Chitin Extraction and Content Measurement in Magnaporthe oryzae

Abstract

Chitin is a linear polysaccharide composed of β (1→4)-linked N-acetylglucosamine (GlcNAc) residues. In fungi, chitin is an important component of the cell wall. Here, we provide a protocol to measure the chitin content of fungal cells using Magnaporthe oryzae as an example.

Background

Chitin is an important component of the cell wall in fungal pathogens and is well known as a pathogen associated molecule pattern (PAMP). Determining the chitin content of a fungal species is important for studying fungal biology and host-pathogen interactions. The Morgan-Elson method based on colorimetric approach has been adapted in yeast to measure cellular chitin levels (Leloir and Cardini, 1953; Bulik et al., 2003 ; Baker et al., 2007 ). However, there is no specific protocol established for Magnaporthe oryzae, which is the causal agent of rice blast, the most important fungal disease in the world. Here, we describe a reliable and simple protocol which was modified from the Morgan-Elson method to test the chitin content of M. oryzae ( Song et al., 2010 ).

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 Na₂HPO₄, 20 mM NaH₂PO₄, 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 is 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 × 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 × 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