• Nuria EscuderoEmail author
  • Sonia Gómez-Vidal
  • Luis V. Lopez-Llorca
Part of the Sustainability in Plant and Crop Protection book series (SUPP)


Fungal proteomics research is growing as a result of the large number of fungal sequenced genomes of well annotated proteins available today. The proteome of any organism is dynamic as proteins differ depending on environmental conditions, unlike genomes which are practically constant for all the cells of an organism. In this chapter we have reviewed the ‘state-of-the-art’ of fungal proteomics, including sample preparation, protein separation and identification. We have given examples of proteomics of entomopathogenic and nematophagous fungi. We have also focused our attention on the proteomic study of Pochonia chlamydosporia carried out to date. In this study, the fungus was grown in chitin or chitosan as the main carbon and nitrogen nutrient sources, and the secretome of the fungus in both conditions analyzed. Proteins were concentrated using TCA/acetone. Two-dimensional, sodium dodecyl sulphate polyacrylamide gel electrophoresis and differential gel electrophoresis separated proteins for size and isolectric point. Some of the proteins overexpressed with chitosan that were identified using MALDI/TOF-TOF and LC-MS, were related with carbohydrate or protein degradation. The recently available complete Pochonia chlamydosporia genome sequence could help with protein identification of fungal secretomes under various conditions.



This research was funded by the Spanish Ministry of Economy and Competitiveness Grant AGL 2015-66833.


  1. Abdallah, C., Dumas-Gaudot, E., Renaut, J. et al. (2012). Gel-based and gel-free quantitative proteomics approaches at a glance. International Journal of Plant Genomics. ID 494572 doi: 10.1155/2012/494572.
  2. Adav, S. S., & Sze, S. K. (2013). Fungal secretome for biorefinery: Recent advances in proteomic technology. Mass Spectrometry Letters, 4, 1–9.CrossRefGoogle Scholar
  3. Baggerman, G., Vierstraete, E., De Loof, A., et al. (2005). Gel based versus gel-free proteomics: A review. Combinatorial Chemistry & High Throughput Screening, 8, 669–677.CrossRefGoogle Scholar
  4. Barros, B. H., da Silva, S. H., dos ReisMarques, E. R., et al. (2010). A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum. Fungal Biology, 114, 572–579.CrossRefPubMedGoogle Scholar
  5. Bhadauria, V., Zhao, W. S., Wang, L. X., et al. (2007). Advances in fungal proteomics. Microbiological Research, 162, 193–200.CrossRefPubMedGoogle Scholar
  6. Bianco, L., & Perrotta, G. (2015). Methodologies and perspectives of proteomics applied to filamentous fungi: From sample preparation to secretome analysis. International Journal of Molecular Sciences, 16, 5803–5829.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bini, L., Calvete, J. J., Hochstrasser, D., et al. (2014). The magic of words. Journal of Proteomics, 107, 1–4.CrossRefPubMedGoogle Scholar
  8. Bonants, P. J. M., Fitters, P. F. L., Thijs, H., et al. (1995). A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs. Microbiology, 141, 775–784.CrossRefPubMedGoogle Scholar
  9. Borrebaeck, C. A. K., Mattiason, B., & Nordbring-Hertz, B. (1984). Isolation and partial characterization of a carbohydrate-binding protein from a nematode-trapping fungus. Journal of Bacteriology, 159, 53–56.PubMedPubMedCentralGoogle Scholar
  10. Bruneau, J.-M., Magnin, T., Tagat, E., Legrand, R., Bernard, M., Diaquin, M., Fudali, C., & Latgé, J.-P. (2001). Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis. Electrophoresis, 22, 2812–2823.CrossRefPubMedGoogle Scholar
  11. Casas-Flores, S., & Herrera-Estrella, A. (2007). Antagonism of plant parasitic nematodes by fungi. In C. P. Kubicek & I. S. Druzhinina (Eds.), Environmental and microbial relationships, 2nd edn, The Mycota IV (pp. 147–157). Heidelberg: Springer.Google Scholar
  12. Damerval, C., De Vienne, D., Zivy, M., et al. (1986). Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis, 7, 52–54.CrossRefGoogle Scholar
  13. De Oliveira, J. M. P. F., & de Graaff, L. H. (2011). Proteomics of industrial fungi: Trends and insights for biotechnology. Applied Microbiology and Biotechnology, 89, 225–237.CrossRefPubMedGoogle Scholar
  14. Doyle, S. (2011). Fungal proteomics: From identification to function. FEMS Microbiology Letters, 321, 1–9.CrossRefPubMedGoogle Scholar
  15. Engelmann, I., & Pujo, N. (2010). Innate immunity in C. elegans. In K. Söderhäll (Ed.), Invertebrate Immunity (pp. 105–121). Heidelberg: Landes Bioscience and Springer Science+Business Media.CrossRefGoogle Scholar
  16. Escudero, N., Ferreira, S. R., Lopez-Moya, F., et al. (2016). Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia. Fungal Biology. doi: 10.1016/j.funbio.2015.12.005.
  17. Esteves, I., Peteira, B., Powers, S., et al. (2009). Effects of osmotic and matric potential on radial growth and accumulation of endogenous reserves in three isolates of Pochonia chlamydosporia. Biocontrol Science and Technology, 19, 185–199.CrossRefGoogle Scholar
  18. Giraldo, M. C., & Valent, B. (2013). Filamentous plant pathogen effectors in action. Nature Reviews. Microbiology, 11, 800–814.CrossRefPubMedGoogle Scholar
  19. Girard, V., Dieryckx, C., Job, C., et al. (2013). Secretomes: The fungal strike force. Proteomics, 13, 597–608.CrossRefPubMedGoogle Scholar
  20. Gómez-Vidal, S., Lopez-Llorca, L. V., Jansson, H.-B., et al. (2006). Endophytic colonization of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi. Micron, 37, 624–632.CrossRefPubMedGoogle Scholar
  21. Gómez-Vidal, S., Tena, M., Lopez-Llorca, L. V., et al. (2008). Protein extraction from Phoenix dactylifera L. leaves, a recalcitrant material, for two-dimensional electrophoresis. Electrophoresis, 29, 448–456.CrossRefPubMedGoogle Scholar
  22. Gómez-Vidal, S., Salinas, J., Tena, M., et al. (2009). Proteomic analysis of date palm (Phoenix dactylifera L.) responses to endophytic colonization by entomopathogenic fungi. Electrophoresis, 30, 2996–3005.CrossRefPubMedGoogle Scholar
  23. Görg, A., & Weiss, W. (2004). Protein profile comparisons of microorganism, cells and tissues using 2D gels. In D. W. Speicher (Ed.), Proteome analysis. Interpreting the genome (pp. 20–74). Amsterdam: Elsevier BV.Google Scholar
  24. Grinyer, J., McKay, M., Herbert, B., et al. (2004). Fungal proteomics: Mapping the mitochondrial proteins of a Trichoderma harzianum strain applied for biological control. Current Genetics, 45, 170–175.CrossRefPubMedGoogle Scholar
  25. Hernández-Macedo, M. L., Ferraz, A., Rodriguez, J., et al. (2002). Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: Differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles. Electrophoresis, 23, 655–661.CrossRefPubMedGoogle Scholar
  26. Iijima, N., Yoshino, H., Ten, L. C., et al. (2002). Two genes encoding fruit body lectins of Pleurotus cornucopiae: Sequence similarity with the lectin of a nematode-trapping fungus. Bioscience, Biotechnology, and Biochemistry, 66, 2083–2089.CrossRefPubMedGoogle Scholar
  27. Iijima, N., Amano, K., Ando, A., et al. (2003). Production of fruiting-body lectins of Pleurotus cornucopiae in methylotrophic yeast Pichia pastoris. Journal of Bioscience and Bioengineering, 95, 416–418.CrossRefPubMedGoogle Scholar
  28. James, P. (1997). Protein identification in the post-genome era: The rapid rise of proteomics. Quarterly Reviews of Biophysics, 30, 279–331.CrossRefPubMedGoogle Scholar
  29. Jansson, H. B., & Friman, E. (1999). Infection-related surface proteins on conidia of the nematophagous fungus Drechmeria coniospora. Mycological Research, 103, 249–256.CrossRefGoogle Scholar
  30. Jansson, H., & Lopez-Llorca, L.V. (2001). Biology of nematophagous fungi. Trichomycetes and other fungal groups: Robert W. Lichtwardt commemoration, pp. 144–173.Google Scholar
  31. Khan, A., Williams, K., Molloy, M. P., et al. (2003). Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels. Protein Expression and Purification, 32, 210–220.CrossRefPubMedGoogle Scholar
  32. Khan, A., Williams, K. L., & Nevalainen, H. K. M. (2004). Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles. Biological Control, 3, 346–352.CrossRefGoogle Scholar
  33. Khan, A., Williams, K., & Soon, J. (2008). Proteomic analysis of the knob-producing nematode-trapping fungus Monacrosporium lysipagum. Mycological Research, 112, 1447–1452.CrossRefPubMedGoogle Scholar
  34. Kim, Y., Nandakumar, M. P., & Marten, M. R. (2007). Proteomics of filamentous fungi. Trends in Biotechnology, 25, 395–400.CrossRefPubMedGoogle Scholar
  35. Larriba, E., Martín-Nieto, J., & Lopez-Llorca, L. V. (2012). Gene cloning, molecular modeling, and phylogenetics of serine protease P32 and serine carboxypeptidase SCP1 from nematophagous fungi Pochonia rubescens and Pochonia chlamydosporia. Canadian Journal of Microbiology, 58, 815–827.CrossRefPubMedGoogle Scholar
  36. Larriba, E., Jaime, M. D. L. A., Carbonell-Caballero, J., et al. (2014). Sequencing and functional analysis of the genome of a nematode egg-parasitic fungus, Pochonia chlamydosporia. Fungal Genetics and Biology, 65(C), 69–80.CrossRefPubMedGoogle Scholar
  37. Liang, L., Wu, H., Liu, Z., et al. (2013). Proteomic and transcriptional analyses of Arthrobotrys oligospora cell wall related proteins reveal complexity of fungal virulence against nematodes. Applied Microbiology and Biotechnology, 97, 8683–8692.CrossRefPubMedGoogle Scholar
  38. Lim, D., Hains, P., Walsh, B., et al. (2001). Proteins associated with the cell envelope of Trichoderma reesei: A proteomic approach. Proteomics, 1, 899–910.CrossRefPubMedGoogle Scholar
  39. Lopez-Llorca, L. (1990). Purification and properties of extracellular proteases produced by the nematophagous fungus Verticillium suchlasporium. Canadian Journal of Microbiology, 36, 530–537.CrossRefGoogle Scholar
  40. Lopez-Llorca, L., & Robertson, W. (1992). Immunocytochemical localization of a 32-kDa protease from the nematophagous fungus Verticillium suchlasporium in infected nematode eggs. Experimental Mycology, 16, 261–267.CrossRefGoogle Scholar
  41. Lopez-Llorca, L. V., Maciá-Vicente, J. G., Jansson, H., et al. (2008). Mode of action and interactions of nematophagous fungi. In A. Ciancio & K. G. Mukerji (Eds.), Integrated Management of Plant Pests and Diseases, vol. 2. Of the series integrated management and biocontrol of vegetable and grain crops nematodes (pp. 51–76). Netherlands: Springer.Google Scholar
  42. Lopez-Llorca, L. V., Gómez-Vidal, S., Monfort, E., et al. (2010). Expression of serine proteases in egg-parasitic nematophagous fungi during barley root colonization. Fungal Genetics and Biology, 47, 342–351.CrossRefPubMedGoogle Scholar
  43. Maciá-Vicente, J. G., Palma-Guerrero, J., Gómez-Vidal, S., et al. (2011). New insights on the mode of action of fungal pathogens of invertebrates for improving their biocontrol performance. In K. G. Davies & Y. Spiegel (Eds.), Biological control of plant-parasitic nematodes: building coherence between microbial ecology and molecular mechanisms, progress in biological control (pp. 203–225). Netherlands: Springer.CrossRefGoogle Scholar
  44. Manalil, N. S., Te’o, V. S. J., Braithwaite, K., et al. (2009). A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae. Current Genetics, 55, 571–581.CrossRefPubMedGoogle Scholar
  45. Manalil, N. S., Te’o, V. S. J., Braithwaite, K., et al. (2010). Comparative analysis of the Metarhizium anisopliae secretome in response to exposure to the greyback cane grub and grub cuticles. Fungal Biology, 114, 637–645.CrossRefPubMedGoogle Scholar
  46. Marouga, R., David, S., & Hawkins, E. (2005). The development of the DIGE system: 2D fluorescence difference gel analysis technology. Analytical and Bioanalytical Chemistry, 382, 669–678.CrossRefPubMedGoogle Scholar
  47. Martínez-Gomariz, M., Perumal, P., Mekala, S., et al. (2009). Proteomics analysis of cytoplasmic and surface proteins from yeast cells, hyphae, and biofilms of Candida albicans. Proteomics, 9, 2230–2252.CrossRefPubMedPubMedCentralGoogle Scholar
  48. Mi, Q., Yang, J., Ye, F., et al. (2010). Cloning and overexpression of Pochonia chlamydosporia chitinase gene pcchi44, a potential virulence factor in infection against nematodes. Process Biochemistry, 45, 810–814.CrossRefGoogle Scholar
  49. Minden, J. S., Dowd, S. R., Meyer, H. E., et al. (2009). Difference gel electrophoresis. Electophoresis, 30, S156–S161.CrossRefGoogle Scholar
  50. Minglian, Z., Minghe, M., & Keqin, Z. (2004). Characterization of a neutral serine protease and its full-length cDNa from the nematode-trapping fungus Arthrobotrys oligospora. Mycologia, 96, 16–22.CrossRefPubMedGoogle Scholar
  51. Morton, C., Hirsch, P., Peberdy, J., et al. (2003). Cloning of and genetic variation in protease VCP1 from the nematophagous fungus Pochonia chlamydosporia. Mycological Research, 107, 38–46.CrossRefPubMedGoogle Scholar
  52. Murad, A. M., Noronha, E. F., Miller, R. N. G., et al. (2008). Proteomic analysis of Metarhizium anisopliae secretion in the presence of the insect pest Callosobruchus maculatus. Microbiology, 154, 3766–3774.CrossRefPubMedGoogle Scholar
  53. Nordbring-Hertz, B., Jansson, H. B., & Tunlid, A. (2006). Nematophagous fungi. In: eLS. Wiley, Chichester. doi: 10.1002/9780470015902.a0000374.pub3.
  54. Palma-Guerrero, J., Jansson, H. B., Salinas, J., et al. (2008). Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi. Journal of Applied Microbiology, 104, 541–553.PubMedGoogle Scholar
  55. Palma-Guerrero, J., Gómez-Vidal, S., Tikhonov, V. E., et al. (2010). Comparative analysis of extracellular proteins from Pochonia chlamydosporia grown with chitosan or chitin as main carbon and nitrogen sources. Enzyme and Microbial Technology, 46, 568–574.CrossRefGoogle Scholar
  56. Qiu, J., Su, Y., Gelbic, I., et al. (2012). Proteomic analysis of proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus. Aschersonia placenta. Canadian Journal of Microbiology, 58, 1327–1334.CrossRefPubMedGoogle Scholar
  57. Quin, L., Liu, X., Li, J., et al. (2009). Protein profile of Nomuraea rileyi spore isolation from infected silkworm. Current Microbiology, 58, 578–585.CrossRefGoogle Scholar
  58. Rodrigues, A. M., Kubitschek-Barreira, P. H., Fernandes, G. F., et al. (2015). Two-dimensional gel electrophoresis data for proteomic profiling of Sporothrix yeast cells. Data in Brief, 2, 32–38.CrossRefPubMedGoogle Scholar
  59. Rosen, S., Ek, B., Rask, L., et al. (1992). Purification and characterization of a surface lectin from the nematode-trapping fungus Arthrobotrys oligospora. Journal of General Microbiology, 138(12), 2663–2672.CrossRefPubMedGoogle Scholar
  60. Salazar, O. (2008). Bacteria and yeast cell disruption using lytic enzymes. In A. Posh (Ed.), Methods in Molecular Biology (pp. 23–34). Clifton NJ: Humana Press.Google Scholar
  61. Santi, L., Silva, W. O. B., Pinto, A. F. M., et al. (2010). Metarhizium anisopliae host-pathogen interaction: Differential immunoproteomics reveals proteins involved in the infection process of arthropods. Fungal Biology, 114, 312–319.CrossRefPubMedGoogle Scholar
  62. Segers, R., Butt, T., Kerry, B., et al. (1994). The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ. Microbiology, 140, 2715–2723.CrossRefPubMedGoogle Scholar
  63. Segers, R., Butt, T., Keen, J., et al. (1995). The subtilisins of the invertebrate mycopathogens Verticillium chlamydosporium and Metarhizium anisopliae are serologically and functionally related. FEMS Microbiology Letters, 126, 227–231.CrossRefPubMedGoogle Scholar
  64. Segers, R., Butt, T. M., Kerry, B. R., et al. (1996). The role of the proteinase VCP1 produced by the nematophagous Verticillium chlamydosporium in the infection process of nematode eggs. Mycological Research, 100, 421–428.CrossRefGoogle Scholar
  65. Shahid, A. A., Rao, Q. A., & Bakhsh, A. (2012). Entomopathogenic fungi as biological controllers: New insights into their virulence and pathogenicity. Archives of Biological Sciences, 64, 21–42.CrossRefGoogle Scholar
  66. Simpson, R. J. (2003). Preparation of cellular and subcellular extracts. In R. J. Simpson (Ed.), Proteins and proteomics. A laboratory manual (pp. 91–142). New York: Cold Spring Harbor Laboratory Press.Google Scholar
  67. St. Leger, R. J., Wang, C., & Fang, W. (2011). New perspectives on insect pathogens. Fungal Biology Reviews, 25, 84–88.CrossRefGoogle Scholar
  68. Stirling, G. R. (2014). Biological control of plant-parasitic nematodes: Soil ecosystem management in sustainable agriculture (2nd ed.). London: CABI.Google Scholar
  69. Su, Y., Guo, Q., Tu, J., et al. (2013). Proteins differentially expressed in conidia and mycelia of the entomopathogenic fungus Metarhizium anisopliae sensu stricto. Canadian Journal of Microbiology, 59, 443–448.CrossRefPubMedGoogle Scholar
  70. Tikhonov, V., Lopez-Llorca, L., Salinas, J., et al. (2002). Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium. Fungal Genetics and Biology, 35, 67–78.CrossRefPubMedGoogle Scholar
  71. Tjalsma, H., Bolhuis, A., Jongbloed, J. D. H., et al. (2000). Signal peptide-dependent protein transport in Bacillus subtilis, a genome-based survey of the secretome. Microbiology and Molecular Biology Reviews, 64, 515–547.CrossRefPubMedPubMedCentralGoogle Scholar
  72. Tunlid, A., Rosen, S., Ek, B., et al. (1994). Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora. Microbiology, 140, 1687–1695.CrossRefPubMedGoogle Scholar
  73. Vega, F. E., Goettel, M. S., Blackwell, M., et al. (2009). Fungal entomopathogens: New insights on their ecology. Fungal Ecology, 2, 149–159.CrossRefGoogle Scholar
  74. Wang, M., Yang, J., & Zhang, K. Q. (2006a). Characterization of an extracellular protease and its cDNA from the nematode-trapping fungus Monacrosporium microscaphoides. Canadian Journal of Microbiology, 52, 130–139.CrossRefPubMedGoogle Scholar
  75. Wang, R. B., Yang, J. K., Lin, C., et al. (2006b). Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Dactylella shizishanna. Letters in Applied Microbiology, 42, 589–594.PubMedGoogle Scholar
  76. Wang, B., Wu, W., & Liu, X. (2007). Purification and characterization of a neutral serine protease with nematicidal activity from Hirsutella rhossiliensis. Mycopathologia, 1163, 169–176.CrossRefGoogle Scholar
  77. Wang, B., Liu, X., Wu, W., et al. (2009). Purification, characterization, and gene cloning of an alkaline serine protease from a highly virulent strain of the nematode-endoparasitic fungus Hirsutella rhossiliensis. Microbiological Research, 164, 665–673.CrossRefPubMedGoogle Scholar
  78. Westermeier, R., & Naven, T. (2002). Expression proteomics. In R. Westermeier & T. Naven (Eds.), Proteomics in practice: A laboratory manual of proteome analysis (pp. 11–160). Weinheim: Wiley-VCH Verlag-GmbH.CrossRefGoogle Scholar
  79. Yang, J., Li, J., Liang, L., et al. (2007a). Cloning and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys conoides. Archives of Microbiology, 188, 167–174.CrossRefPubMedGoogle Scholar
  80. Yang, J., Liang, L., Zhang, Y., et al. (2007b). Purification and cloning of a novel serine protease from the nematode-trapping fungus Dactylellina varietas and its potential roles in infection against nematodes. Applied Microbiology and Biotechnology, 75, 557–565.CrossRefPubMedGoogle Scholar
  81. Yang, J. K., Ye, F. P., Mi, Q. L., et al. (2008). Purification and cloning of an extracellular serine protease from the nematode-trapping fungus Monacrosporium cystosporium. Journal of Microbiology and Biotechnology, 18, 852–858.PubMedGoogle Scholar
  82. Yang, J., Wang, L., Ji, X., et al. (2011). Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation. PLoS Pathogens, 7(9), e1002179.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Nuria Escudero
    • 1
    • 2
    Email author
  • Sonia Gómez-Vidal
    • 1
    • 3
  • Luis V. Lopez-Llorca
    • 1
  1. 1.Laboratory of Plant Pathology, Multidisciplinary Institute for Environmental Studies (MIES) Ramón Margalef, Department of Marine Sciences and Applied BiologyUniversity of AlicanteAlicanteSpain
  2. 2.Department of Agri-Food Engineering and BiotechnologyUniversitat Politècnica de CatalunyaCatalunyaSpain
  3. 3.Research Technical ServicesUniversity of AlicanteAlicanteSpain

Personalised recommendations