Measuring Secretion of Capsidiol in Leaf Tissues of Nicotiana benthamiana
烟草叶组织分泌辣椒素的测定
发布: 2018年08月05日第8卷第15期 DOI: 10.21769/BioProtoc.2954 浏览次数: 5252
评审: Arsalan DaudiMichael EnosKangquan Yin
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参见作者原研究论文
The authors used this protocol in:
May 2016
Abstract
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)
Background
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
- Pipette tips (e.g., PIPETMAN Diamond Tips, Gilson)
- Needleless syringe (e.g., TerumoTM Tuberculin Syringes 1 ml, Terumo Medical, catalog number: SS-01T )
- 50 ml tubes (e.g., 50 ml Falcon centrifuge tube)
- 200 ml round-bottom flask
- 2 ml tubes (e.g., microcentrifuge tube, 2 ml with lid, BRAND, catalog number: 780550 )
- Leaves of 4-5 weeks old wild-type or gene-silenced Nicotiana benthamiana
Notes:- For Virus-induced gene silencing (VIGS) of N. benthamiana, see Zhang and Liu, (2014).
- Similar method can be applied for other plant species (e.g., see Khare et al., 2017).
- For Virus-induced gene silencing (VIGS) of N. benthamiana, see Zhang and Liu, (2014).
- INF1 elicitor produced by P. infestans or in E. coli
Notes:- For the method of INF1 preparation, see Takemoto et al. (2005) or Shibata et al. (2010).
- Other elicitors may be used as well.
- For the method of INF1 preparation, see Takemoto et al. (2005) or Shibata et al. (2010).
- Capsidiol
Note: Capsidiol is not commercially available. For the method of capsidiol purification, see Matsukawa et al. (2013). - 99.5% Cyclohexane (Wako Pure Chemical Industries, catalog number: 034-05006 )
- 99.5% Cyclohexane/Ethyl acetate (1:1, v/v) (Ethyl acetate, Wako Pure Chemical Industries, catalog number: 051-00351 )
- 99.5% acetonitrile (Wako Pure Chemical Industries, catalog number: 019-08631 )
- Liquid nitrogen
- Methanol (Wako Pure Chemical Industries, catalog number: 131-01826 )
Equipment
- Pipettes (e.g., PIPETMAN P, Gilson)
- Electronic scale (e.g., Mettler-Toledo International, model: AG245 Analytical balance)
- Freezer
- Rotary evaporator with water bath (e.g., Yamato Scientific, model: RE-301-BW )
- Centrifugal concentrator (e.g., TOMY DIGITAL BIOLOGY, model: CC-105 with Vacuum Pump)
- Microtube mixer (e.g., TAITEC, model: E-36 )
- Sonicator Bath (e.g., SND, model: US-101 )
- Mortar and pestle
- Microcentrifuge (e.g., KUBOTA, model: 3740 )
- High-performance liquid chromatography (HPLC) system (e.g., Prominence Modular HPLC system, Shimadzu, Japan)
- ODS column (e.g., Nomura Chemical, model: Develosil ODS-UG-3 )
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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.
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分类
植物科学 > 植物免疫 > 宿主-细菌相互作用
细胞生物学 > 细胞分离和培养 > 细胞分离
分子生物学 > DNA > DNA 克隆
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