Applied Microbiology and Biotechnology

, Volume 65, Issue 1, pp 46–55 | Cite as

Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity

  • Belen Suarez
  • Manuel Rey
  • Pablo Castillo
  • Enrique Monte
  • Antonio Llobell
Biotechnologically Relevant Enzymes and Proteins


Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism. Several proteins with proteolytic activity were detected in culture filtrates from T. harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity. The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7–4.9. The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity. Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1. The protein sequence showed <43% identity to mammal trypsins and 47–57% to other fungal trypsin-like proteins described thus far. Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media. The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations. This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T. harzianum CECT 2413. A role for PRA1 in the protection of plants against pests and pathogens provided by T. harzianum CECT 2413 is proposed.


Chitin Trichoderma Culture Filtrate Fungal Cell Wall Trichoderma Strain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Ahman J, Johansson T, Olsson M, Punto PJ, van den Hondel CAMJJ, Tunlid A (2002) Improving the pathogenicity of a nematode-trapping fungus by genetic engineering of a subtilisin with nematotoxic activity. Appl Environ Microbiol 68:3408–3415CrossRefPubMedGoogle Scholar
  2. Ait-Lahsen H, Soler A, Rey M, de la Cruz J, Monte E, Llobell A (2001) Molecular and antifungal properties of an exo-α-glucanase, AGN13.1, from the biocontrol fungus Trichoderma harzianum. App Environ Microbiol 67:5833–5839CrossRefGoogle Scholar
  3. Benitez T, Limon MC, Delgado-Jarana J, Rey M (1998) Glucanolytic and other enzymes and their genes. In: Kubicek CP, Harman GE (eds) Trichoderma and Gliocladium. Enzymes, biological control and commercial applications, vol 2. Taylor and Francis, London, pp 101–127Google Scholar
  4. Bertagnolli BL, Dal Soglio FK, Sinclair JB (1996) Extracellular enzyme profiles of the fungal pathogen Rhizoctonia solani isolate 2B-12 and of two antagonists, Bacillus megaterium strain B153-2-2 and Trichoderma harzianum isolate Th008. I. Possible correlations with inhibition of growth and biocontrol. Physiol Mol Plant Pathol 48:145–160CrossRefGoogle Scholar
  5. Biely P, Tenkanen M (1998) Enzymology of hemicellulose degradation. In: Kubicek CPHarman GE (eds) Trichoderma and Gliocladium. Enzymes, biological control and commercial applications, vol 2. Taylor and Francis, London, pp 25–47Google Scholar
  6. Bonants PJ, Fitters PF, Thijs H, den Belder E, Waalwijk C, Henfling JW. (1995) A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs. Microbiology 141:775–784PubMedGoogle Scholar
  7. Bradford M (1976) A rapid and sesitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Brants A, Brown CR, Earle ED (2000) Trichoderma harzianum endochitinase does not provide resistance to Meloidogyne hapla in transgenic tobacco. J Nematol 32:289–296Google Scholar
  9. Campbell CL, Madden LV (1990) Introduction to plant disease epidemiology. Wiley, New YorkGoogle Scholar
  10. Carlile AJ, Bindschedler LV, Bailey AM, Bowyer P, Clarkson JM, Cooper RM (2000) Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum. Mol Plant Microbe Interact 13:538–550PubMedGoogle Scholar
  11. Cortes C, Gutierrez A, Olmedo V, Inbar J, Chet I, Herrera-Estrella A (1998) The expression of genes involved in parasitism by Trichoderma harzianum is triggered by a diffusible factor. Mol Gen Genetics 260:218–225CrossRefGoogle Scholar
  12. De la Cruz J, Hidalgo-Gallego A, Lora JM, Benitez T, Pintor Toro JA, Llobell A (1992) Isolation and characterization of three chitinases from Trichoderma harzianum. Eur J Biochem 206:856–867Google Scholar
  13. De Marco JL, Felix CR (2002) Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches’ bromm disease. BMC Biochem 3:3–9CrossRefPubMedGoogle Scholar
  14. Donzelli BGG, Harman GE (2001) Interaction of ammonium, glucose, and chitin, and chitin regulates the expression of cell wall-degrading enzymes in Trichoderma atroviride strain P1. Appl Environ Microbiol 67:5643–5647Google Scholar
  15. Elad Y, Kapat A (1999) The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea. Eur J Plant Pathol 105:177–189CrossRefGoogle Scholar
  16. Fleet G, Phaff HJ (1974) Glucanases in Schizosaccharomyces: isolation and properties of the cell wall associated β-1,3-glucanases. J Biol Chem 249:1717–1728PubMedGoogle Scholar
  17. Flores A, Chet I, Herrera-Estrella A (1997) Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1. Curr Genet 31:30–37CrossRefPubMedGoogle Scholar
  18. Garcia-Carreño FL, Dimes LE, Haard NF (1993) Substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal Biochem 214:65–69CrossRefPubMedGoogle Scholar
  19. Geremia RA, Goldman GH, Jacobs D, Ardiles W, Vila SB, van Montagu M, Herrera-Estrella A (1993) Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum. Mol Microbiol 8:603–613PubMedGoogle Scholar
  20. Goldman GH, Pellizon CH, Marins M, McInervey JO, Goldman MHS (1998) Trichoderma spp. genome and gene structure. In: Kubicek CP, Harman GE (eds) Trichoderma and Gliocladium. Basic biology, taxonomy and genetics, vol 1. Taylor and Francis, London, pp 209–224Google Scholar
  21. Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research, 2nd edn. Wiley, New YorkGoogle Scholar
  22. Harman GE, Björkman T (1998) Potential and existing uses of Trichoderma and Gliocladium for plant disease control and plant growth enhancement. In: Kubicek CP, Harman GE (eds) Trichoderma and Gliocladium. Enzymes, biological control and commercial applications, vol 2. Taylor and Francis, London, pp 229–265Google Scholar
  23. Higgins DG, Sharp PM (1988) CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73:237–244PubMedGoogle Scholar
  24. Hjeljord I, Tronsmo A (1998) Trichoderma and Gliocladium in biological control: an overview. In: Kubicek CP, Harman GE (eds) Trichoderma and Gliocladium. Enzymes, biological control and commercial applications, vol 2. Taylor and Francis, London, pp 131–151Google Scholar
  25. Holwerda BC, Rogers JC (1992) Purification and characterization of Aleurain. A plant thiol protease functionally homologous to mammalian cathepsin H. Plant Physiol 99:848–855Google Scholar
  26. Kapteyn JC, Montijn RC, Vink E, de la Cruz J, Llobell A, Douwes JE, Shimoi H, Lipke PN, Klis FM (1996) Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked β-1,3/β-1,6-glucan heteropolymer. Glycobiology 3:337–345Google Scholar
  27. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotides sequences. J Mol Evol 2:87–90Google Scholar
  28. Lee YP, Takahashi T (1966) An improved colorimetric determination of amino acids with the use of ninhydrin. Anal Biochem 14:71–77Google Scholar
  29. Lorito M (1998) Chitinolytic enzymes and their genes. In: Kubicek CP, Harman GE (eds) Trichoderma and Gliocladium. Enzymes, biological control and commercial applications, vol 2. Taylor and Francis, London, pp 73–99Google Scholar
  30. Markaryan A, Lee JD, Sirakova TD, Kolattukudy PE (1996) Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides. J Bacteriol 178:2211–2215PubMedGoogle Scholar
  31. Meera MS, Shivana MB, Kageyama K, Hyakumachi M (1994) Plant growth promoting fungi frim zoysiagrass rhizosphere as potential inducers of systemic resistance in cucumbers. Phytopahology 84:1399–1406Google Scholar
  32. Murphy JM, Walton JD (1996) Three extracellular proteases form Cochliobolus carbonum: cloning and targeted disruption of ALP1. Mol Plant Microbe Interact 9:290–297PubMedGoogle Scholar
  33. Nico AI, Rapoport HF, Jimenez-Diaz RM, Castillo P (2002) Incidence and population density of plant-parasitic nematodes associated with olive planting stocks at nurseries in Southern Spain. Plant Dis 86:1075–1079Google Scholar
  34. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6Google Scholar
  35. Olmedo-Monfil V, Mendoza-Mendoza A, Gomez I, Cortes C, Herrera-Estrella A (2002) Multiple environmental signals determine the transcriptional activation of the mycoparasitism related gene prb1 in Trichoderma atroviride. Mol Gen Genom 267:703–712CrossRefGoogle Scholar
  36. Penttilä M, Nevalainen H, Ratto M, Salminen E, Knowles J (1987) A versatile transformation system for the filamentous fungus Trichoderma reesei. Gene 61:155–164PubMedGoogle Scholar
  37. Perona JJ, Hedstrom L, Rutter WJ, Fletterick RJ (1995) Structural origins of substrate discrimination in trypsin and chymotrypsin. Biochemistry 34:1489–1499PubMedGoogle Scholar
  38. Rao MS, Reddy PP, Nagesh M (1996) Evaluation of plant based formulations of Trichoderma harzianum for the management of Meloidogyne incognita on egg plant. Nematol Mediterr 26: 59–62Google Scholar
  39. Reader U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20Google Scholar
  40. Rodriguez Kabana R, Kelley WD, Curl EA (1978) Proteolytic activity of Trichoderma viride in mixed culture with Sclerotium rolfsii in soil. Can J Microbiol. 24:487–490Google Scholar
  41. Rypniewski WR, Hastrup S, Betzel C, Dauter M, Dauter Z, Papendorf G, Branner S, Wilson KS (1993) The sequence and X-ray structure of the trypsin from Fusarium oxysporum. Protein Eng 6:341–348PubMedGoogle Scholar
  42. Saifullah, Thomas BJ (1996) Studies on the parasitism of Globodera rostochiensis by Trichoderma harzianum using low temperature scanning electron microscopy. Afro-Asian J Nematol 6:117–122Google Scholar
  43. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, NYGoogle Scholar
  44. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci 74:5463–5467PubMedGoogle Scholar
  45. Screen SE, St. Leger RJ (2000) Cloning, expression, and substrate specificity of a fungal chymotrypsin. J Biol Chem 275:6689–6694CrossRefPubMedGoogle Scholar
  46. Segers R, Butt TM, Kerry BR, Peberdy JF (1994) The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ. Microbiology 140:2715–2723PubMedGoogle Scholar
  47. Sharon E, Bar-Eyal I, Chet I, Herrera-Estrella A, Kleifeld O, Spiegel Y (2001) Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum. Phytopahology 91:687–693Google Scholar
  48. Smithson SL, Paterson IC, Bailey AM, Screen SE, Hunt BA, Cobb BD, Cooper RM, Charnley AK, Clarkson JM (1995) Cloning and characterisation of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae. Gene 166:161–165CrossRefPubMedGoogle Scholar
  49. Spiegel Y, Chet I (1998) Evaluation of Trichoderma spp. as a biocontrol agent against soilborne fungi and plant-parasitic nematodes in Israel. Integr Pest Manage Rev 3:169–494CrossRefGoogle Scholar
  50. St. Leger RJ, Joshi L, Bidochka MJ, Roberts DW (1995) Protein synthesis in Metarhizium anisopliae growing on host. Mycol Res 99:1934–1040Google Scholar
  51. St. Leger RJ, Joshi L, Bidochka MJ, Rizzo NW, Roberts DW (1996) Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles. Appl Environ Microbiol 62:1257–1264PubMedGoogle Scholar
  52. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignement by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedGoogle Scholar
  53. Uchikoba T, Mase T, Arima K, Yonezawa H, Kaneda M (2001) Isolation and characterization of a trypsin-like protease from Trichoderma viride. Biol Chem 382:1509–1513PubMedGoogle Scholar
  54. Urtz BE, Rice WC (2000) Purification and characterization of a novel protease form Bauveria bassiana. Mycol Res 104:180–186Google Scholar
  55. Viterbo A, Montero M, Ramot O, Friesem D, Monte E, Llobell A, Chet I (2002) Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T. harzianum T-203). Curr Genet 42:114–122CrossRefPubMedGoogle Scholar
  56. Windham GL, Windham MT, Pederson GA (1993) Interaction of Trichoderma harzianum, Meloidogyne incognita, and Meloidogyne arenariaon Trifolium repens. Nematropica 23:99–103Google Scholar
  57. Yedidia I, Benhamou N, Chet I (1999) Induction of defense responses in cucumber plants (Cucumis sativus L.) by the biocontrol agent Trichoderma harzianum. Appl Environ Microbiol 65:1061–1070PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Belen Suarez
    • 1
  • Manuel Rey
    • 2
  • Pablo Castillo
    • 3
  • Enrique Monte
    • 4
  • Antonio Llobell
    • 1
    • 5
  1. 1.1IBVF-CIC Isla de la CartujaCSIC/Universidad de SevillaSevillaSpain
  2. 2.Newbiotechnic S.ASevillaSpain
  3. 3.Instituto de Agricultura SostenibleCSICCordobaSpain
  4. 4.Centro Hispano-Luso de Investigaciones AgrariasUniversidad de SalamancaSalamancaSpain
  5. 5.Centro de Investigaciones Científicas Isla de la CartujaInstituto de Bioquímica Vegetal y FotosíntesisSevillaSpain

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