Advertisement

Current Genetics

, Volume 27, Issue 1, pp 83–89 | Cite as

Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum

  • Irene García
  • José M. Lora
  • Jesús de la Cruz
  • Tahía Benítez
  • Antonio Llobell
  • José A. Pintor-Toro
Original Paper

Abstract

A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to aminoacid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced aminoacid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.

Key words

Trichoderma Chitinase Mycoparasitism Biological control 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adams DJ, Causier BE, Mellor KJ, Keer V, Milling R, Dadi J (1993) Regulation of chitinase and chitin synthase in fungi. Int Symp Chitin Enzymol, Senigallia (Italy), pp 1–2Google Scholar
  2. Adams PB (1990) The potential of mycoparasites for biological control of plant diseases. Annu Rev Phytopathol 28:59–83Google Scholar
  3. Bartnicki-García S (1968) Cell wall chemistry, morphogenesis and taxonomy of fungi. Annu Rev Microbiol 22:87–109Google Scholar
  4. Bauw G De Loose M, Inze D, Van Montagu M, Vandekerchove J (1987) Alteration in the phenotype of plant cells studied by amino-terminal amino-acid sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts. Proc Natl Acad Sci USA 84:4806–4811Google Scholar
  5. Blaak H, Stoch S, Schrempf H (1993) The Streptomyces olivaceoviridens exochitinase gene (chi O1) and its regulation. Int Symp Chitin Enzymol, Senigallia (Italy), p 29Google Scholar
  6. Blaiseau PL, Lafay JF (1992) Primary structure of a chitinase-encoding gene (chi1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases. Gene 120:243–248Google Scholar
  7. Butler AR, O'Donnell RW, Martin VJ, Gooday GW, Stark MJR (1991) Kluyveromyces lactis toxin has an essential chitinase activity. Eur J Biochem 199:483–488Google Scholar
  8. Cabib E (1987) The synthesis and degradation of chitin. In: Meisler A (ed) Advances in enzymology and related areas of molecular biology. Wiley, New York, pp 59–101Google Scholar
  9. Chen AC, Mayer RT, De Loach JR (1982) Purification and characterization of chitinase from the stable fly, Stomoxys calcitrans. Arch Biochem Biophys 216:314–321Google Scholar
  10. Chérif M, Benhamou N (1990) Cytochemical aspects of chitin breakdown during the parasitic action of a Trichoderma sp. on Fusarium oxysporum f. sp. radicislycopersici. Phytopathology 80: 1406–1414Google Scholar
  11. Chet I, Schichler H, Haran S, Appenheim AB (1993) Cloned chitinases and their role in biological control of plant pathogenic fungi. Int Sym Chitin Enzymol, Senigallia (Italy), pp 47–48Google Scholar
  12. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K (1993) Plant chitinases. Plant J 3:31–40Google Scholar
  13. Dawson C, Belloch C, García-López MD, Uruburu F (1990) Catalogue of strains, Spanish type culture collection. Universidad Valencia, ValenciaGoogle Scholar
  14. 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 206:859–867Google Scholar
  15. De la Cruz J, Rey M, Lora JM, Hidalgo-Gallego A, Dominguez F, Pintor-Toro JA, Llobell A, Benitez T (1993) Carbon source control on β-glucanases, chitobiase and chitinase from Trichoderma harzianum. Arch Microbiol 159:316–322Google Scholar
  16. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis for the VAX. Nucleic Acids Res 12:387–395Google Scholar
  17. Elad Y, Chet I, Henis Y (1982) Degradation of plant pathogenic fungi by Trichoderma harzianum. Can J Microbiol 28:719–725Google Scholar
  18. Heijne G von (1983) Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 133:17–21Google Scholar
  19. Howell CR (1987) Relevance of mycoparasitism in the biological control of Rhizoctonia solani by Gliocladium virens. Phytopathology 77:992–997Google Scholar
  20. Humphreys AM, Gooday GW (1984) Properties of chitinase activity from Mucor mucedo: evidence for a membrane-bound zymogenic form. J Gen Microbiol 130:1359–1366Google Scholar
  21. Jones J, Grady K, Suslow T, Bedbrook J (1986) Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. EMBO J 5:467–473Google Scholar
  22. Kohl J, Schlosser E (1992) Antagonism against Rhizoctonia solani and cellulolytic activity of strains of Trichoderma sp. In: James EC, Papavizas GC, Cook RJ (eds) Biological control of plant diseases. NATO ASI Series, 230. Plenum Press, New York London, pp 331–332Google Scholar
  23. Kuranda MJ, Robbins PW (1991) Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J Biol Chem 266:19758–19767Google Scholar
  24. Malcolm BA, Rosenberg S, Corey MJ, Allen JS, Baetselier A, Kirsch JF (1989) Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme. Proc Natl Acad Sci USA 86:133–137Google Scholar
  25. Metraux JP, Burkhart W, Moyer M, Dincher S, Middlesteadt W, Williams S, Payne G, Carnes M, Ryals J (1989) Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase. Proc Natl Acad Sci USA 86:896–900Google Scholar
  26. Murray MG, Thomson WF (1980) Rapid isolation of high-molecular-weight plant DNA. Nucleic Acids Res 8:4321–4325Google Scholar
  27. Papavizas CG (1985) Trichoderma and Gliocladium: biology, ecology and potential for biocontrol. Annu Rev Phytopathol 23:23–54Google Scholar
  28. Ridout CJ, Coley-Smith JR, Lynch JM (1986) Enzyme activity and electrophoretic profile of extracellular protein induced in Trichoderma spp. by cell walls of Rhizoctonia solani. J Gen Microbiol 132:2345–2352Google Scholar
  29. Rouvinen J, Bergfors T, Teeri T, Knowles JKC, Jones TA (1990) Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science 249:380–386Google Scholar
  30. Samac DA, Hironaka CM, Yallaly PE, Shah DM (1990) Isolation and characterization of the genes encoding basic and acidic chitinase in Arabidopsis thaliana. Plant Physiol 93:907–914Google Scholar
  31. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, cold Spring Harbor, New YorkGoogle Scholar
  32. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  33. Stark MJR, Boyd A (1986) The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them. EMBO J 5:1995–2002Google Scholar
  34. Sundheim L (1992) Effect of chitinase-encoding genes in biocontrol of Pseudomonas spp. In: James EC, Papavizas GC, Cook RJ (eds) Biological control of plant diseases. NATO ASI Series, 230. Plenum Press, New York London, pp 331–332Google Scholar
  35. Tsujibo H, Orikoshi H, Tanno H, Fujimoto K, Miyamoto K, Imada C, Okami Y, Inamori Y (1993) Cloning, sequence, and expression of a chitinase gene from a marine bacterium Alteromonas sp. strain O-7. J Bacteriol 175:176–181Google Scholar
  36. Wadsworth GJ, Redinbaug MG, Scandalios JG (1988) A procedures for the small-scale isolation of plant RNA suitable for RNA-blot analysis. Anal Biochem 172:279–283Google Scholar
  37. Watanabe T, Oyanagi W, Suzuki H, Tanaka H (1990) Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type-III homology units of fibronectin. J Biol Chem 265:15659–15665Google Scholar
  38. Watanabe T, Oyanagi W, Suzuki H, Tanaka H (1992) Structure of the gene encoding chitinase-D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class-III plant chitinases. J Bacteriol 174:408–414Google Scholar
  39. Watanabe T, Kobori K, Miyashita K, Fujii T Sakai H, Uchida M, Tanaka H (1993) Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J Biol Chem 268:18567–18572Google Scholar
  40. Yanai K, Takaya N, Kojima N, Horiuchi H, Ohta A, Takagi M (1992) Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes. J Bacteriol 174: 7398–7406Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Irene García
    • 1
  • José M. Lora
    • 1
  • Jesús de la Cruz
    • 2
    • 3
  • Tahía Benítez
    • 4
  • Antonio Llobell
    • 2
    • 3
  • José A. Pintor-Toro
    • 1
  1. 1.Instituto de Recursos Naturales y AgrobiologíaCSICSevillaSpain
  2. 2.Instituto de Bioquímica Vegetal y FotosíntesisCSICSevillaSpain
  3. 3.Universidad de SevillaSevillaSpain
  4. 4.Departamento de GenéticaUniversidad de SevillaSevillaSpain

Personalised recommendations