Growth Assay and Detection of TRP and Indole Derivatives in Piriformospora indica Culture Supernatant by LC-MS/MS

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

1. 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.
       
      
2. 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.