Welcome guest, Sign in

Home

X
Loading

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Chitin Extraction and Content Measurement in Magnaporthe oryzae

Microbiology > Microbial biochemistry > Carbohydrate
Authors: Xinyu Liu
Xinyu LiuAffiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
Bio-protocol author page: a4123
 and Zhengguang Zhang
Zhengguang ZhangAffiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
For correspondence: zhgzhang@njau.edu.cn
Bio-protocol author page: a4208
Vol 7, Iss 5, 3/5/2017, 959 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2164

[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.

Keywords: Chitin, Cell wall, GlcNAc, Pathogenicity, Magnaporthe oryzae

[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 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.


How to cite: Liu, X. and Zhang, Z. (2017). Chitin Extraction and Content Measurement in Magnaporthe oryzae. Bio-protocol 7(5): e2164. DOI: 10.21769/BioProtoc.2164; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Ask the Authors:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
How to cite
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook