Chitin Extraction and Content Measurement in Magnaporthe oryzae

Materials and Reagents

1. Microtube
   
2. Microtiter plates (Corning, Costar^®, catalog number: 42592 )
   
3. Miracloth (EMD Millipore, catalog number: 475855 )
   
4. Cells of Magnaporthe oryzae
   
5. Potassium hydroxide (KOH) (Sangon Biotech, catalog number: A610441 )
   
6. 10x phosphate buffered saline (PBS, 1.35 M NaCl, 47 mM KCl, 100 mM Na₂HPO_4, 20 mM NaH₂PO_4, pH = 7.4) (Beyotime, catalog number: ST476 )
   
7. Streptomyces plicatus chitinase (Sigma-Aldrich, catalog number: C6137 )
   
8. Sodium borate (Sangon Biotech, catalog number: A100390 )
   
9. GlcNAc (Sigma-Aldrich, catalog number: PHR1432-1G )
   
10. Sodium hydrogen (Na₂HPO₄) (Sangon Biotech, catalog number: A501727 )
    
11. Citric acid (Sangon Biotech, catalog number: A501702 )
    
12. p-dimethylaminobenzaldehyde (Sigma-Aldrich, catalog number: D2004 )
    
13. Hydrochloric acid (HCl)
    
14. Acetic acid (Sangon Biotech, catalog number: A501931 )
    
15. Biotin
    
16. Pyridoxin
    
17. Thiamine
    
18. Riboflavin
    
19. p-aminobenzoic acid
    
20. nicotinic acid
    
21. ZnSO₄·7H₂O
    
22. H₃BO₃
    
23. MnCl₂·4H₂O
    
24. FeSO₄·7H₂O
    
25. CoCl₂·6H₂O
    
26. CuSO₄·5H₂O
    
27. Na₂MnO₄·2H₂O
    
28. Na₄EDTA
    
29. NaNO₃
    
30. KCl
    
31. MgSO₄·7H₂O
    
32. KH₂PO₄
    
33. D-glucose
    
34. Peptone
    
35. Yeast extract
    
36. Casamino acid
    
37. Agar
    
38. McIlvaine’s buffer (see Recipes)
    
39. Ehrlich’s solution (see Recipes)
    
40. Vitamin solution (see Recipes)
    
41. Trace elements (see Recipes)
    
42. 20x nitrate salts (see Recipes)
    
43. CM medium (see Recipes)
    

Equipment

1. Freeze dryer (Marin Christ German)
   
2. Vortex mixer
   
3. Water bath (SHEL Lab, model: W6M-2 )
   
4. Centrifuge (Eppendorf centrifuge) (Eppendorf, model: 5418 )
   
5. Incubator shaker (Crystal Technology & Industries, model: IS-RSD3 )
   
6. pH/ATU electrode (Sartorius, German)
   
7. Microplate reader (Molecular Devices, model: VersaMax ELISA )
   
8. PCR instrument (Takara Bio, model: TP600 )
   
9. Eppendorf micropipette (1,000 μl, 100 μl, 10 μl)
   
10. Dark glass bottle
    

Software

1. SPSS 2.0 (Chicago, IL, USA)

Procedure

1. All of the strains are cultured on solid CM medium for 7 days at 28 °C. The agar culture is cut into 1 x 1 mm squares and the squares are cultured in liquid CM for another 2 days.
   
2. Filtered through one layer of Miracloth to collect mycelium from liquid CM. Then the mycelium are quickly lyophilized by a freeze dryer for 24 h. 5 mg mycelium is mixed with 1 ml 6% KOH in each 2 ml microtube using a vortex mixer and then incubated in a water bath at 80 °C for 90 min.
   
3. Samples are centrifuged at 16,000 x g for 10 min, and the suspension is discarded.
   
4. Each pellet is washed with 1 ml 1x PBS (which is diluted from 10x PBS) for three times and then centrifuged at 16,000 x g for 5 min to discard the suspension.
   
5. Each pellet is resuspended with 0.5 ml of McIlvaine’s buffer (see Recipes) (Baker et al., 2007). 100 μl chitinase is added into each sample. And then samples are incubated at 37 °C for 16 h in the dark, at 220 rpm in an incubator shaker.
   
6. Chitinase-treated samples are mixed with equal volume of 0.27 M sodium borate (pH = 9.0) and incubated at 100 °C for 10 min in a PCR instrument.
   
7. After being cooled down to room temperature, 200 μl of each sample is added to 1 ml Ehrlich’s solution (see Recipes) and then incubate at 37 °C for 30 min in an incubator shaker.
   
8. 100 μl of each sample is transferred into a well of a microtiter plate with low-evaporation and the absorbance is measured at 585 nm by a microplate reader. Standard curves are prepared from stocks of 0.1 to 2.0 mM (0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM and 2.0 mM) GlcNAc.
   

Data analysis

Standard curves are prepared from stocks of 0.1 to 2.0 mM GlcNAc. Each result is presented at least three replicated measurements. The significance of differences between treatments is statistically evaluated using SDs and one-way analysis of variance (ANOVA) in SPSS 2.0 (Chicago, IL, USA). Data for two specific different treatments are compared statistically using ANOVA, followed by an F-test if the ANOVA result is significant at P < 0.05 or P < 0.01.

Notes

1. Both McIlvaine’s buffer and Ehrlich’s solution need to be prepared just before use.
   
2. In step 1, 25 squares are enough and the volume of the liquid CM is 70 ml.
   
3. In step 2, samples are incubated in a water bath at 80 °C for 90 min. Samples need to be vortexed every 15 min.
   
4. In step 6, after being extracted at 100 °C for 10 min, all the samples should be cooled on ice to room temperature immediately.
   
5. In step 6, after being extracted at 100 °C for 10 min in the PCR instrument, the solution is clear and transparent
   
6. In step 7, the Ehrlich’s solution is clear and does not need to be centrifuged.
   
7. If the samples are difficult to suspend, break the pellet by an injector or a micropipette.
   

Recipes

1. McIlvaine’s buffer
   0.2 M Na₂HPO₄
   0.1 M citric acid (pH = 6.0)
   
2. Ehrlich’s solution
   10 g p-dimethylaminobenzaldehyde in 12.5 ml concentrated HCl (37%) and 87.5 ml glacial acetic acid
   
3. Vitamin solution (100 ml)
   0.01 g Biotin
   0.01 g Pyridoxin
   0.01 g Thiamine
   0.01 g Riboflavin
   0.01 g p-aminobenzoic acid
   0.01 g nicotinic acid
   Add ddH₂O to 100 ml and store in a dark glass bottle at 4 °C
   
4. Trace elements (100 ml)
   2.2 g ZnSO₄·7H₂O
   1.1 g H₃BO₃
   0.5 g MnCl₂·4H₂O
   0.5 g FeSO₄·7H₂O
   0.17 g CoCl₂·6H₂O
   0.16 g CuSO₄·5H₂O
   0.15 g Na₂MnO₄·2H₂O
   5 g Na₄EDTA
   Add ddH₂O to 100 ml and adjust the pH to 5.8, store at 4 °C
   
5. 20x nitrate salts (1 L)
   120 g NaNO₃
   10.4 g KCl
   10.4 g MgSO₄·7H₂O
   30.4 g KH₂PO₄
   Add ddH₂O to 1 L and store at 4 °C
   
6. CM medium
   10 g D-glucose
   2 g peptone
   1 g yeast extract
   1 g casamino acid
   1 ml vitamin solution
   1 ml trace elements
   50 ml 20x nitrate salts
   Add ddH₂O to 1 L
   For solid media add 15 g agar and autoclave at 121 °C for 20 min
   

Acknowledgments

This research was supported by the key program of Natural Science Foundation of China (Grant No. 31530063, ZZ), National Science Foundation for Distinguished Young Scholars of China (Grant No. 31325022 to ZZ), Natural Science Foundation of China (Grant No. 31271998, ZZ), and the especially appointed professorship (Jiangsu, China).

References

1. Baker, L. G., Specht, C. A., Donlin, M. J. and Lodge, J. K. (2007). Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans. Eukaryot Cell 6(5): 855-867.
   
2. Bulik, D. A., Olczak, M., Lucero, H. A., Osmond, B. C., Robbins, P. W. and Specht, C. A. (2003). Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress. Eukaryot Cell 2(5): 886-900.
   
3. Leloir, L. F. and Cardini, C. E. (1953). The biosynthesis of glucosamine. Biochim Biophys Acta 12(1-2): 15-22.
   
4. Song, W., Dou, X., Qi, Z., Wang, Q., Zhang, X., Zhang, H., Guo, M., Dong, S., Zhang, Z., Wang, P. and Zheng, X. (2010). R-SNARE homolog MoSec22 is required for conidiogenesis, cell wall integrity, and pathogenesis of Magnaporthe oryzae. PLoS One 5(10): e13193.