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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）

MH Magdalena Hilbert
LV Lars M. Voll
JH Jörg Hofmann
AZ Alga Zuccaro

发布: 2013年06月20日第3卷第12期 DOI: 10.21769/BioProtoc.800 浏览次数: 13316

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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

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

植物科学 > 植物生理学 > 胞内共生

微生物学 > 微生物-宿主相互作用 > 真菌

生物化学 > 其它化合物 > 植物激素 > 乙烯

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