生长 注意:所有步骤都应使用防护服,因为台盼蓝溶液和水合氯醛是高毒性化学品。
    1. 在50ml锥形管中收获Hpa 接种的幼苗的空间部分 并用台盼蓝溶液覆盖收获的幼苗 注意:每个调查行至少要采集50个子叶。
    2. 在通风橱中的水浴中煮沸1分钟。
    3. 在室温下孵育1小时。
    4. 用水合氯醛溶液替代台盼蓝溶液,在室温下在Labo摇床上放置过夜
    5. 用60%甘油代替水合氯醛溶液。
    6. 使用立体显微镜计数每个子叶的孢子囊数目

      图2. Typan蓝色染色的子叶。抗性(左)和 接种后7天的拟南芥的易感(右)种质 与 Hpa 。使用Col-0植物(左)和Ws-2 eds1-1突变体(右) 作为拟南芥 分离物的抗性和易感种质 Emoy2。黄色箭头表示过敏反应 (HR),通常与植物相关的程序性细胞死亡 抗病性。红色箭头表示孢子囊菌。

食谱

  1. 台盼蓝溶液
    10ml乳酸
    10ml甘油 10克酚
    10ml无菌水
    10mg台盼蓝
    通过用乙醇(1:1v/v)稀释上述溶液并在室温下储存来制备工作溶液。
  2. 水合氯醛溶液
    向瓶中加入约200ml无菌水和500g水合氯醛,在通风橱中搅拌过夜

致谢

我们的工作得到了盖茨比基金会的支持( http://www.gatsby.org.uk/) ,JSPS KAKENHI 15K18651(SA)和24228008(KS)和RIKEN特殊博士后研究奖学金(SA)。我们感谢Timothy Westlake对手稿的帮助。

参考文献

  1. Asai,S.,Rallapalli,G.,Piquerez,S.J.,Caillaud,M.C.,Furzer,O.J.,Ishaque,N.,Wirthmueller,L.,Fabro,G.,Shirasu,K.and Jones,J.D。(2014)。 在阿拉伯芥中表达谱分析 /霜霉病相互作用揭示了高度表达的效应子其减弱对水杨酸的反应。 PLoS Pathog 10(10):e1004443。
  2. Coates,M.E。和Beynon,J.L。(2010)。 Hyaloperonospora Arabidopsidis 作为病原体模型。 Annu Rev Phytopathol 48:329-345。
  3. Holt,B.F.,3rd,Belkhadir,Y。和Dangl,J.L。(2005)。 抗病性控制植物免疫系统中的抗病性蛋白质稳定性。 309(5736):929-932。
  4. Parker,J.E.,Holub,E.B.,Frost,L.N.,Falk,A.,Gunn,N.D。和Daniels,M.J。(1996)。 eds1的表征,在拟南芥中突变,抑制对寄生霜霉菌的抗性通过几个不同的RPP基因。 植物细胞 8(11):2033-2046。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Asai, S., Shirasu, K. and Jones, J. D. (2015). Hyaloperonospora arabidopsidis (Downy Mildew) Infection Assay in Arabidopsis. Bio-protocol 5(20): e1627. DOI: 10.21769/BioProtoc.1627.
  2. Asai, S., Rallapalli, G., Piquerez, S. J., Caillaud, M. C., Furzer, O. J., Ishaque, N., Wirthmueller, L., Fabro, G., Shirasu, K. and Jones, J. D. (2014). Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid. PLoS Pathog 10(10): e1004443.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。