Skip to main content
SpringerLink
Log in

Cloning and characterization of the Thcut1 gene encoding a cutinase of Trichoderma harzianum T34

  • Research Article
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

The cloning and characterization of the Thcut1 gene, which encodes a cutinase protein of the biocontrol fungus Trichoderma harzianum T34, is reported. Cutinases, which are secreted enzymes that hydrolyse cutin, belong to a class of serine esterases able to hydrolyze fatty acid esters and emulsified triglycerides. The Thcut1 gene was isolated by screening of a genomic DNA library from EST 2104, generated from a T. harzianum T34 cDNA library constructed under mycoparasitic and nutrient stress conditions, as a probe. Thcut1 shows similarity with fungal cutinase genes and is present as a single copy in the genome of T. harzianum. RNA blot analyses revealed that Thcut1 mRNA is strongly induced in vitro by olive oil and the cutin monomer 16-hydroxy-hexadecanoic acid and that it is repressed by glucose. Significant transcript levels were also detected when strawberry plants or pectin were present in the media and in the absence of glucose. Expression of the Thcut1 gene in Pichia pastoris gave rise to transformants with high esterase activity and a high level of secretion of the THCUT1 protein. Recombinant cutinase secretion at flask level indicated that P. pastoris transformants could be applied to set up the production of this enzyme at industrial scale.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Abu Bakar FD, Abdul Murab AM, Hamid AM, Zamrod Z, Mahadi NM (2005) Induction and expression of cutinase activity during saprophytic growth of the fungal plant pathogen, Glomerella cingulata. Asia Pac J Mol Biol Biotechnol 13:63–69

    Google Scholar 

  • Alfano G, Lewis Ivey ML, Cakir C, Bos JIB, Miller SA, Madden LV, Kamoun S, Hoitink HAJ (2007) Systemic modulation of gene expression in tomato by Trichoderma hamatum 382. Phytopathology 97:429–437

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Carvalho CML, Aires-Barros MR, Cabral JMS (1998) Cutinase structure, function and biocatalytic applications. Electron J Biotechnol 1:160–173

    Article  Google Scholar 

  • Cortés C, Gutiérrez 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 Genet 260:218–225

    Article  PubMed  Google Scholar 

  • Deising H, Nicholson RL, Haug M, Howard RJ, Mendgen K (1992) Adhesion pad formation and the involvement of cutinase and esterases in the attachment of uredospores to the host cuticule. Plant Cell 4:1101–1111

    Article  PubMed  CAS  Google Scholar 

  • Dickman MB, Patil SS (1986) Cutinase deficient mutants of Colletotrichum gloesporioides are non-pathogenic to papaya fruit. Physiol Mol Plant Pathol 28:235–242

    CAS  Google Scholar 

  • Felsenstein J (1985) Confidence-limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  • Gorner W, Durchschlag E, Martinez-Pastor MT, Estruch F, Ammerer G, Hamilton B, Ruis H, Schuller C (1998) Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev 12:586–597

    Article  PubMed  CAS  Google Scholar 

  • Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species—opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56

    Article  PubMed  CAS  Google Scholar 

  • Heinen W (1960) The enzymic degradation of cutin. Acta Bot Neerl 9:167–190

    CAS  Google Scholar 

  • Ilmen M, Thrane C, Penttilä M (1996) The glucose repressor gene cre1 of Trichoderma: isolation and expression of a full-length and a truncated mutant form. Mol Gen Genet 251:451–460

    PubMed  CAS  Google Scholar 

  • Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202

    Article  PubMed  CAS  Google Scholar 

  • Kleman-Leyer KM, Siika-Aho M, Teeri TT, Kirk K (1996) The cellulases endoglucanase I and cellobiohydrolase II of Trichoderma reesei act synergistically to solubilize native cotton cellulose but not to decrease its molecular size. Appl Environ Microbiol 62:2883–2887

    PubMed  CAS  Google Scholar 

  • Kolattukudy PE (1985) Enzymatic penetration of the plant cuticle by fungal pathogens. Annu Rev Phytopathol 23:223–250

    Article  CAS  Google Scholar 

  • Köller W, Parker DM (1989) Purification and characterisation of cutinase from Venturia inequalis. Phytopathology 79:278–283

    Article  Google Scholar 

  • Kulmburg P, Mathieu M, Dowzer C, Kelly J, Felenbok B (1993) Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in Aspergillus nidulans. Mol Microbiol 7:847–857

    Article  PubMed  CAS  Google Scholar 

  • Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    Article  PubMed  CAS  Google Scholar 

  • Lacks SA, Springhorn SS (1980) Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J Biol Chem 255:7467–7473

    PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 22:680–685

    Article  Google Scholar 

  • Lauwereys M, de Geus P, de Meutter J, Stanssenw P, Matthyssens G (1991) In: Alberghina L, Schmid RD, Verger R (eds) Lipases—structure, mechanism and genetic engineering, vol 16. VCH, Weinheim, pp 243–251

  • Li D, Ashby AM, Johnstone K (2003) Molecular evidence that the extracellular cutinase Pbc1 is required for pathogenicity of Pyrenopeziza brassicae on oilseed rape. Mol Plant Microbe Interact 16:545–552

    Article  PubMed  CAS  Google Scholar 

  • Lora JM, de la Cruz J, Llobell A, Benítez T, Pintor-Toro JA (1995) Molecular characterization and heterologous expression of an endo-beta-1, 6-glucanase gene from the mycoparasitic fungus Trichoderma harzianum. Mol Gen Genet 247:639–645

    Article  PubMed  CAS  Google Scholar 

  • Maiti IB, Kolattukudy PE (1979) Prevention of fungal infection of plants by specific inhibition of cutinase. Science 205:507–508

    Article  PubMed  CAS  Google Scholar 

  • Mizutani C, Sethumadhavan K, Howley P, Bertoniere N (2002) Effect of a non-ionic surfactant on Trichoderma cellulase treatments of regenerated cellulose and cotton yarns. Cellulose 9:83–89

    Article  CAS  Google Scholar 

  • Mohamed SA, Farid NM, Hossiny EN, Bassuiny RI (2006) Biochemical characterization of an extracellular polygalacturonase from Trichoderma harzianum. J Biotechnol 127:54–64

    Article  PubMed  CAS  Google Scholar 

  • Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–35

    Article  PubMed  CAS  Google Scholar 

  • 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 10:1–6

    Article  PubMed  CAS  Google Scholar 

  • Oksanen T, Pere J, Buchert J, Viikari L (1997) The effect of Trichoderma reesei cellulases and hemicellulases on the paper technical properties of never-dried bleached kraft pulp. Cellulose 4:329–339

    Article  CAS  Google Scholar 

  • Penttilä M, Nevalainen H, Ratto M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164

    Article  PubMed  Google Scholar 

  • Purdy RE, Kolattukudy PE (1973) Depolymerization of a hydroxy fatty acid biopolymer, cutin, by an extracellular enzyme from Fusarium solani f. pisi: isolation and some properties of the enzyme. Arch Biochem Biophys 159:61–69

    Article  PubMed  CAS  Google Scholar 

  • Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20

    Article  CAS  Google Scholar 

  • Ravagnani A, Gorfinkiel L, Langdon T, Diallinas G, Adjadj E, Demais S, Gorton D, Arst HN Jr, Scazzocchio C (1997) Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA. EMBO J 16:3974–3986

    Article  PubMed  CAS  Google Scholar 

  • Rey M, Llobell A, Monte E, Scala F, Lorito M (2004) Genomics of Trichoderma. In: Khachatourians GG (ed) Fungal genomics applied mycology and biotechnology, vol 4. Elsevier Science, Amsterdam, pp 225–248

    Google Scholar 

  • Rogers LM, Flaishman MA, Kolattukudy PE (1994) Cutinase gene disruption in Fusarium solani f sp pisi decreases its virulence on pea. Plant Cell 6:935–945

    Article  PubMed  CAS  Google Scholar 

  • Royer JC, Nakas JP (1990) Interrelationship of xylanase induction and cellulase induction of Trichoderma longibrachiatum. Appl Environ Microbiol 56:2535–2539

    PubMed  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sanz L, Montero M, Grondona I, Vizcaíno JA, Llobell A, Hermosa R, Monte E (2004) Cell wall-degrading isoenzyme profiles of Trichoderma biocontrol strains show correlation with rDNA taxonomic species. Curr Genet 46:277–286

    Article  PubMed  CAS  Google Scholar 

  • Sanz L, Montero M, Redondo J, Llobell A, Monte E (2005) Expression of an alpha-1,3-glucanase during mycoparasitic interaction of Trichoderma asperellum. FEBS J 272:493–499

    Article  PubMed  CAS  Google Scholar 

  • Sarda L, Desnuelle P (1958) Action de la lipase pancreatique sur les esters en emulsion. Biochem Biophys Acta 30:513–521

    Article  PubMed  CAS  Google Scholar 

  • Sears IB, O’Connor J, Rossanese OW, Glick BS (1998) A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris. Yeast 14:783–790

    Article  PubMed  CAS  Google Scholar 

  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  PubMed  CAS  Google Scholar 

  • Shoresh M, Harman GE (2008) The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach. Plant Physiol 10.1104, 108.123810

    Google Scholar 

  • Suárez MB, Vizcaíno JA, Llobell A, Monte E (2007) Characterization of genes encoding novel peptidases in the biocontrol fungus Trichoderma harzianum CECT 2413 using the TrichoEST functional genomics approach. Curr Genet 51:331–342

    Article  PubMed  Google Scholar 

  • Sweigard JA, Chumley FG, Valent B (1992a) Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea. Mol Gen Genet 232:174–182

    PubMed  CAS  Google Scholar 

  • Sweigard JA, Chumley FG, Valent B (1992b) Disruption of a Magnaporthe grisea cutinase gene. Mol Gen Genet 232:183–190

    PubMed  CAS  Google Scholar 

  • Swofford L (2002) PAUP*: phylogenetic analysis using parsimony (* and other methods). Version 4., Sinauer Associates, Sunderland, Massachusetts.

  • Tanksley SD, Rick CM (1980) Genetics of esterases in species of Lycopersicon. Theor Appl Genet 56:209–219

    Article  CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmouqin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  PubMed  CAS  Google Scholar 

  • van der Hijden HT, Marugg JD, Warr JF, Klugkist J, Musters W, Hondmann DHA (1994) Enzyme-containing surfactants compositions. Patent WO 9403578

  • van Kan JAL, van’t Klooster JW, Wagemakers CAM, Dees DCT, van der Vlugt-Bergmans CJB (1997) Cutinase A of Botrytis cinerea is expressed, but not essential during penetration of gerbera and tomato. Mol Plant Microbe Interact 10:30–38

    Article  PubMed  Google Scholar 

  • Verger R, de Haas GH (1976) Interfacial enzyme kinetics of lipolysis. Annu Rev Biophys Bioeng 5:77–117

    Article  PubMed  CAS  Google Scholar 

  • Vizcaíno JA, González FJ, Suárez MB, Redondo J, Heinrich J, Delgado-Jarana J, Hermosa R, Gutiérrez S, Monte E, Llobell A, Rey M (2006) Generation, annotation, analysis of ESTs from Trichoderma harzianum CECT 2413. BMC Genomics 7:193

    Article  PubMed  Google Scholar 

  • Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM (1997) Isolation of the Pichia pastoris glyceraldehyde-3-P-dehydrogenase gene and regulation and use of its promoter. Gene 186:37–44

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Authors wish to acknowledge the financial support of the Spanish Ministry of Education and Science (AGL2008-0512/AGR), and Junta de Castilla y León (research group GR67 project). We want to recognize the work carried out by J. Álvarez for his technical support. We also wish to thank Dr. Benjamin S. Glick of the Department of Molecular Genetics and Cell Biology of the University of Chicago, for the kind concession of the pIB2 plasmid. We are also grateful to the members of the Proteomics Unit (PROTEORED) for advice and technical help on mass spectrometry and to N. Skinner for supervising the English version of this manuscript.

Author information

Authors and Affiliations

  1. Departamento de Microbiología y Genética, Spanish-Portuguese Centre of Agricultural Research (CIALE), Universidad de Salamanca, Río Duero, 12, 37185, Villamayor, Salamanca, Spain

    M. Belén Rubio, Rosa Hermosa & Enrique Monte

  2. Area de Microbiología. Escuela Universitaria de Ciencias de la Salud, Universidad de León, Campus de Ponferrada, Avda. Astorga s/n, 24400, Ponferrada, Spain

    Rosa E. Cardoza & Santiago Gutiérrez

  3. Departamento de Biología Molecular, Universidad de León, Campus de Ponferrada, Avda. Astorga s/n, 24400, Ponferrada, Spain

    Santiago Gutiérrez

Authors
  1. M. Belén Rubio
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rosa E. Cardoza
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rosa Hermosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Santiago Gutiérrez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Enrique Monte
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Santiago Gutiérrez.

Additional information

Communicated by U. Kück.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material (DOC 123 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rubio, M.B., Cardoza, R.E., Hermosa, R. et al. Cloning and characterization of the Thcut1 gene encoding a cutinase of Trichoderma harzianum T34. Curr Genet 54, 301–312 (2008). https://doi.org/10.1007/s00294-008-0218-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-008-0218-6

Keywords

Search

Navigation