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In vitro CLE Peptide Bioactivity Assay on Plant Roots
植物根部CLE肽的生物活性的体外实验   

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Plant Physiology
Jan 2015

Abstract

Plant CLAVATA3/ESR (CLE)-related proteins play diverse roles in plant growth and development including regulating the development of root meristem. The mature functional forms of CLE peptides are typically 12-13 amino acids (aa) in length that are derived from the conserved C-termini of their precursor proteins. Genes encoding small secreted peptides sharing similarity to plant CLE proteins have recently been cloned from plant-parasitic nematodes, pests that infect many important crops. It is demonstrated that exogenous application of synthetic 12-14 aa CLE peptides corresponding to the CLE domain of their precursor proteins can suppress plant root growth. This protocol is to evaluate the bioactivity of CLE peptides originated from plant-parasitic nematodes by measuring the growth of plant roots or the size of root apical meristem (RAM) after CLE peptide treatment. Plants used in the study included Arabidopsis and potato.

Keywords: CLE peptide (CLE肽), Plant-parasitic nematode (植物寄生线虫), Root apical meristem (根顶端分生组织), Potato root growth (马铃薯根生长)

Materials and Reagents

  1. Micropore tape (Thermo Fisher Scientific, catalog number: 19-027-761 )
  2. Sterile square petri dishes (VWR International, catalog number: 60872-310 )
  3. Sterile 6-well plates (Greiner Bio-One GmbH, catalog number: 657185 )
  4. 1.7 ml micro centrifuge tubes (Laboratory Products Sales, catalog number: L211511 )
  5. 50 ml conical centrifuge tubes
  6. Microscope slides (Laboratory Products Sales, catalog number: M130400 )
  7. Sterile steel blades (Staples, catalog number: 111322 )
  8. Arabidopsis thaliana (Columbia-0) seeds (https://www.arabidopsis.org/abrc/index.jsp)
  9. Potato true seeds (Solanum tuberosum) (http://www.ars-grin.gov/nr6/)
  10. Potato plant (Solanum tuberosum cv. Désirée) (http://www.ars-grin.gov/nr6/)
  11. CLE peptides (> 70% purity) (Selleck Chemicals LLC) (www.selleckchem.com)
  12. Bleach (6% sodium hypochlorite) (Clorox Company)
  13. Sterile distilled water
  14. Agarose (Thermo Fisher Scientific, catalog number: BP160-100 )
  15. Timentin (PhytoTechnology Laboratories®, catalog number: T869 )
  16. Thiamine hydrochloride (C12H17ClN4OS.HCl) (Sigma-Aldrich, catalog number: T1270 )
  17. MS salt (Caisson Laboratories, catalog number: MSP01-50LT )
  18. Inositol (Thermo Fisher Scientific, catalog number: AC122261000 )
  19. 2-(4-Morpholino)ethane Sulfonic Acid (MES) (Thermo Fisher Scientific, catalog number: BP300-100 )
  20. Sodium Phosphate Monobasic Monohydrate (NaH2PO4.H2O) (Thermo Fisher Scientific, catalog number: S369-500 )
  21. Sucrose (Thermo Fisher Scientific, catalog number: S5-12 )
  22. Gelrite (Thermo Fisher Scientific, catalog number: CAS 71010-52-1 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: CAS 71010-52-1 ”.
  23. Agar (Thermo Fisher Scientific, catalog number: BP1423-500 )
  24. Propagation medium (1 L) (see Recipes)
  25. ½ MS medium (1 L) (see Recipes)
  26. 0.1% agarose (see Recipes)
  27. Sodium phosphate buffer (see Recipes)

Equipment

  1. Sterile forceps and scalpel (sterilized by heat treatment using a Bunsen burner)
  2. 360° Multi-functional tube rotator (Thermo Fisher Scientific, Barnstead Thermolyne, model: Labquake Shaker Rotisserie )
  3. Growth chamber (Percival Scientific, model: I-66LLVL )
  4. Biosafety cabinet (NuAire, model: Class II Type A/B3 )
  5. Olympus BX-50 microscope
  6. Digital CCD camera (QImaging, model: Retiga EXi )

Software

  1. MetaMorph imaging analysis software
    (http://core.phmtox.msu.edu/Scheduling/ItemDocs/9/MetaMorph_Software_Basic_Analysis_Guide.pdf)

Procedure

  1. Peptide preparation (performed under sterile conditions)
    1. Dissolve synthesized CLE peptides in filter-sterilized 50 mM sodium phosphate buffer to make a final stock concentration of 10 mM.
    2. Aliquot 200 µl of the peptide stock into 1.7 ml microfuge tubes and store them in a -80 °C freezer for later uses.

  2. Peptide assay with plant seeds (performed under sterile conditions)
    1. Measure out approximately 100 seeds (Arabidopsis or potato) for each peptide treatment and transfer the seeds to a labeled 1.7 ml microcentrifuge tube.
    2. Add 1 ml 50% bleach to the tube and incubate the seeds in bleach for 15 min (for Arabidopsis seeds) or 5 min (for potato true seeds) on a rotator. During the incubation time, if seeds clump in the tube bottom, resuspend the seeds by gentle shaking and then put the tube back on the rotator.
    3. Remove the bleach solution and rinse the seeds with 1.5 ml sterile distilled water for three times (5 min each on a rotator) to remove any trace of bleach.
    4. Remove sterile water after last wash. For Arabidopsis seeds, add 1 ml of sterile 0.1% agarose solution to the tube, resuspend the seeds by gentle shaking and store the tube at 4 °C for 3 d (with light or in darkness) before plating on the plates. For potato true seeds, plate them directly on agar plates.
    5. Add the peptide stock solution to 30 ml of ½ MS agar medium to achieve desirable peptide concentrations (e.g., 5 µM or 10 µM). After mixing up, pour the medium into a square plate and use the agar plate until the medium solidified. Agar plates containing the same volume of 50 mM sodium phosphate buffer as the added peptide stock will be used as controls.
    6. Plate approximately 12 seeds on the top of a square plate.
    7. Place the plates vertically in a growth chamber and culture at 24 °C under a 16-h/8-h light/dark cycle.
    8. Measure the root length (using a ruler) from the base of the hypocotyl to the tip of the primary root at several time points (e.g., 7 d and 14 d) after plating (Figure 1), or measure the size of the root apical meristem (RAM) of each root tip (Figure 2). Roots of 10 seedlings were measured at each time point.


      Figure 1. Effects of 10 µM GrCLE1-1 peptide (RVTPGGPDPLHN) on the growth of Arabidopsis and potato roots 10 d after peptide treatment


      Figure 2. Effects of 10 µM GrCLE1-1 peptide (RVTPGGPDPLHN) on the development of root apical meristem of Arabidopsis and potato roots 14 d after peptide treatment

  3. Peptide assay on tissue-cultured potato plantlets (performed under sterile conditions)
    1. Plate internode stem segments (approximately 1 cm long) cut using sterile steel blade from in vitro grown potato plantlets into each well of a 6-well plate that contains 6 to 7 ml of propagation medium with certain concentrations of the tested CLE peptide and timentin (100 μg/ml). The top part of the internode segment should be exposed in the air. For the test using internode stem pieces, usually higher peptide concentrations (e.g., 5 μM) need to be used in order to observe an effect on root growth.
    2. Label plates and seal them with micropore tapes.
    3. Cultivate plates in a growth chamber at 24 °C under a 16-h/8-h light/dark cycle for two weeks and then measure the RAM size of root tips.

  4. Measurement of the RAM size of root tips
    1. Use a steel blade to cut off the primary root tips (approximately 2 cm long) from peptide-treated plant seedlings.
    2. Mount the root tips immediately in water onto a glass slide and examine under an Olympus BX-50 microscope equipped with Nomarski optics.
    3. Capture the images using a QImaging Retiga EXi digital CCD camera.
    4. Measure microscopically the root apical meristem region of individual primary roots covering the distance from the position around the quiescent center to the position where elongated root cells immediately follow cytoplasm-dense meristematic cells (Figure 2).
    5. Analyze the collected data using the MetaMorph imaging analysis software (The manual can be found in the following link: http://core.phmtox.msu.edu/Scheduling/ItemDocs/9/MetaMorph_Software_Basic_Analysis_Guide.pdf).

Recipes

  1. Propagation medium (1 L)
    4.3 g MS salt
    0.17 g NaH2PO4.H2O
    0.10 g inositol
    0.4 mg thiamine HCl
    30 g sucrose
    2.5 g gelrite
    Adjust pH to 6.0 with KOH and autoclave (121 °C, 20 min)
  2. ½ MS medium (1 L)
    2.15 g MS salt
    10 g sucrose
    0.5 g MES
    15 g agar
    Adjust pH to 5.8 with KOH and autoclave (121 °C, 20 min)
  3. 0.1% agarose
    0.1 g agarose in 100 ml distilled water
    Autoclave and store at room temperature
  4. 50 mM sodium phosphate buffer
    44 mM NaH2PO4
    6 mM Na2HPO4
    Adjust pH to 6.0 with NaOH and filter sterilize

Acknowledgments

This work was supported by funding from USDA-ARS and the USDA-NRI Competitive Grants program.

References

  1. Chen, S., Lang, P., Chronis, D., Zhang, S., De Jong, W. S., Mitchum, M. G. and Wang, X. (2015). In planta processing and glycosylation of a nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-like effector and its interaction with a host CLAVATA2-like receptor to promote parasitism. Plant Physiol 167(1): 262-272.
  2. Chronis, D., Chen, S., Lang, P., Tran, T., Thurston, D. and Wang, X. (2014). In vitro nematode infection on potato plant. Bio-protocol 4(1): e1016.
  3. Fiers, M., Golemiec, E., Xu, J., van der Geest, L., Heidstra, R., Stiekema, W. and Liu, C. M. (2005). The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway. Plant Cell 17(9): 2542-2553.
  4. Lu, S. W., Chen, S., Wang, J., Yu, H., Chronis, D., Mitchum, M. G. and Wang, X. (2009). Structural and functional diversity of CLAVATA3/ESR (CLE)-like genes from the potato cyst nematode Globodera rostochiensis. Mol Plant Microbe Interact 22(9): 1128-1142.
  5. Miyawaki, K., Tabata, R. and Sawa, S. (2013). Evolutionarily conserved CLE peptide signaling in plant development, symbiosis, and parasitism. Curr Opin Plant Biol 16(5): 598-606.

简介

植物CLAVATA3/ESR(CLE)相关蛋白质在植物生长和发育中发挥多种作用,包括调节根分生组织的发育。 CLE肽的成熟功能形式通常为12-13个氨基酸(aa)长度,其衍生自其前体蛋白的保守C-末端。 最近从植物寄生线虫,感染许多重要作物的害虫克隆了编码与植物CLE蛋白相似的小分泌肽的基因。 证明外源应用对应于其前体蛋白的CLE结构域的合成12-14aa CLE肽可以抑制植物根生长。 该协议是通过测量CLE肽处理后植物根的生长或根尖分生组织(RAM)的大小来评价源自植物寄生线虫的CLE肽的生物活性。 用于研究的植物包括拟南芥和马铃薯。

关键字:CLE肽, 植物寄生线虫, 根顶端分生组织, 马铃薯根生长

材料和试剂

  1. Micropore带(Thermo Fisher Scientific,目录号:19-027-761)
  2. 无菌方培养皿(VWR International,目录号:60872-310)
  3. 无菌6孔板(Greiner Bio-One GmbH,目录号:657185)
  4. 1.7ml微量离心管(Laboratory Products Sales,目录号:L211511)
  5. 50ml锥形离心管
  6. 显微镜载玻片(实验室产品销售,目录号:M130400)
  7. 无菌钢刀片(Staples,目录号:111322)
  8. 拟南芥(哥伦比亚-0 )种子( https://www.arabidopsis.org/abrc/index.jsp
  9. 马铃薯真实种子( Solanum tuberosum )( http://www.ars-grin .gov/nr6/
  10. 马铃薯植物( Solanum tuberosum cv。Désirée)( http://www.ars -grin.gov/nr6/
  11. CLE肽(纯度> 70%)(Selleck Chemicals LLC)( www.selleckchem.com
  12. 漂白剂(6%次氯酸钠)(Clorox公司)
  13. 无菌蒸馏水
  14. 琼脂糖(Thermo Fisher Scientific,目录号:BP160-100)
  15. Timentin( Phyto Technology Laboratories ?,目录号:T869)
  16. 盐酸硫胺素(C 12 H 17 H 17 ClN 4 OS i.HCl)(Sigma-Aldrich,目录号:T1270 )
  17. MS盐(Caisson Laboratories,目录号:MSP01-50LT)
  18. 肌醇(Thermo Fisher Scientific,目录号:AC122261000)
  19. 2-(4-吗啉基)乙烷磺酸(MES)(Thermo Fisher Scientific,目录号:BP300-100)
  20. 磷酸二氢钠一水合物(NaH 2 PO 4 PO 4,H 2 O 2)(Thermo Fisher Scientific,目录号: S369-500)
  21. 蔗糖(Thermo Fisher Scientific,目录号:S5-12)
  22. Gelrite(Thermo Fisher Scientific,目录号:CAS 71010-52-1)
    注意:目前,它是"Sigma-Aldrich,目录号:CAS 71010-52-1"。
  23. 琼脂(Thermo Fisher Scientific,目录号:BP1423-500)
  24. 繁殖培养基(1升)(参见配方)
  25. ?MS培养基(1 L)(参见配方)
  26. 0.1%琼脂糖(参见配方)
  27. 磷酸钠缓冲液(见配方)

设备

  1. 无菌镊子和手术刀(使用本生灯进行热处理消毒)
  2. 360°多功能管旋转器(Thermo Fisher Scientific,Barnstead Thermolyne,型号:Labquake Shaker Rotisserie)
  3. 生长室(Percival Scientific,型号:I-66LLVL)
  4. 生物安全柜(NuAire,型号:II类A/B3)
  5. 奥林巴斯BX-50显微镜
  6. 数字CCD摄像机(QImaging,型号:Retiga EXi)

软件

  1. MetaMorph成像分析软件
    http://core.phmtox.msu.edu/Scheduling/ItemDocs/9/MetaMorph_Software_Basic_Analysis_Guide.pdf

程序

  1. 肽制备(在无菌条件下进行)
    1. 将合成的CLE肽溶于过滤灭菌的50mM钠中 磷酸盐缓冲液中,使最终母液浓度为10mM
    2. 将200μl肽储备液分装到1.7 ml微量离心管中,并储存在-80°C冰箱中备用。

  2. 用植物种子进行的肽测定(在无菌条件下进行)
    1. 每个测量约100个种子(拟南芥或马铃薯) 肽处理并将种子转移至标记的1.7ml 微量离心管。
    2. 加入1毫升50%漂白剂到管和 在漂白剂中孵育种子15分钟(对于拟南芥种子)或5分钟 ?(用于马铃薯真实种子)。在孵化时间,如果 种子在管底部,通过轻轻摇动重悬种子 然后将管子放回旋转器。
    3. 取出漂白剂 溶液并用1.5ml无菌蒸馏水冲洗种子 三次(在旋转器上每次5分钟)以除去任何痕量的漂白剂
    4. 最后一次清洗后取出无菌水。对于拟南芥种子,添加1 ml的无菌0.1%琼脂糖溶液到管中,重悬种子 轻轻摇动并将管在4℃下储存3天(用光或进行) 黑暗)。对于马铃薯真正的种子,板 他们直接在琼脂板上
    5. 添加肽储备溶液 30ml 1/2MS琼脂培养基以获得期望的肽浓度 (例如,5μM或10μM)。混合后,将介质倒入一个正方形 并使用琼脂平板直到培养基凝固。琼脂板 含有与加入的相同体积的50mM磷酸钠缓冲液 ?肽库将用作对照
    6. 在正方形板的顶部镀上约12粒种子。
    7. 将板垂直放置在生长室中,并在24℃下在16小时/8小时光/暗循环下培养。
    8. 从底部测量根长度(使用标尺) 下胚轴在几个时间点(例如,)处到主根的顶端 ?d和14 d)电镀后(图1),或测量根部的尺寸 每个根尖的顶端分生组织(RAM)(图2)。 10棵幼苗的根 ?在每个时间点测量

      图1. 10μM的作用 GrCLE1-1肽(RVTPGGPDPLHN)对拟南芥和马铃薯生长的影响 根治疗后10 d肽治疗


      图2. 10μM的影响 GrCLE1-1肽(RVTPGGPDPLHN)对根尖的发育 拟南芥分生组织和肽处理后14天的马铃薯根部

  3. 对组织培养的马铃薯苗(在无菌条件下进行)进行肽测定
    1. 板节间茎段(约1厘米长)切割使用 无菌钢刀片从体外生长的马铃薯苗进入每个孔 的含有6至7ml传代培养基的6孔板 某些浓度的测试的CLE肽和特美汀(100 μg/ml)。节间段的顶部应暴露在 空气。对于使用节间茎片的试验,通常是较高的肽 浓度(例如,<5μM)需要使用以便观察 对根生长的影响
    2. 标签板并用微孔胶带密封
    3. 在生长室中在24℃下在16小时/8小时培养平板 亮/暗循环两个星期,然后测量根的RAM大小 提示。

  4. 测量根尖的RAM大小
    1. 使用钢刀片切断来自肽处理的植物幼苗的主要根尖(约2cm长)
    2. 将根尖立即置于水中的玻璃载玻片上 在装有Nomarski光学元件的Olympus BX-50显微镜下进行检查
    3. 使用QImaging Retiga EXi数字CCD相机捕获图像。
    4. 显微镜下测量根尖分生组织区 个体主根覆盖距离周围的位置 静息中心到细长根细胞的位置 紧随细胞质密度分生细胞(图2)
    5. 使用MetaMorph成像分析分析收集的数据 软件(手册可在以下链接中找到: http://core .phmtox.msu.edu/Scheduling/ItemDocs/9/MetaMorph_Software_Basic_Analysis_Guide.pdf )。

食谱

  1. 繁殖培养基(1 L)
    4.3克MS盐
    0.17g NaH 2 PO 4 subO 2。 H O 0.10克肌醇
    0.4mg硫胺素HCl 30克蔗糖 2.5克gelrite
    用KOH和高压釜(121℃,20分钟)调节pH至6.0
  2. ?MS培养基(1 L)
    2.15g MS盐
    10g蔗糖 0.5 g MES
    15克琼脂
    用KOH和高压釜(121℃,20分钟)将pH调节至5.8
  3. 0.1%琼脂糖 0.1g琼脂糖在100ml蒸馏水中的溶液 高压灭菌并在室温下贮存
  4. 50mM磷酸钠缓冲液 44mM NaH 2 PO 4>/
    6mM Na 2 HPO 4
    用NaOH调节pH至6.0,过滤灭菌

致谢

这项工作得到了USDA-ARS和USDA-NRI竞争性拨款计划的资助。

参考文献

  1. Chen,S.,Lang,P.,Chronis,D.,Zhang,S.,De Jong,W. S.,Mitchum,M.G.and Wang,X.(2015)。 在植物中处理和糖基化线虫CLAVATA3/ENDOSPERM SURROUNDING REGION-类似效应子及其与宿主CLAVATA2样受体的相互作用以促进寄生。植物生理学167(1):262-272。
  2. Chronis,D.,Chen,S.,Lang,P.,Tran,T.,Thurston,D.and Wang,X.(2014)。 体外线虫对马铃薯植株的感染。 生物协议 4(1):e1016。
  3. Fiers,M.,Golemiec,E.,Xu,J.,van der Geest,L.,Heidstra,R.,Stiekema,W.and Liu,C.M。(2005)。 14个氨基酸的CLV3,CLE19和CLE40肽触发根分生组织的消耗> Arabidopsis 通过CLAVATA2依赖性通路。 植物细胞 17(9):2542-2553。
  4. Lu,S.W.,Chen,S.,Wang,J.,Yu,H.,Chronis,D.,Mitchum,M.G.and Wang,X.(2009)。 来自马铃薯胞囊线虫的CLAVATA3/ESR(CLE)样基因的结构和功能多样性> Globodera rostochiensis 。 Mol Plant Microbe Interact 22(9):1128-1142。
  5. Miyawaki,K.,Tabata,R。和Sawa,S。(2013)。 进化保守的CLE肽信号在植物发育,共生和寄生中的作用。 Curr Opin Plant Biol 16(5):598-606。
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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. Chen, S. and Wang, X. (2015). In vitro CLE Peptide Bioactivity Assay on Plant Roots. Bio-protocol 5(24): e1689. DOI: 10.21769/BioProtoc.1689.
  2. Chen, S., Lang, P., Chronis, D., Zhang, S., De Jong, W. S., Mitchum, M. G. and Wang, X. (2015). In planta processing and glycosylation of a nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-like effector and its interaction with a host CLAVATA2-like receptor to promote parasitism. Plant Physiol 167(1): 262-272.

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