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In vitro Cell Wall Stress Assay for Fusarium oxysporum

尖孢镰刀菌细胞壁应激的体外实验

Elena Pérez-Nadales Elena Pérez-Nadales
AP Antonio Di Pietro

发布: 2016年09月05日第6卷第17期 DOI: 10.21769/BioProtoc.1915 浏览次数: 11106

评审: Zhaohui LiuEmmanuel ZavalzaAnonymous reviewer(s)

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Abstract

In this protocol we describe a cell wall stress assay for the fungal pathogen F. oxysporum, based on exposure to the two anionic dyes Calcofluor White (CFW) and Congo Red (CR). Both compounds have been used to exert stress upon the fungal cell wall in vitro (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Leach et al., 2012; Heilmann et al., 2013; Garcia et al., 2015). CFW perturbs chitin assembly, whereas CR interferes with β-glucan synthesis, resulting in cell wall-weakening and activation of the cell wall stress response (Ram and Klis, 2006; Kopecka and Gabriel, 1992; Roncero and Duran, 1985). Presumably, the signaling pathways and cell wall changes associated with this response reflect cell wall homeostasis during normal growth as well as cell wall remodeling events in response to stresses encountered during the fungus-host interactions. The conditions for preparation of CFW and CR culture medium specified in this protocol are based on the paper by Ram and Klis entitled “Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red”, published in Nature protocols (Ram and Klis, 2006). This paper established the optimum conditions for preparation of CFW and CR stock solutions and suggested maintaining the culture medium at a constant pH to avoid acidification, protonation and precipitation of these dyes. This cell wall stress assay has been widely used in our group for the characterization of F. oxysporum mutants in mitogen activated protein kinase (MAPK) signaling pathway genes involved in cell wall integrity (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Turra et al., 2014).

Materials and Reagents

  1. Round (90 mm diameter) or square (120 x 120 mm) Petri dishes
  2. Autoclavable, filtration material [e.g., Miracloth (Merck Millipore, catalog number: 475855 )]
  3. Calcofluor white fluorescent brightener (Sigma-Aldrich, catalog number: F-3543 )
  4. Congo red (Sigma-Aldrich, catalog number: 860956 )
  5. Distilled water
  6. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
  7. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  8. Potatoes
  9. Cloth strainer
  10. Potato dextrose agar (PDA) (Scharlab, catalog number: 01-483-500 )
  11. Yeast extract (Merck Millipore, catalog number: 1119261000 )
  12. Peptone (Merck Millipore, catalog number: 1119311000 )
  13. Glucose (VWR, Normapur®, catalog number: 50-99-7 )
  14. Agar (BD, BactoTM, catalog number: 214010 )
  15. MgSO4·7H2O (Merck Millipore, catalog number: 1058865000 )
  16. KH2PO4 (Merck Millipore, catalog number: 1048731000 )
  17. KCl (Merck Millipore, catalog number: 1049330500 )
  18. NaNO3 (Merck Millipore, catalog number: 1065371000 )
  19. Sucrose (Merck Millipore, catalog number: 1076875000 )
  20. Agar (Thermo Fisher Scientific, OxoidTM, catalog number: LP0011 )
  21. 2-(N-morpholino) ethanesulfonic acid (MES), monohydrate (Sigma-Aldrich, catalog number: 69892 )
  22. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  23. Potato dextrose broth (PDB) (see Recipes)
  24. CFW and CR stock solutions (see Recipes)
    1. CFW stock solutions (20 mg/ml)
    2. CR stock solutions (50 mg/ml)
  25. Fungal growth agar medium (see Recipes)
    1. YPD agar medium
    2. PDA agar medium
    3. Puhalla’s minimal agar medium (MM)

Note: CFW and CR are hazardous and potentially carcinogenic so caution must be taken to avoid skin contact or inhalation of these compounds.

Equipment

  1. Protection gloves and coat
  2. Flasks and magnetic stirrer for preparation of solutions
  3. Neubauer chamber for cell counting
  4. Microwave
  5. Autoclave
  6. Fungal growth incubator
  7. Water bath
  8. Laminar flow hood
  9. Heat plate and cooking pot to boil potatoes
  10. pH meter

Note: No other special equipment is required.

Software

  1. SPSS 15.0 for Windows® (LEAD Technologies Inc.)

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Pérez-Nadales, E. and Di Pietro, A. (2016). In vitro Cell Wall Stress Assay for Fusarium oxysporum. Bio-protocol 6(17): e1915. DOI: 10.21769/BioProtoc.1915.

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分类

微生物学 > 抗微生物试验 > 抗真菌试验

微生物学 > 微生物-宿主相互作用 > 真菌

生物化学 > 其它化合物 > 抗菌剂

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