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Growth Assay and Detection of TRP and Indole Derivatives in Piriformospora indica Culture Supernatant by LC-MS/MS
TRP对印度梨形孢(P. indica)生长的影响与其代谢产物IAA及吲哚类衍生物的检测(LC-MS/MS)   

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Abstract

The mutualistic root endophyte Piriformospora indica colonizes a wide range of plants and the colonization of root cells by this fungus is very often associated with beneficial effects to its host, such as growth promotion and increased biotic and abiotic stress tolerance. These traits may be based on general mechanisms and signaling pathways common to many different plant species. One such mechanism could be the recruitment of phytohormone pathways by P. indica. It is known, that many mutualistic microorganisms are able to synthesize and secrete phytohormones during the interaction with their host plants. This protocol has been successfully utilized to analyze tryptophan (TRP)-dependent biosynthesis of indole-3-acetic acid (IAA) and its indole derivatives by P. indica as well as their influence on the growth of this fungus (Hilbert et al., 2012).

Materials and Reagents

  1. Indole derivative:
    TRP (Sigma-Aldrich, catalog number: T0254-500g )
    IAD (indole-3-acetaldehyde) (Sigma-Aldrich, catalog number: I1000-100mg )
    IAA (Sigma-Aldrich, catalog number: I5148-2g )
  2. Neubauer improved counting chamber (Marienfeld-Superior)
  3. Sterile scalpel
  4. Sterile drigalski spatula
  5. Miracloth filter 15 cm x 15 cm (Merck KGaA, catalog number: 475855 )
  6. Ruler
  7. 0.3 ml polypropylene snap ring microvials
  8. 0.9% NaCl
  9. 0.002% (v/v) Tween water 20
  10. 90% methanol (HPLC grade)
  11. Acetonitrile (HPLC grade)
  12. Acetic acid (HPLC grade)
  13. Chlamydospores solution
  14. Microelements (MnCl2.4H2O, H3BO3, ZnSO4.7H2O, KI, Na2MO4.2H2O, CuSO4.5H2O)
  15. Glucose
  16. Peptone
  17. Yeast extract
  18. Casamine acids
  19. Agar
  20. Complete medium (CM medium) (see Recipes)
  21. Buffer A (see Recipes)
  22. Buffer B (see Recipes)

Equipment

  1. 1.5 ml Eppendorf tubes
  2. 50 ml Falcon tube
  3. Petri dishes (12 mm diameter)
  4. Incubator (e.g. B. Braun Biotech International, Certomat BS-1)
  5. Biofuge Primo R (Heraeus, Germany) (Rotor, catalog number: 7590 )
  6. Phenomenex Luna 250 x 4.6 mm C18 RP-HPLC column (Phenomenex)
  7. Phenomenex Luna 10 x 4.6 mm C18 RP-HPLC guard column (Phenomenex)
  8. ICS3000 HPLC system (Dionex)
  9. QTrap 3200 triple quadrupole mass spectrometer (Applied Biosystems SCIEX)
  10. Polypropylene snap ring microvial

Procedure

I.   Growth Assay

  1. Collect spores from 3 to 4 weeks old Piriformospora indica plate cultures (see Figure 1).


    Figure 1. Four-week-old P. Indica agar plate

    1. Pour approximately 5 ml sterile 0.002% Tween water 20 on 3-4 weeks old P. indica plate under sterile condition at room temperature (RT).
    2. Scratch plate with sterile Drigalski spatula and/or scalpel and mix.
    3. Pour spore solution through miracloth filter and collect flow through in 50 ml Falcon tube.
    4. Centrifuge for 7 min at 3,500 rpm discard supernatant.
    5. Wash pellet with 5-10 ml 0.002% Tween water 20.
    6. Centrifuge for 7 min at 3,500 rpm, discard supernatant.
    7. Wash pellet with 5-10 ml 0.002% Tween water 20.
    8. Centrifuge for 7 min at 3,500 rpm, discard supernatant.
    9. Resuspend spore pellet in 10 ml 0.002% Tween water 20, count spores with counting chamber (e.g. Neubauer improved) and dilute to requested spore concentration (e.g. 500,000 spores/ml)
  2. Inoculate 50 ml CM medium (Pham et al., 2004) supplemented with appropriate indole derivative (e.g. 2.5 mM TRP; 250 μM IAD or 1, 10, 100 μM IAA) with 400 μl chlamydospores solution (500,000 spores/ml) and cultivate for 7 days at 28 °C in the dark (alternatively wrap flasks with aluminium foil). Use mock inoculated flask as a negative control.
  3. Separate supernatant from mycelium using miracloth filter (check the mass of each filter before).
  4. Wash mycelium with 0.9% NaCl and let the whole miracloth filter with fungal biomass dry overnight in oven (85 °C).
  5. Measure the dry fungal biomass (= mass of miracloth filter with dried fungal biomass – mass of empty miracloth filter).
  6. Place 5 mm agar plugs from the 3 to 4 weeks old Piriformospora indica plate culture in the middle of a CM agar plate supplemented with the appropriate indole derivative (2.5 mM TRP, 250 μM IAD or 1, 10, 100 μM IAA). Use CM agar plate as control.
  7. Use ruler to measure colony diameter after 14 days of cultivation at 28 °C in the dark.
     

II.  Detection of tryptophan and indole derivatives in culture supernatant by LC-MS/MS

  1. Use a 15 μl aliquot of P. indica culture supernatant obtained from section I point 2 of the procedure.
  2. Add 1 ml of 90% methanol.
  3. Vortex briefly and dilute an aliquot 1:10 in 90% methanol into a 0.3 ml polypropylene snap ring microvial.
  4. Analyze 10 μl of the 1:10 dilution by LC-MS/MS.
  5. IAA and ILA are separated on an ICS3000 HPLC system equipped with a Phenomenex Luna 250 x 4.6 mm C18 RP-HPLC column with the following gradient
    1. 0 to 5 min                 hold at 80% buffer A, 20% buffer B
    2. 5 to 26 min               hold at 54% buffer A, 46% buffer B
    3. 26 to 27 min             ramp to 10% buffer A, 90% buffer B
    4. 32 to 34 min             ramp to 80% buffer A, 20% buffer B
    5. 32 to 34 min             ramp to 80% buffer A, 20% buffer B
    6. 34 to 45 min             equilibrate with 80% buffer A, 20% buffer B
  6. Subject the HPLC eluate to coupled electrospray ionization in the negative ionization mode and to subsequent tandem MS analysis on the QTrap 3200 mass spectrometer with the following settings:
    1. dwell time                                             75 ms
    2. declustering potential (DP)                   -22 V (IAA), -30 V (ILA)
    3. entrance potential (EP)                         -7 V (IAA), -55 V (ILA)
    4. collision energy (CE)                            -15 V (IAA), -18 V (ILA)
    5. collision energy (CE)                            -15 V (IAA), -18 V (ILA)
    6. collision cell exit potential (CXP)           0 V (IAA),  -4 V (ILA)
  7. Quantitate IAA using the m/z transitions 174/130 and 174/128.
  8. Quantitate ILA using the m/z transitions 204/128 and 204/158.
  9. Employ commercially available authentic substances as references.

Recipes

  1. Complete medium (CM medium; Pham et al., 2004)
    1. CM medium (1 L)
      50 ml 20x salt solution
      20 g glucose 
      2 g peptone
      1 g yeast extract
      1 g casamine acids
      1 ml microelements
      15 g agar
    2. 20x salt solution
      120 g NaNO3
      10.4 g KCl
      10.4 g MgSO4.7H2O
      30.4 g KH2PO4
    3. Microelements
      6 g MnCl2.4H2O
      1.5 g H3BO3
      2.65 g ZnSO4.7H2O
      750 mg KI
      2.4 mg Na2MO4.2H2O
      130 mg CuSO4.5H2O
  2. Buffer A
    0.75% acetic acid, pH 2.55 (adjust with acetic acid, if necessary)
  3. Buffer B
    acetonitrile/0.75% acetic acid, pH 2.55 (adjust with acetic acid, if necessary)

Acknowledgments

This protocol is adapted from Hilbert et al. (2012).

References

  1. Hilbert, M., Voll, L. M., Ding, Y., Hofmann, J., Sharma, M. and Zuccaro, A. (2012). Hilbert, M., Voll, L. M., Ding, Y., Hofmann, J., Sharma, M. and Zuccaro, A. (2012). Indole derivative production by the root endophyte Piriformospora indica is not required for growth promotion but for biotrophic colonization of barley roots. New Phytol 196(2): 520-534.
  2. Pham,G. H., Singh, A., Malla, R., Kumari, R., Prasad, R., Sachdev, M., Luis, P., Kaldorf, M., Peskan, T., Herrmann, S. (2004). Interaction of P. indica with other microorganisms and plants. In: Varma A, Abbott L, Werner D, Hampp R, eds. Plant Surface Microbiol  Heidelberg, Germany: Springer, 237-265. 

简介

共生根内生真菌(Pyriformospora indica)殖民广泛的植物,并且这种真菌对根细胞的定植通常与对其宿主的有益效果相关,例如生长促进和增加的生物和非生物胁迫耐受性 。 这些性状可以基于许多不同植物物种共有的一般机制和信号传导途径。 一种这样的机制可以是通过P募集植物激素途径。 indica 。 已知许多共生微生物能够在与其宿主植物相互作用期间合成和分泌植物激素。 该方案已经成功地用于通过P分析吲哚-3-乙酸(IAA)及其吲哚衍生物的色氨酸(TRP)依赖性生物合成。 蚜虫以及它们对这种真菌生长的影响(Hilbert等人,2012)。

材料和试剂

  1. 吲哚衍生物:
    TRP(Sigma-Aldrich,目录号:T0254-500g) IAD(吲哚-3-乙醛)(Sigma-Aldrich,目录号:I1000-100mg) IAA(Sigma-Aldrich,目录号:I5148-2g)
  2. Neubauer改进了计数室(Marienfeld-Superior)
  3. 无菌手术刀
  4. 无菌drigalski铲子
  5. Miracloth过滤器15cm×15cm(Merck KGaA,目录号:475855)
  6. 标尺
  7. 0.3 ml聚丙烯卡环微型管
  8. 0.9%NaCl
  9. 0.002%(v/v)吐温水20
  10. 90%甲醇(HPLC级)
  11. 乙腈(HPLC级)
  12. 乙酸(HPLC级)
  13. 衣原体溶液
  14. 微量元素(MnCl <2> 4H 2 O,H 3 BO 3,ZnSO 4,NH 4 sub 2 SO 4,Na 2 O,KI,Na 2,MO 4,SO 4,SO 4,SO 4,SO 4, > 4 5H 2 O)
  15. 葡萄糖
  16. 蛋白胨
  17. 酵母提取物
  18. 酪蛋白酸
  19. Agar
  20. 完全培养基(CM培养基)(参见配方)
  21. 缓冲区A(参见配方)
  22. 缓冲液B(参见配方)

设备

  1. 1.5 ml Eppendorf管
  2. 50ml Falcon管
  3. 培养皿(直径12mm)
  4. 孵化器(例如 B. Braun Biotech International,Certomat BS-1)
  5. Biofuge Primo R(Heraeus,Germany)(Rotor,目录号:7590)
  6. Phenomenex Luna 250×4.6mm C18R-HPLC柱(Phenomenex)
  7. Phenomenex Luna 10×4.6mm C18s RP-HPLC保护柱(Phenomenex)
  8. ICS3000 HPLC系统(Dionex)
  9. QTrap 3200三重四极杆质谱仪(Applied Biosystems SCIEX)
  10. 聚丙烯卡环微通道

程序

I.   生长测定

  1. 从3至4周龄的梨孢镰孢平板培养物中收集孢子(参见图1)。


    图1.四周龄的 p。 Indica 琼脂平板

    1. 在3-4周龄的P上倒约5ml无菌的0.002%Tween水20。 无菌条件下在室温(RT)下培养
    2. 用无菌Drigalski抹刀和/或解剖刀刮擦板并混合
    3. 通过miracloth过滤器倒入芽孢溶液,收集流经50ml Falcon管
    4. 在3,500rpm离心7分钟,弃去上清液
    5. 用5-10ml 0.002%吐温水20洗涤沉淀。
    6. 以3500rpm离心7分钟,弃去上清液
    7. 用5-10ml 0.002%吐温水20洗涤沉淀。
    8. 以3500rpm离心7分钟,弃去上清液
    9. 在10ml 0.002%Tween水中重悬孢子沉淀20,用计数室计数孢子(例如改进Neubauer)并稀释至所需孢子浓度(例如 500,000孢子/ml) br />
  2. 接种补充有适当的吲哚衍生物(例如2.5mM TRP;250μMIAD或1,10,100μMIAA)的50ml CM培养基(Phamem et al。,2004)用400μl孢子孢子溶液(500,000孢子/ml),并在28℃在黑暗中(或者用铝箔包裹的烧瓶)培养7天。使用模拟接种的烧瓶作为阴性对照
  3. 使用miracloth过滤器从菌丝体分离上清液(检查每个过滤器的质量)
  4. 用0.9%NaCl洗涤菌丝体,并使整个miracloth过滤器与真菌生物质在烘箱(85℃)中干燥过夜。
  5. 测量干燥真菌生物量(=具有干燥真菌生物质的miracloth过滤器的质量 - 空miracloth过滤器的质量)。
  6. 在补充有合适的吲哚衍生物(2.5mM TRP,250μMIAD或1μM,10μM)的CM琼脂平板的中间放置来自3至4周龄的梨孢镰孢平板培养物的5mm琼脂塞, 100μMIAA)。使用CM琼脂板作为对照。
  7. 在28℃黑暗中培养14天后,用直尺测量菌落直径。
     

II。  通过LC-MS/MS检测培养上清液中的色氨酸和吲哚衍生物

  1. 使用15μl等份的em。 腋窝培养上清液
  2. 加入1ml 90%甲醇
  3. 短暂涡旋并将90%甲醇中的1:10稀释液稀释到0.3ml聚丙烯弹性环微孔中。
  4. 通过LC-MS/MS分析10μl的1:10稀释液。
  5. 在配备有Phenomenex Luna 250×4.6mm C18s-RP-HPLC柱的ICS3000 HPLC系统上分离IAA和ILA,具有以下梯度
    1. 0至5分钟               保持在80%缓冲液A,20%缓冲液B中
    2. 5至26分钟             保持在54%缓冲液A,46%缓冲液B
    3. 26至27分钟           升至10%缓冲液A,90%缓冲液B
    4. 32至34分钟           升至80%缓冲液A,20%缓冲液B
    5. 32至34分钟           升至80%缓冲液A,20%缓冲液B
    6. 34至45分钟           用80%缓冲液A,20%缓冲液B平衡
  6. 使HPLC洗脱液在负电离模式下进行偶联的电喷雾电离,并在QTrap 3200质谱仪上进行随后的串联质谱分析,具有以下设置:
    1. 停留时间                                           75毫秒
    2. 去簇电位(DP)                 -22 V(IAA),-30 V(ILA)
    3. 入口潜力(EP)                       -7V(IAA), - 55V(ILA)
    4. 碰撞能量(CE)                           -15 V(IAA),-18 V(ILA)
    5. 碰撞能量(CE)                           -15 V(IAA),-18 V(ILA)
    6. 碰撞池出口潜力(CXP)         0V(IAA),-4V(ILA)
  7. 使用m/z跃迁174/130和174/128定量IAA
  8. 使用m/z转换204/128和204/158定量ILA。
  9. 使用商业可得的正宗物质作为参考

食谱

  1. 完全培养基(CM培养基; Pham等人,2004)
    1. CM介质(1L)
      50ml 20x盐溶液
      20克葡萄糖
      2 g蛋白胨
      1g酵母提取物
      1克酪蛋白酸
      1 ml微量元素
      15克琼脂
    2. 20x盐溶液
      120克NaNO 3
      10.4克KCl
      10.4g MgSO 4·7H 2 O·h/v 30.4g KH 2 PO 4 sub/
    3. 微元件
      6g MnCl 2·4H 2 O 2·h/v 1.5g H sub 3 BO sub 3
      2.65g ZnSO 4·7H 2 O·h/v 750 mg KI
      2.4mg Na 2 MO 4+ 2NH 2 O
      130mg CuSO 4·5H 2 O
  2. 缓冲区A
    0.75%乙酸,pH2.55(如果需要,用乙酸调节)
  3. 缓冲区B
    乙腈/0.75%乙酸,pH2.55(如果需要,用乙酸调节)

致谢

该协议改编自Hilbert等人(2012)。

参考文献

  1. Hilbert,M.,Voll,L.M.,Ding,Y.,Hofmann,J.,Sharma,M。和Zuccaro,A。(2012)。 Hilbert,M.,Voll,LM,Ding,Y.,Hofmann,J.,Sharma,M.和Zuccaro,A。(2012)。根内生真菌(Piriformospora indica)的吲哚衍生物生产不是生长促进所必需的,而是用于大麦根的生物营养定植。 New Phytol 196(2):520-534。
  2. Pham,G。 H.,Singh,A.,Malla,R.,Kumari,R.,Prasad,R.,Sachdev,M.,Luis,P.,Kaldorf,M.,Peskan,T.,Herrmann, 。 p的交互。 indic与其他微生物和植物。 In:Varma A,Abbott L,Werner D,Hampp R,eds。 植物表面微生物 Heidelberg,Germany:Springer,237-265。 
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hilbert, M., Voll, L. M., Hofmann, J. and Zuccaro, A. (2013). Growth Assay and Detection of TRP and Indole Derivatives in Piriformospora indica Culture Supernatant by LC-MS/MS. Bio-protocol 3(12): e800. DOI: 10.21769/BioProtoc.800.
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