The cell wall proteome from two strains of Pseudocercospora fijiensis with differences in virulence

  • Yamily Y. Burgos-Canul
  • Blondy Canto-Canché
  • Maxim V. Berezovski
  • Gleb Mironov
  • Víctor M. Loyola-Vargas
  • Ana Paulina Barba de Rosa
  • Miguel Tzec-Simá
  • Ligia Brito-Argáez
  • Mildred Carrillo-Pech
  • Rosa Grijalva-Arango
  • Gilberto Muñoz-Pérez
  • Ignacio Islas-FloresEmail author
Original Paper


Pseudocercospora fijiensis causes black Sigatoka disease, the most important threat to banana. The cell wall is crucial for fungal biological processes, including pathogenesis. Here, we performed cell wall proteomics analyses of two P. fijiensis strains, the highly virulent Oz2b, and the less virulent C1233 strains. Strains were starved from nitrogen to mimic the host environment. Interestingly, in vitro cultures of the C1233 strain grew faster than Oz2b in PDB medium, suggesting that C1233 survives outside the host better than the highly virulent Oz2b strain. Both strains were submitted to nitrogen starvation and the cell wall proteins were isolated and subjected to nano-HPLC–MS/MS. A total of 2686 proteins were obtained from which only 240 had a known function and thus, bioinformatics analyses were performed on this group. We found that 90 cell wall proteins were shared by both strains, 21 were unique for Oz2b and 39 for C1233. Shared proteins comprised 24 pathogenicity factors, including Avr4 and Ecp6, two effectors from P. fijiensis, while the unique proteins comprised 16 virulence factors in C1233 and 11 in Oz2b. The P. fijiensis cell wall proteome comprised canonical proteins, but thirty percent were atypical, a feature which in other phytopathogens has been interpreted as contamination. However, a comparison with the identities of atypical proteins in other reports suggests that the P. fijiensis proteins we detected were not contaminants. This is the first proteomics analysis of the P. fijiensis cell wall and our results expands the understanding of the fundamental biology of fungal phytopathogens and will help to decipher the molecular mechanisms of pathogenesis and virulence in P. fijiensis.

Graphic abstract


Cell wall proteome Fungal cell wall isolation Pathogenicity factors Pseudocercospora fijiensis 



The authors would like to thank Dr. Marco A. Villanueva, for his critical reading, as well as to the three anonymous reviewers, whose comments have helped to improve the quality of the data analysis in the manuscript. We would also wish to acknowledge E. Góngora-Castillo and A. Enríquez-Valencia for their advice regarding bioinformatics analysis. Y. Burgos-Canul was supported by scholarship No. 255427 for Ph. D. studies from CONACYT México. This work was funded by Grants 220957 from CONACyT and 247355 from FOMIX.

Author contributions

YB-C, II-F and BC-C conceived, designed and wrote the paper; VL-V and APB gave support in the design of proteomic experiments; YB-C, performed cell wall isolation and protein extractions; MB and GM performed MS analysis, searched for protein IDs in the Uniprot database and edited the preliminary list of proteins; LB-A, MC-P and GM-P, provided P. fijiensis strains and technically supported the research; MT-S assisted with infection experiments, photography and sample preparation, II-F and BC-C; supervised all the work. All authors participated in the data analysis, wrote and read the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

11274_2019_2681_MOESM1_ESM.xlsx (17 kb)
Supplementary file1 (XLSX 16 kb)
11274_2019_2681_MOESM2_ESM.xlsx (16 kb)
Supplementary file2 (XLSX 15 kb)
11274_2019_2681_MOESM3_ESM.xlsx (14 kb)
Supplementary file3 (XLSX 13 kb)
11274_2019_2681_MOESM4_ESM.xlsx (15 kb)
Supplementary file4 (XLSX 15 kb)


  1. Aguilar-Barragan A, García-Torres AE, Odriozola-Casas O, Macedo-Raygoza G, Ogura T, Manzo-Sánchez G, James AC, Islas-Flores I, Beltrán-García MJ (2014) Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México. Braz J Microbiol 45:359–364CrossRefGoogle Scholar
  2. Alkan N, Espeso EA, Prusky D (2013) Virulence regulation of phytopathogenic fungi by pH. Antioxid Redox Signal 19:1012–1025. CrossRefPubMedGoogle Scholar
  3. Almeida F, Wolf JM, Casadevall A (2015) Virulence-associated enzymes of Cryptococcus neoformans. Eukaryot Cell 14:1173–1185. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ao J, Aldabbous ME, Notaro MJ, Lojacono M, Free SJ (2016) A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling. Fungal Genet Biol 94:47–53. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Araújo DS, de Sousa Lima P, Baeza LC, Parente AFA, Melo Bailão A, Borges CL, de Almeida Soares CM (2017) Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins. Biochim Biophys Acta 1865:1304–1314. CrossRefGoogle Scholar
  6. Bellosta P, Zhang Q, Goff SP, Basilico C (1997) Signaling through the ARK tyrosine kinase receptor protects from apoptosis in the absence of growth stimulation. Oncogene 15:2387–3297. CrossRefPubMedGoogle Scholar
  7. Bowman SM, Free SJ (2006) The structure and synthesis of the fungal cell wall. BioEssays 28:799–808. CrossRefPubMedGoogle Scholar
  8. Bruneau JM, Magnin T, Tagat E, Legrand R, Bernard M, Diaquin M, Fudali C, Latge JP (2001) Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis. Electrophoresis 22:2812–2823.;2-Q CrossRefPubMedGoogle Scholar
  9. Castillo L, Calvo E, Martínez AI, Ruiz-Herrera J, Valentín E, Lopez JA, Sentandreu R (2008) A study of the Candida albicans cell wall proteome. Proteomics 8:3871–3881. CrossRefPubMedGoogle Scholar
  10. Chang TC, Salvucci A, Crous PW, Stergiopoulos I (2016) Comparative genomics of the Sigatoka disease complex on banana suggests a link between parallel evolutionary changes in Pseudocercospora fijiensis and Pseudocercospora eumusae and increased virulence on the banana host. PLoS Genet 12:e1005904. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chitty JL, Blake K, Blundell R, Andre YQ, Thompson M, Robertson AA, Butler M, Cooper M, Kappler U, Williams S, Kobe B, Fraser JA (2017) Cryptococcus neoformans ADS lyase is an enzyme essential for virulence whose crystal structure reveals features exploitable in antifungal drug design. J Biol Chem 292:11829–11839. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Chu J, Li WF, Cheng W, Lu M, Zhou KH, Zhu HQ, Li FG, Zhou CZ (2015) Comparative analyses of secreted proteins from the phytopathogenic fungus Verticillium dahliae in response to nitrogen starvation. Biochim Biophys Acta 1854:437–448. CrossRefPubMedGoogle Scholar
  13. Crous PW, Groenewald JZ, Slippers B, Wingfield MJ (2016) Global food and fibre security threatened by current inefficiencies in fungal identification. Philos Trans R Soc B 371:1–7. CrossRefGoogle Scholar
  14. de Groot PWJ, de Boer AD, Brandt BW, Valentín E (2016) 5 The ascomycetous cell wall: from a proteomic perspective. growth, differentiation and sexuality. In: Wendland J (ed) The Mycota (a comprehensive treatise on fungi as experimental systems for basic and applied research). Springer, Cham, pp 81–101Google Scholar
  15. DOE Joint Genome Institute Accessed 15 Nov 2017
  16. Eisenhaber B, Schneider G, Wildpaner M, Eisenhaber F (2004) A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans, Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe. Am J Mol Biol 337:243–253. CrossRefGoogle Scholar
  17. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Am J Mol Biol 300:1005–1016. CrossRefGoogle Scholar
  18. Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989. CrossRefPubMedGoogle Scholar
  19. Escobar-Tovar L, Guzmán-Quesada M, Sandoval-Fernández JA, Gómez-Lim MA (2015) Comparative analysis of the in vitro and in planta secretomes from Mycosphaerella fijiensis isolates. Fungal Biol 119:447–470. CrossRefPubMedGoogle Scholar
  20. Fang X, Barbetti MJ (2014) Differential protein accumulations in isolates of the strawberry wilt pathogen Fusarium oxysporum f. sp. fragariae differing in virulence. J Proteom 108:223–237. CrossRefGoogle Scholar
  21. Fankhauser NMP (2005) Khogpi: identification of GPI-anchor signals by a Kohonen self organizing map. Bioinformatics 21:1846–1852. CrossRefPubMedGoogle Scholar
  22. Fouré E (1982) Les Cercosporiose du bananier et leur traitemant. Comportament des varietés. Estude de la sensibilité varietale des bananiers et plantains a Mycosphaerella fijiensis Morelet au Gabon (maladies de raises noires). I Incubation et evolution de la maladie. Fruits 37:749–771Google Scholar
  23. Friedman DB (2007) Quantitative proteomics for two-dimensional gels using difference gel electrophoresis. In: Matthiesen R (ed) Mass spectrometry data analysis in proteomics. Humana Press, New York, pp 219–239Google Scholar
  24. Goh J, Jeon J, Lee YH (2017) ER retention receptor, MoERR1 is required for fungal development and pathogenicity in the rice blast fungus, Magnaporthe oryzae. Sci Rep 7:1259. CrossRefPubMedPubMedCentralGoogle Scholar
  25. González-Fernández R, Aloria K, Valero-Galván J, Redondo I, Arizmendi JM, Jorrín-Novo JV (2014) Proteomic analysis of mycelium and secretome of different Botrytis cinerea wild-type strains. J Proteom 97:195–221. CrossRefGoogle Scholar
  26. Gupta R, Brunak S (2002) Prediction of glycosylation across the human proteome and the correlation to protein function. Pac Symp Biocomput 7:310–322Google Scholar
  27. Kall L, Krogh A, Sonnhammer EL (2004) A combined transmembrane topology and signal peptide prediction method. Am J Mol Biol 338:1027–1036. CrossRefGoogle Scholar
  28. Kantún-Moreno N, Vázquez-Eúan R, Tzec-Simá M, Peraza-Echeverría L, Grijalva-Arango R, Rodríguez-García C, James AC, Ramírez-Prado J, Islas-Flores I, Canto-Canché B (2013) Genome-wide in silico identification of GPI proteins in Mycosphaerella fijiensis and transcriptional analysis of two GPI-anchored beta-1,3-glucanosyltransferases. Mycologia 105:285–296. CrossRefPubMedGoogle Scholar
  29. Karkowska-Kuleta J, Kozik A (2015) Cell wall proteome of pathogenic fungi. Acta Biochim Pol 62:339–351. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Ku JW, Gan YH (2018) Modulation of bacterial virulence and fitness by host glutathione. Curr Opin Microbiol 47:8–13. CrossRefPubMedGoogle Scholar
  31. Leiva-Mora M, Alvarado-Capó Y, Acosta-Suárez M, Cruz Martín M, Sánchez-García C, Roque B (2010) Protocolo para la inoculación artificial de plantas de Musa spp. con Mycosphaerella fijiensis y evaluación de su respuesta mediante variables epifitiológicas y componentes de la resistencia. Biotecnol Veg 10:79–88Google Scholar
  32. Li E, Ling J, Wang G, Xiao J, Yang Y, Mao Z, Wang X, Xie B (2015) Comparative proteomics analyses of two races of Fusarium oxysporum f. sp. conglutinans that differ in pathogenicity. Sci Rep 5:13663. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Liu L, Free SJ (2016) Characterization of the Sclerotinia sclerotiorum cell wall proteome. Mol Plant Pathol 17:985–995. CrossRefPubMedGoogle Scholar
  34. Longo LVG, da Cunha JPC, Sobreira TJP, Puccia R (2014) Proteome of cell wall-extracts from pathogenic Paracoccidioides brasiliensis: comparison among morphological phases, isolates, and reported fungal extracellular vesicle proteins. EuPA Open Proteom 3:216–228. CrossRefGoogle Scholar
  35. Maddi A, Bowman SM, Free SJ (2009) Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans. Fungal Genet Biol 46:768–781. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Manikandan R, Harish S, Karthikeyan G, Raguchander T (2018) Comparative proteomic analysis of different isolates of Fusarium oxysporum f. sp. lycopersici to exploit the differentially expressed proteins responsible for virulence on tomato plants. Front Microbiol 9:420. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Manteau S, Abouna S, Lambert B, Legendre L (2003) Differential regulation by ambient pH of putative virulence factors secretion by the phytopathogenic fungus Botrytis cinerea. FEMS Microbiol Ecol 43:359–366. CrossRefPubMedGoogle Scholar
  38. Maria de Jesus BC, Escoute J, Madeira JP, Romero RE, Nicole M, Oliveira LC, Hamelin C, Lartaud M, Verdeil JL (2011) Reactive oxygen species and cellular interactions between Mycosphaerella fijiensis and banana. Trop Plant Biol 4:134–143. CrossRefGoogle Scholar
  39. Moyrand F, Janbon G (2004) UGD1, encoding the Cryptococcus neoformans UDP-glucose dehydrogenase, is essential for growth at 37 degrees C and for capsule biosynthesis. Eukaryot Cell 3:1601–1608. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Msanne J, Chen M, Luttgeharm KD, Bradley AM, Mays ES, Paper JM, Boyle DL, Cahoon RE, Schrick K, Cahoon EB (2015) Glucosylceramides are critical for cell-type differentiation and organogenesis, but not for cell viability in Arabidopsis. Plant J 84:188–201. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Noar RD, Daub ME (2016) Transcriptome sequencing of Mycosphaerella fijiensis during association with Musa acuminata reveals candidate pathogenicity genes. BMC Genom 17:690. CrossRefGoogle Scholar
  42. Oh Y, Robertson SL, Parker J, Muddiman DC, Dean RA (2017) Comparative proteomic analysis between nitrogen supplemented and starved conditions in Magnaporthe oryzae. Proteome Sci 15:1–12. CrossRefGoogle Scholar
  43. Oliveros JC (2007) Venny. An interactive tool for comparing lists with Venn's diagrams. Accessed 2 Feb 2018
  44. Pan Y, Ye T, Gao Z (2017) Cloning and functional analysis of succinate dehydrogenase gene PsSDHA in Phytophthora sojae. Microb Pathog 108:40–48. CrossRefPubMedGoogle Scholar
  45. Peraza-Echeverría L, Rodríguez-García CM, Zapata-Salazar DM (2008) A rapid, effective method for profuse in vitro conidial production of Mycosphaerella fijiensis. Australas Plant Pathol 37:460–463. CrossRefGoogle Scholar
  46. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. CrossRefPubMedGoogle Scholar
  47. Pierleoni A, Martelli PL, Casadio R (2008) PredGPI: a GPI-anchor predictor. BMC Bioinform 9:392. CrossRefGoogle Scholar
  48. Pitarch A, Nombela C, Gil C (2008) Cell wall fractionation for yeast and fungal proteomics. In: Posch A (ed) 2D PAGE: sample preparation and fractionation. Humana Press, Totowa, pp 217–239CrossRefGoogle Scholar
  49. Plaumann PL, Schmidpeter J, Dahl M, Taher L, Koch C (2018) A dispensable chromosome is required for virulence in the hemibiotrophic plant pathogen Colletotrichum higginsianum. Front Microbiol 9:1005. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Portal O, Izquierdo Y, De Vleesschauwer D, Sánchez-Rodríguez A, Mendoza-Rodríguez M, Acosta-Suárez M, Ocaña B, Jiménez E, Höfte M (2011) Analysis of expressed sequence tags derived from a compatible Mycosphaerella fijiensis–banana interaction. Plant Cell Rep 30:913–928. CrossRefPubMedGoogle Scholar
  51. Prados-Rosales R, Luque-Garcia JL, Martínez-López R, Gil C, Di Pietro A (2009) The Fusarium oxysporum cell wall proteome under adhesion-inducing conditions. Proteomics 9:4755–4769. CrossRefPubMedGoogle Scholar
  52. Riccillo PM, Muglia CI, de Bruijn FJ, Roe AJ, Booth IR, Aguilar OM (2000) Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance. J Bacteriol 182:1748–1753. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Rodríguez-García CM, Canché-Gómez AD, Sáenz-Carbonell L, Peraza-Echeverría L, Canto-Canché B, Islas-Flores I, Peraza-Echeverría S (2016) Expression of MfAvr4 in banana leaf sections with black leaf streak disease caused by Mycosphaerella fijiensis: a technical evaluation. Austral Plant Pathol 45:481–488. CrossRefGoogle Scholar
  54. Sacristán S, García-Arenal F (2008) The evolution of virulence and pathogenicity in plant pathogen populations. Mol Plant Pathol 9:369–384. CrossRefPubMedGoogle Scholar
  55. Sánchez-Fresneda R, Martínez-Esparza M, Maicas S, Argüelles JC, Valentín E (2014) In Candida parapsilosis the ATC1 gene encodes for an acid trehalase involved in trehalose hydrolysis, stress resistance and virulence. PLoS ONE 9:e99113. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Sánchez-Vallet A, Mesters JR, Thomma BP (2015) The battle for chitin recognition in plant-microbe interactions. FEMS Microbiol Rev 39:171–183. CrossRefPubMedGoogle Scholar
  57. Snoeijers SS, Pérez-García A, Joosten MHAJ, De Wit PJGM (2000) The effect of nitrogen on disease development and gene expression in bacterial and fungal plant pathogens. Eur J Plant Pathol 106:493–506. CrossRefGoogle Scholar
  58. Sprockett DD, Piontkivska H, Blackwood CB (2011) Evolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens. Gene 479:29–36. CrossRefPubMedGoogle Scholar
  59. Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KT, Lavrsen K, Dabelsteen S, Pedersen NB, Marcos-Silva L, Gupta R, Bennett EP, Mandel U, Brunak S, Wandall HH, Levery SB, Clausen H (2013) Precision mapping of the human O-GalNAc glycoproteome through simple cell technology. EMBO J 32:1478–1488. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Stergiopoulos I, van den Burg HA, Okmen B, Beenen HG, van Liere S, Kema GH, de Wit PJ (2010) Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots. Proc Natl Acad Sci USA 107:7610–7615. CrossRefPubMedGoogle Scholar
  61. Sun Y, Yi X, Peng M, Zeng H, Wuang D, Tong Z, Chang L, Jin X, Wang X (2014) Proteomics of Fusarium oxysporum race 1 and race 4 reveals enzymes involved in carbohydrate metabolism and ion transport that might play important roles in banana Fusarium wilt. PLoS ONE 9:1–20. CrossRefGoogle Scholar
  62. Surico G (2013) The concepts of plant pathogenicity, virulence/avirulence and effector proteins by a teacher of plant pathology. Phytopathol Mediterr 52:399–417. CrossRefGoogle Scholar
  63. Ünal CM, Steinert M (2014) Microbial peptidyl-prolyl cis/trans isomerases (PPIases): virulence factors and potential alternative drug targets. Microbiol Mol Biol Rev 78:544–571. CrossRefPubMedPubMedCentralGoogle Scholar
  64. Uniprot Accessed 7 Nov 2017
  65. Urban M, Cuzick A, Rutherford K, Irvine A, Pedro H, Pant R, Sadanadan V, Khamari L, Billal S, Mohanty S, Hammond-Kosack KE (2016) PHI-base: a new interface and further additions for the multi-species pathogen-host interactions database. Nucleic Acids Res 45:D604–D610. CrossRefPubMedPubMedCentralGoogle Scholar
  66. Vylkova S (2017) Environmental pH modulation by pathogenic fungi as a strategy to conquer the host. PLoS Pathog 13:e1006149. CrossRefPubMedPubMedCentralGoogle Scholar
  67. Xu S, Chen J, Liu L, Wang X, Huang X, Zhai Y (2007) Proteomics associated with virulence differentiation of Curvularia lunata in maize in China. J Integr Plant Biol 49:487–496. CrossRefGoogle Scholar
  68. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2018) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:293–297. CrossRefGoogle Scholar
  69. Zhang J, Xin L, Shan B, Chen W, Xie M, Yuen D, Zhang W, Zhang Z, Lajoie GA, Ma B (2012) PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification. Mol Cell Proteom 11(M111):010587. CrossRefGoogle Scholar
  70. Zilm PS, Bagley CJ, Rogers AH, Milne IR, Gully NJ (2007) The proteomic profile of Fusobacterium nucleatum is regulated by growth pH. Microbiology 153:148–159. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Unidad de Bioquímica y Biología Molecular de PlantasCentro de Investigación Científica de YucatánMéridaMexico
  2. 2.Unidad de BiotecnologíaCentro de Investigación Científica de YucatánMéridaMexico
  3. 3.Department of Chemistry and Biomolecular SciencesUniversity of OttawaOttawa, ONCanada
  4. 4.IPICYT, Instituto Potosino de Investigación Científica y TecnológicaSan Luis PotosíMexico
  5. 5.Unidad de Recursos NaturalesCentro de Investigación Científica de YucatánMéridaMexico

Personalised recommendations