European Journal of Plant Pathology

, Volume 143, Issue 4, pp 823–831 | Cite as

Purification of a novel hydrophobin PN1 involved in antibacterial activity from an edible mushroom Pleurotus nebrodensis

  • Rui Ying Zhang
  • Dan Dan Hu
  • Jin Gang Gu
  • Jin Xia Zhang
  • Paul H. Goodwin
  • Qing Xiu Hu
Article

Abstract

Due to their hydrophobic characteristics, hydrophobins function in a broad range of processes in the growth and development of filamentous fungi. In this study, a novel hydrophobin, PN1, was purified from the fruiting bodies of Pleurotus nebrodensis by first removing other proteins from mycelial cell walls with hot SDS solution. The molecular mass of PN1 was approximately 10.7 kDa. The N-terminal AA sequence was QESPVNQCNTGSIEECKTVQ, which did not match that of any other mushroom hydrophobin described thus far. Levels of hydrophobin PN1 protein in the tissue of fruiting bodies was decreased by fruiting body development under low temperatures. Low levels of hydrophobin PN1 was correlated to the formation of water-soaked blotches on fruiting body tissues that appeared dark and translucent, and were usually sunken. In addition, hydrophobin PN1 levels were correlated with protecting fruiting bodies against surface bacterial colonization and multiplication. Low levels of hydrophobin PN1 resulted in water-soaked blotches that had high levels of surface bacteria belonging to multiple species when compared with nonsymptomatic tissue of fruiting bodies with higher hydrophobin PN1. These results show a direct link between mushroom hydrophobins and resistance of mushrooms to bacterial attack.

Keywords

Pleurotus nebrodensis Hydrophobins Edible mushroom Resistance Water-soaked blotch Bacteria 

References

  1. Aimanianda, V., Bayry, J., Bozza, S., Kniemeyer, O., Perruccio, K., Elluru, S. R., et al. (2009). Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature, 460(7259), 1117–1121.CrossRefPubMedGoogle Scholar
  2. Ando, A., Harada, A., Miura, K., & Tamai, Y. (2001). A gene encoding a hydrophobin, fvh1, is specifically expressed after the induction of fruiting in the edible mushroom Flammulina velutipes. Current Genetics, 39(3), 190–197.CrossRefPubMedGoogle Scholar
  3. Asgeirsddttir, S. A., De Vries, O. M. H., & Wessels, J. G. H. (1998). Identification of three differentially expressed hydrophobins in Pleurotus ostreatus (oyster mushroom). Microbiology, 144(11), 2961–2969.CrossRefGoogle Scholar
  4. Asgeirsdottir, S. A., Halsall, J. R., & Casselton, L. A. (1997). Expression of two closely linked hydrophobin genes of Coprinus cinereus is monokaryon-specific and down-regulated by the oid-1 mutation. Fungal Genetics and Biology, 22(1), 54–63.CrossRefPubMedGoogle Scholar
  5. Beckerman, J. L., & Ebbole, D. J. (1996). MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition. Molecular Plant-Microbe Interactions, 9(6), 450–456.CrossRefPubMedGoogle Scholar
  6. Bell-Pedersen, D., Dunlap, J. C., & Loros, J. J. (1996). Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene. Molecular and Cellular Biology, 16(2), 513–521.PubMedCentralCrossRefPubMedGoogle Scholar
  7. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254.CrossRefPubMedGoogle Scholar
  8. Butler, M. J., Gardiner, R. B., & Day, A. W. (2005). Degradation of melanin or inhibition of its synthesis: are these a significant approach as a biological control of phytopathogenic fungi? Biological Control, 32(2), 326–336.CrossRefGoogle Scholar
  9. Chen, Z. C. (1996). The domestication and cultivation of Pleurotus ferulae in China. Acta Edulis Fungi, 3(4), 11–14.Google Scholar
  10. Chun, J., Lee, J.-H., Jung, Y., Kim, M., Kim, S., Kim, B. K., et al. (2007). EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. International Journal of Systematic and Evolutionary Microbiology, 57(10), 2259–2261.CrossRefPubMedGoogle Scholar
  11. De Groot, P. W., Schaap, P. J., Sonnenberg, A. S., Visser, J., & Van Griensven, L. J. (1996). The Agaricus bisporus hypA gene encodes a hydrophobin and specifically accumulates in peel tissue of mushroom caps during fruit body development. Journal of Molecular Biology, 257(5), 1008–1018.CrossRefPubMedGoogle Scholar
  12. De Groot, P. W., Roeven, R. T., Van Griensven, L. J., Visser, J., & Schaap, P. J. (1999). Different temporal and spatial expression of two hydrophobin-encoding genes of the edible mushroom Agaricus bisporus. Microbiology, 145(5), 1105–1113.CrossRefPubMedGoogle Scholar
  13. Degani, O., Lev, S., & Ronen, M. (2013). Hydrophobin gene expression in the maize pathogen Cochliobolus heterostrophus. Physiological and Molecular Plant Pathology, 83, 25–34.CrossRefGoogle Scholar
  14. Di Pietro, A., García-Maceira, F. I., Méglecz, E., & Roncero, M. I. G. (2001). A MAP kinase of the vascular wilt fungus Fusarium oxysporum is essential for root penetration and pathogenesis. Molecular Microbiology, 39(5), 1140–1152.CrossRefPubMedGoogle Scholar
  15. Duffy, B., Schouten, A., & Raaijmakers, J. M. (2003). Pathogen self-defense: mechanisms to counteract microbial antagonism. Annual Review of Phytopathology, 41(1), 501–538.CrossRefPubMedGoogle Scholar
  16. Jia, S. M., & Qin, M. (2006). Domestication and cultivation of Pleurotus nebrodensis in China. Edible Fungi of China, 25(3), 3–7.Google Scholar
  17. Kawai, G., Babasaki, K., & Neda, H. (2008). Taxonomic position of a Chinese Pleurotus “Bai-Ling-Gu”: it belongs to Pleurotus eryngii (DC.: Fr.) Quél. and evolved independently in China. Mycoscience, 49(1), 75–87.CrossRefGoogle Scholar
  18. Kim, S., Ahn, I. P., Rho, H. S., & Lee, Y. H. (2005). MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization. Molecular Microbiology, 57(5), 1224–1237.CrossRefPubMedGoogle Scholar
  19. Klimes, A., & Dobinson, K. F. (2006). A hydrophobin gene, VDH1, is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae. Fungal Genetics and Biology, 43(4), 283–294.CrossRefPubMedGoogle Scholar
  20. Kobayashi, D. Y., & Crouch, J. A. (2009). Bacterial/fungal interactions: from pathogens to mutualistic endosymbionts. Annual Review of Phytopathology, 47(1), 63–82.CrossRefPubMedGoogle Scholar
  21. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.CrossRefPubMedGoogle Scholar
  22. Lau, G., & Hamer, J. E. (1996). Regulatory genes controlling MPG1 expression and pathogenicity in the rice blast fungus Magnaporthe grisea. The Plant Cell Online, 8(5), 771–781.CrossRefGoogle Scholar
  23. Lee, Y. H., & Dean, R. A. (1993). cAMP regulates infection structure formation in the plant pathogenic fungus Magnaporthe grisea. The Plant Cell Online, 5(6), 693–700.CrossRefGoogle Scholar
  24. Lugones, L. G., Bosscher, J. S., Scholtmeyer, K., De Vries, O. M., & Wessels, J. G. (1996). An abundant hydrophobin (ABH1) forms hydrophobic rodlet layers in Agaricus bisporus fruiting bodies. Microbiology, 142(5), 1321–1329.CrossRefPubMedGoogle Scholar
  25. Lugones, L. G., Wosten, H. A., & Wessels, J. G. (1998). A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom). Microbiology, 144(8), 2345–2353.CrossRefPubMedGoogle Scholar
  26. Lugones, L. G., Wösten, H. A. B., Birkenkamp, K. U., Sjollema, K. A., Zagers, J., & Wessels, J. G. H. (1999). Hydrophobins line air channels in fruiting bodies of Schizophyllum commune and Agaricus bisporus. Mycological Research, 103(5), 635–640.CrossRefGoogle Scholar
  27. Lugones, L. G., De Jong, J. F., De Vries, O. M., Jalving, R., Dijksterhuis, J., & Wosten, H. A. (2004). The SC15 protein of Schizophyllum commune mediates formation of aerial hyphae and attachment in the absence of the SC3 hydrophobin. Molecular Microbiology, 53(2), 707–716.CrossRefPubMedGoogle Scholar
  28. Ma, A., Shan, L., Wang, N., Zheng, L., Chen, L., & Xie, B. (2007). Characterization of a Pleurotus ostreatus fruiting body-specific hydrophobin gene, Po.hyd. Journal of Basic Microbiology, 47(4), 317–324.CrossRefPubMedGoogle Scholar
  29. Ma, A., Shan, L., Wang, H., Du, Z., & Xie, B. (2008). Partial characterization of a hydrophobin protein Po.HYD1 purified from the oyster mushroom. Pleurotus ostreatus World Journal of Microbiology and Biotechnology, 24(4), 501–507.CrossRefGoogle Scholar
  30. Minenko, E., Vogel, R. F., & Niessen, L. (2014). Significance of the class II hydrophobin FgHyd5p for the life cycle of Fusarium graminearum. Fungal Biology, 118(4), 385–393.CrossRefPubMedGoogle Scholar
  31. Mou, C. J., Cao, Y. Q., & Ma, J. L. (1987). A new variety of Pleurotus eryngii and its cultural charcters. Acta Mycologica Sinica, 6(3), 153–156.Google Scholar
  32. Neuhoff, V., Arold, N., Taube, D., & Ehrhardt, W. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis, 9(6), 255–262.CrossRefPubMedGoogle Scholar
  33. Nielsen, P. S., Clark, A. J., Oliver, R. P., Huber, M., & Spanu, P. D. (2001). HCf-6, a novel class II hydrophobin from Cladosporium fulvum. Microbiological Research, 156(1), 59–63.CrossRefPubMedGoogle Scholar
  34. Penas, M. M., Asgeirsdottir, S. A., Lasa, I., Culianez-Macia, F. A., Pisabarro, A. G., Wessels, J. G., et al. (1998). Identification, characterization, and In situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus. Applied and Environmental Microbiology, 64(10), 4028–4034.PubMedCentralPubMedGoogle Scholar
  35. Penas, M. M., Rust, B., Larraya, L. M., Ramirez, L., & Pisabarro, A. G. (2002). Differentially regulated, vegetative-mycelium-specific hydrophobins of the edible basidiomycete Pleurotus ostreatus. Applied and Environmental Microbiology, 68(8), 3891–3898.PubMedCentralCrossRefPubMedGoogle Scholar
  36. Penas, M. M., Aranguren, J., Ramirez, L., & Pisabarro, A. G. (2004). Structure of gene coding for the fruit body-specific hydrophobin Fbh1 of the edible basidiomycete Pleurotus ostreatus. Mycologia, 96(1), 75–82.CrossRefPubMedGoogle Scholar
  37. Soanes, D. M., Kershaw, M. J., Cooley, R. N., & Talbot, N. J. (2002). Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea. Molecular Plant-Microbe Interactions, 15(12), 1253–1267.CrossRefPubMedGoogle Scholar
  38. Talbot, N. J., Kershaw, M. J., Wakley, G. E., De Vries, O., Wessels, J., & Hamer, J. E. (1996). MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. The Plant Cell, 8(6), 985–999.PubMedCentralCrossRefPubMedGoogle Scholar
  39. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25(24), 4876–4882.PubMedCentralCrossRefPubMedGoogle Scholar
  40. Tolaas, A. G. (1915). A bacterial disease of cultivated mushroom. Phytopathology, 5, 51–54.Google Scholar
  41. Van Wetter, M.-A., Wösten, H. A. B., & Wessels, J. G. H. (2000). SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune. Molecular Microbiology, 36(1), 201–210.CrossRefPubMedGoogle Scholar
  42. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2), 697–703.PubMedCentralPubMedGoogle Scholar
  43. Wessels, J. G. H. (1994). Developmental regulation of fungal cell wall formation. Annual Review of Phytopathology, 32(1), 413–437.CrossRefGoogle Scholar
  44. Wessels, J. G. H., De Vries, O. M. H., Ásgeirsdóttir, S. A., & Schuren, F. H. J. (1991a). Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in Schizophyllum. The Plant Cell, 3(8), 793–799.PubMedCentralCrossRefPubMedGoogle Scholar
  45. Wessels, J. G. H., De Vries, O. M. H., Asgeirsdottir, S. A., & Springer, J. (1991b). The thn mutation of Schizophyllum commune, which suppresses formation of aerial hyphae, affects expression of the Sc3 hydrophobin gene. Journal of General Microbiology, 137(10), 2439–2445.CrossRefPubMedGoogle Scholar
  46. Wösten, H. A. B. (2001). Hydrophobins: multipurpose proteins. Annual Review of Microbiology, 55(1), 625–646.CrossRefPubMedGoogle Scholar
  47. Yamada, M., Sakuraba, S., Shibata, K., Inatomi, S., Okazaki, M., & Shimosaka, M. (2005). Cloning and characterization of a gene coding for a hydrophobin, Fv-hyd1, specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes. Applied Microbiology and Biotechnology, 67(2), 240–246.CrossRefPubMedGoogle Scholar
  48. Yu, L., Zhang, B., Szilvay, G. R., Sun, R., Janis, J., Wang, Z., et al. (2008). Protein HGFI from the edible mushroom Grifola frondosa is a novel 8 kDa class I hydrophobin that forms rodlets in compressed monolayers. Microbiology, 154(6), 1677–1685.CrossRefPubMedGoogle Scholar
  49. Zhang, R. Y., Zuo, X. M., & Jiang, R. B. (2007). Isolation and identification of the pathogenic bacteria causing brown blotch disease of oyster mushroom. Edible Fungi of China, 26(5), 58–60.Google Scholar
  50. Zhang, R. Y., Hu, D. D., Gu, J. G., Zuo, X. M., & Hu, Q. X. (2010). Analysis of the cultivation experience and problems for Pleurotus nebrodensis. Edible Fungi of China, 29(2), 63–65.Google Scholar
  51. Zhang, R. Y., Hu, D. D., Zuo, X. M., Gu, J. G., & Hu, Q. X. (2012). Preliminary study on the wet blotch disease of Pleurotus nebrodensis. Acta Edulis Fungi, 19(2), 106–110.Google Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2015

Authors and Affiliations

  • Rui Ying Zhang
    • 1
  • Dan Dan Hu
    • 2
  • Jin Gang Gu
    • 1
  • Jin Xia Zhang
    • 1
  • Paul H. Goodwin
    • 3
  • Qing Xiu Hu
    • 1
  1. 1.Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, and Institute of Agricultural Resources and Regional PlanningChinese Academy of Agricultural SciencesBeijingChina
  2. 2.Beijing Academy of Science and TechnologyBeijingChina
  3. 3.School of Environmental SciencesUniversity of GuelphGuelphCanada

Personalised recommendations