Current Genetics

, Volume 58, Issue 3, pp 165–177 | Cite as

Genome-wide analysis of cell wall-related genes in Tuber melanosporum

  • Raffaella Balestrini
  • Fabiano Sillo
  • Annegret Kohler
  • Georg Schneider
  • Antonella Faccio
  • Emilie Tisserant
  • Francis Martin
  • Paola Bonfante
Research Article


A genome-wide inventory of proteins involved in cell wall synthesis and remodeling has been obtained by taking advantage of the recently released genome sequence of the ectomycorrhizal Tuber melanosporum black truffle. Genes that encode cell wall biosynthetic enzymes, enzymes involved in cell wall polysaccharide synthesis or modification, GPI-anchored proteins and other cell wall proteins were identified in the black truffle genome. As a second step, array data were validated and the symbiotic stage was chosen as the main focus. Quantitative RT-PCR experiments were performed on 29 selected genes to verify their expression during ectomycorrhizal formation. The results confirmed the array data, and this suggests that cell wall-related genes are required for morphogenetic transition from mycelium growth to the ectomycorrhizal branched hyphae. Labeling experiments were also performed on T. melanosporum mycelium and ectomycorrhizae to localize cell wall components.


Tuber melanosporum Fungal genome Cell wall Ectomycorrhizae Symbiotic interactions 

Supplementary material

294_2012_374_MOESM1_ESM.doc (56 kb)
Table S1 (DOC 55 kb)
294_2012_374_MOESM2_ESM.doc (235 kb)
Table S2 (DOC 235 kb)
294_2012_374_MOESM3_ESM.doc (38 kb)
Table S3 (DOC 38 kb)
294_2012_374_MOESM4_ESM.pdf (52 kb)
Table S4. GPI-prediction by using the fungal-specific big-PI algorithm (Eisenhaber et al. 2004) and checking for a predicted signal peptide (Bendtsen et al. 2004). There is experimental evidence for alternative or secondary GPI-anchor attachment sites (Eisenhaber et al. 1999). As such, the predictor reports a main and a secondary site (1: and 2:), each one classified with a letter (P, predicted; S, twilight zone prediction; I, physical properties are not right, but profile matches; N, neither physical properties nor profile matches). The best hit is shown first and is either P or S: if the first one is P, the second one can be P, S or N, or if the first one is S it can be S, I or N. An additional step to check for potential transmembrane domains (TM) in the mature protein has been performed. (PDF 52 kb)
294_2012_374_MOESM5_ESM.xls (36 kb)
Table S5. Expression data for the GPI-anchored protein subset. The individual sheets contain the array and the Solexa/Illumina data as explained in the Supplemental file. (XLS 36 kb)


  1. Aimanianda V, Bayry J, Bozza S, Kniemeyer O, Perruccio K, Elluru SR, Clavaud C, Paris S, Brakhage AA, Kaveri SV, Romani L, Latge JP (2009) Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature 460:1117–1121PubMedCrossRefGoogle Scholar
  2. Amicucci A, Balestrini R, Kohler A, Barbieri E, Saltarelli R, Faccio A, Roberson RW, Bonfante P, Stocchi V (2011) Hyphal and cytoskeleton polarization in Tuber melanosporum: a genomic and cellular analysis. Fungal Genet Biol 48:561–572PubMedCrossRefGoogle Scholar
  3. Balestrini R, Hahn MG, Bonfante P (1996) Location of cell-wall components in ectomycorrhizae of Corylus avellana and Tuber magnatum. Protoplasma 191:55–69CrossRefGoogle Scholar
  4. Balestrini R, Mainieri D, Soragni E, Garnero L, Rollino S, Viotti A, Ottonello S, Bonfante P (2000) Differential expression of chitin synthase III and IV mRNAs in ascomata of Tuber borchii Vittad. Fungal Genet Biol 31:219–232PubMedCrossRefGoogle Scholar
  5. Balestrini R, Bianciotto V, Bonfante P (2011) Mycorrhizae in: Huang, Li, Sumner (eds) Handbook of Soil Sciences, Volume I. Taylor and Francis Group, Boca Raton, pp 24/29–24/40Google Scholar
  6. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795PubMedCrossRefGoogle Scholar
  7. Borgia PT, Iartchouk N, Riggle PJ, Winter KR, Koltin Y, Bulawa CE (1996) The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development. Fungal Genet Biol 20:193–203PubMedCrossRefGoogle Scholar
  8. Borkovich KA, Alex LA, Yarden O et al (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68:1–108PubMedCrossRefGoogle Scholar
  9. Bowman SM, Free SJ (2006) The structure and synthesis of the fungal cell wall. BioEssays 28:799–808PubMedCrossRefGoogle Scholar
  10. Brendel V, Bucher P, Nourbakhsh IR, Blaisdell BE, Karlin S (1992). “Methods and algorithms for statistical analysis of protein sequences.” Proceedings of the National Academy of Sciences of the United States of America 89 (6) (March 15):2002–2006Google Scholar
  11. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238PubMedCrossRefGoogle Scholar
  12. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116CrossRefGoogle Scholar
  13. Choquer M, Boccara M, Goncalves IR, Soulié M-C, Vidal-Cros A (2004) Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six euascomycetes genomes. Eur J Biochem 271:2153–2164PubMedCrossRefGoogle Scholar
  14. Coronado JE, Mneimneh S, Epstein SL, Qiu WG, Lipke PN (2007) Conserved processes and lineage-specific proteins in fungal cell wall evolution. Eukaryot Cell 6:2269–2277PubMedCrossRefGoogle Scholar
  15. Cserzo M, Eisenhaber F, Eisenhaber B, Simon I (2004) TM or not TM: transmembrane protein prediction with low false positive rate using DAS-TMfilter. Bioinformatics 20:136–137PubMedCrossRefGoogle Scholar
  16. de Groot PWJ, Brandt BW, Horiuchi H, Ramd AFJ, de Koster CG, Klis FM (2009) Comprehensive genomic analysis of cell wall genes in Aspergillus nidulans. Fungal Genet Biol 46:S72–S81PubMedCrossRefGoogle Scholar
  17. Denoeud F, Aury JM, Da Silva C, Noel B, Rogier O, Delledonne M, Morgante M, Valle G, Wincker P, Scarpelli C, Jaillon O, Artiguenave F (2008) Annotating genomes with massive-scale RNA sequencing. Genome Biol 9:R175PubMedCrossRefGoogle Scholar
  18. Duo-Chuan L (2006) Review of fungal chitinases. Mycopathologia 161:345–360PubMedCrossRefGoogle Scholar
  19. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797PubMedCrossRefGoogle Scholar
  20. Eisenhaber B, Bork P, Eisenhaber F (1999) Prediction of potential GPI-modification sites in proprotein sequences. J Mol Biol 292:741–758PubMedCrossRefGoogle Scholar
  21. 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. J Mol Biol 337:243–253PubMedCrossRefGoogle Scholar
  22. Gruber S, Vaaje-Kolstad G, Matarese F, López-Mondéjar R, Kubicek CP, Seidl-Seiboth V (2011) Analysis of subgroup C of fungal chitinases containing chitin-binding and LysM modules in the mycoparasite Trichoderma atroviride. Glycobiology 21:122–133PubMedCrossRefGoogle Scholar
  23. Käll L, Krogh A, Sonnhammer ELL (2004) A combined transmembrane topology and signal peptide prediction method. J Mol Biol 338:1027–1036PubMedCrossRefGoogle Scholar
  24. Karlsson M, Stenlid J (2008) Comparative evolutionary histories of the fungal chitinase gene family reveal non-random size expansions and contractions due to adaptive natural selection. Evol Bioinform 4:47–60Google Scholar
  25. Kubicek C, Baker S, Gamauf C, Kenerley C, Druzhinina I (2008) Purifying selection and birth-and-death evolution in the class II hydrophobin gene families of the ascomycete Trichoderma/Hypocrea. BMC Evol Biol 8:4PubMedCrossRefGoogle Scholar
  26. Kulkarni RD, Kelkar HS, Dean RA (2003) An eight-cysteine-containing CFEM domain unique to a group of fungal membrane proteins. TIBS 28:118–121PubMedGoogle Scholar
  27. Kurita T, Noda Y, Takagi T, Osumi M, Yoda K (2011) Kre6 protein essential for yeast cell wall β-1,6-glucan synthesis accumulates at sites of polarized growth. J Biol Chem 286:7429–7438PubMedCrossRefGoogle Scholar
  28. Laurent P, Voiblet C, Tagu D, de Carvalho D, Nehls U, De Bellis R, Balestrini R, Bauw G, Bonfante P, Martin F (1999) A novel class of ectomycorrhiza-regulated cell wall polypeptides in Pisolithus tinctorius. Mol Plant Microbe Interact 12:862–871PubMedCrossRefGoogle Scholar
  29. Lesage G, Bussey H (2006) Cell wall assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 70:317–343PubMedCrossRefGoogle Scholar
  30. Martin F, Laurent P, de Carvalho D, Voiblet C, Balestrini R, Bonfante P, Tagu D (1999) Cell wall proteins of the ectomycorrhizal basidiomycete Pisolithus tinctorius: identification, function, and expression in symbiosis. Fungal Genet Biol 27:161–174PubMedCrossRefGoogle Scholar
  31. Martin F, Kohler A, Murat C, Balestrini R et al (2010) Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis. Nature 464:1033–1038PubMedCrossRefGoogle Scholar
  32. Martin-Urdiroz M, Roncero MIG, Gonzalez-Reyes JA, Ruiz-Roldan C (2008) ChsVb, a Class VII chitin synthase involved in septation, is critical for pathogenicity in Fusarium oxysporum. Eukaryot Cell 7:112–121PubMedCrossRefGoogle Scholar
  33. Miozzi L, Balestrini R, Bolchi A, Novero M, Ottonello S, Bonfante P (2005) Phospholipase A2 up‐regulation during mycorrhiza formation in Tuber borchii. New Phytol 167:229–238PubMedCrossRefGoogle Scholar
  34. Montijn RC, Vink E, Muller WH, Verkleij AJ, Van Den Ende H, Henrissat B, Klis FM (1999) Localization of synthesis of β-1,6-glucan in Saccharomyces cerevisiae. J Bacteriol 181:7414–7420PubMedGoogle Scholar
  35. Ooi HS, Kwo CY, Wildpaner M, Sirota FL, Eisenhaber B, Maurer-Stroh S, Wong WC, Schleiffer A, Eisenhaber F, Schneider G (2009) ANNIE: integrated de novo protein sequence annotation. Nucleic Acids Res 37:435–440CrossRefGoogle Scholar
  36. Pardo M, Monteoliva L, Vazquez P, Martinez R, Molero G, Nombela C, Gil C (2004) PST1 and ECM33 encode two yeast cell surface GPI proteins important for cell wall integrity. Microbiology 150:4157–4170PubMedCrossRefGoogle Scholar
  37. Pel HJ, De Winde JH, Archer DB et al (2007) Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 51388. Nat Biotech 25:221–231CrossRefGoogle Scholar
  38. Ragni E, Fontaine T, Gissi C, Latgè JP, Popolo L (2007) The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis. Yeast 24:297–308PubMedCrossRefGoogle Scholar
  39. Ruiz-Herrera J, Ortiz-Castellanos L, Martínez A-I, León-Ramírez C, Sentandreu R (2008) Analysis of the proteins involved in the structure and synthesis of the cell wall of Ustilago maydis. Fungal Genet Biol 45:S71–S76PubMedCrossRefGoogle Scholar
  40. Seidl V (2008) Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiological functions. Fungal Biol Rev 22:36–42CrossRefGoogle Scholar
  41. Seidl-Seiboth V, Gruber S, Sezerman U, Schwecke T, Albayrak A, Neuhof T, Von Döhren H, Baker SE, Kubicek CP (2011) Novel hydrophobins from Trichoderma define a new hydrophobin subclass: protein properties, evolution, regulation and processing. J Mol Evol 72:339–351PubMedCrossRefGoogle Scholar
  42. Shahinian S, Bussey H (2000) β-1,6-Glucan synthesis in Saccharomyces cerevisiae. Mol Microbiol 35:477–489PubMedCrossRefGoogle Scholar
  43. Soragni E, Bolchi A, Balestrini R, Gambaretto C, Percudani R, Bonfante P, Ottonello S (2001) A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii. EMBO J 20:5079–5090PubMedCrossRefGoogle Scholar
  44. Tagu D, De Bellis R, Balestrini R, de Vries OMH, Piccoli G, Stocchi V, Bonfante P, Martin F (2001) Immunolocalization of hydrophobin HYDPt-1 from the ectomycorrhizal basidiomycete Pisolithus tinctorius during colonization of Eucalyptus globulus roots. New Phytol 149:127–135CrossRefGoogle Scholar
  45. Takeshita N, Yamashita S, Ohta A, Horiuchi H (2006) Aspergillus nidulans class V and VI chitin synthases CsmA and CsmB, each with a myosin motor-like domain, perform compensatory functions that are essential for hyphal tip growth. Mol Microbiol 59:1380–1394PubMedCrossRefGoogle Scholar
  46. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCrossRefGoogle Scholar
  47. Thevissen K, Idkowiak-Baldys J, Im YJ, Takemoto J, François IE, Ferket KK, Aerts AM, Meert EM, Winderickx J, Roosen J, Cammue BP (2005) SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis. FEBS Lett 579:1973–1977PubMedCrossRefGoogle Scholar
  48. Treitschke S, Doehlemann G, Schuster M, Steinberg G (2010) The myosin motor domain of fungal chitin synthase V is dispensable for vesicle motility but required for virulence of the maize pathogen Ustilago maydis. Plant Cell 22:2476–2494PubMedCrossRefGoogle Scholar
  49. Tsigos I, Bouriotis V (1995) Purification and characterization of chitin deacetylase from Colletotrichum lindemuthianum. J Biol Chem 270:26286–26291PubMedCrossRefGoogle Scholar
  50. Tsigos I, Martinou A, Kafetzopoulos D, Bouriotis V (2000) Chitin deacetylases: new, versatile tools in biotechnology. Trends Biotech 18:305–312CrossRefGoogle Scholar
  51. Tusnády GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850PubMedCrossRefGoogle Scholar
  52. van den Burg HA, Harrison SJ, Joosten MH, Vervoort J, de Wit PJ (2006) Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection. Mol Plant Microbe Interact 19:1420–1430PubMedCrossRefGoogle Scholar
  53. Weber I, Aßmann D, Thines E, Steinberg G (2006) Polar localizing class v myosin chitin synthases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis. Plant Cell 18:225–242PubMedCrossRefGoogle Scholar
  54. Whiteford JR, Spanu PD (2002) Hydrophobins and the interactions between fungi and plants. Mol Plant Pathol 3:391–400PubMedCrossRefGoogle Scholar
  55. Zampieri E, Balestrini R, Kohler A, Abbà S, Martin F, Bonfante P (2011) The Perigord black truffle responds to cold temperature with an extensive reprogramming of its transcriptional activity. Fungal Genet Biol 48:585–591PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Raffaella Balestrini
    • 1
    • 2
  • Fabiano Sillo
    • 1
    • 2
  • Annegret Kohler
    • 3
  • Georg Schneider
    • 4
  • Antonella Faccio
    • 1
    • 2
  • Emilie Tisserant
    • 3
  • Francis Martin
    • 3
  • Paola Bonfante
    • 1
    • 2
  1. 1.Istituto per la Protezione delle Piante del CNR, UOS TorinoTurinItaly
  2. 2.Dipartimento di Biologia VegetaleUniTOTurinItaly
  3. 3.UMR 1136, INRA-Nancy Université, Interactions Arbres/MicroorganismesChampenouxFrance
  4. 4.Bioinformatics InstituteA*StarSingaporeSingapore

Personalised recommendations