Abstract
Trichoderma harzianum is a soil-borne filamentous fungus that exhibits biological control properties. T. harzianum can prevent the growth of pathogenic fungi on many types of plant crops, providing a chemically benign alternative to fungicidal agents currently on the market. A proteomic approach was taken to separate and identify proteins from a strain of T. harzianum with well established biocontrol properties. We developed a method of extracting proteins under acidic conditions that increased the solubilisation of alkaline proteins and eliminated acidic cell wall artefacts from micro-organisms in general. Combined with the use of protease inhibitors, this sample preparation method resulted in hundreds of proteins from T. harzianum being extracted and separated by two-dimensional gel electrophoresis. Proteins were identified by a combination of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and liquid chromatography mass spectrometry (LC MS/MS). Manual de novo sequencing was conducted to obtain sequence tags on unidentified proteins. A total of 25 protein spots were positively identified from a whole-cell protein reference map of T. harzianum.
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References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Chet I (1987) Innovative approaches to plant disease control. Wiley, New York, pp 137–160
Cruz J de la, 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 206:859–867
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 8:603–613
Gunasekera TS, Holland RJ, Gillings MR, Briscoe DA Neethling DC, Williams KL, Nevalainen KM (2000) Phenotypic and genetic characterization of Paecilomyces lilacinus strains with biocontrol activity against root-knot nematodes. Can J Microbiol 46:775–783
Harman GE, Kubicek CP (1998) Trichoderma and Gliocladium, vol 2. Enzymes, biological control and commercial application. Taylor and Francis, London, pp 153–171
Harman G E, Hayes CK, Lorito M, Broadway RM, Di Pietro A, Peterbauer CK, Tronsmo A (1993) Chitinolytic enzymes of Trichoderma harzianum: purification of chitobiosidase and endochitinase. Phytopathology 83:313–318
Harman GE, Latorre B, Agosin E, San Martin R, Riegel DG, Nielsen PA, Tronsmo A, Pearson RC (1996) Biological and integrated control of Botrytis bunch rot of grape using Trichoderma spp. Biol Control 7:259–266
Herbert B, Galvani M, Hamdan M, Olivieri E, McCarthy J, Pedersen S, Righetti PG (2001) Reduction and alkylation of proteins in preparation of two-dimensional map analysis: why, when, and how? Electrophoresis 22:2046–573
Herbert B, Hopwood F, Oxley D, McCarthy J, Laver M, Grinyer J, Goodall A, Williams K, Castagna A, Righetti PG (2003a) Beta-elimination: an unexpected artefact in proteome analysis. Proteomics 3:826–831
Herbert BR, Grinyer J, McCarthy JT, Isaacs M, Harry EJ, Nevalainen H, Hunt S, Schulz B, Laver M, Goodall AR, Packer J, Harry J (2003b) Improved two-dimensional electrophoresis of microorganisms after acidic extraction. J Proteome Res (in press)
Kronstad JW (2000) Fungal pathology. Kluwer, Dordrecht, pp 33–64
Kullnig C, Mach RL, Lorito M, Kubicek CP (2000) Enzyme diffusion from Trichoderma atroviride (= T. harzianum P1) to Rhizoctonia solani is a prerequisite for triggering of Trichoderma ech42 gene expression before mycoparasitic contact. Appl Environ Microbiol 66:2232–2234
Lora JM, De la Cruz J, Llobell A, Benitez T, Pintor-Tora 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
Lorito M, Hayes CK, Di Pietro A, Woo SL, Harman GE (1994a) Purification, characterization and synergistic activity of a glucan-1,3-β-glucosidase and an N-acetyl-β-d-glucosaminidase from Trichoderma harzianum. Phytopathology 84:398–405
Lorito M, Hayes CK, Zoina A, Scala F, Del Sorbo G, Woo SL, Harman GE (1994b) Potential of genes and gene products from Trichoderma sp. and Gliocladium sp. for the development of biological pesticides. Mol Biotechnol 2:209–217
Lorito M, Farkas V, Rebuffat S, Bodo B, Kubicek CP (1996) Cell wall synthesis is a major target of mycoparasitic antagonism by Trichoderma harzianum. J Bacteriol 178:6382–6385
Luche S, Santoni V, Rabilloud T (2003) Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 3:249–253
Molloy MP, Herbert BR, Walsh BJ, Tyler MI, Traini M, Sanchez JC, Hochstrasser DF, Williams KL, Gooley AA (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19:837–844
Molloy MP, Phadke ND, Chen H, Tyldesley R, Garfin DE, Maddock JR, Andrews PC (2002) Profiling the alkaline membrane proteome of Caulobacter crescentus with two-dimensional electrophoresis and mass spectrometry. Proteomics 2:899–910
Papavizas GC (1985) Trichoderma and Gliocladium: biology, ecology, and potential for biocontrol. Annu Rev Phytopathol 23:23–54
Pappin DJC, Hojrup P, Bleasby AJ (1993) Rapid identification of proteins by peptide mass fingerprinting. Curr Biol 3:327–332
Peberdy JF (1994) Protein secretion in filamentous fungi—trying to understand a highly productive black box. Trends Biotechnol 12:50–57
Pedersen SK, Harry JL, Sebastian L, Baker J, Traini MD, McCarthy JT, Manoharan A, Wilkins MR, Gooley AA, Righetti PG, Packer NH, Williams KL, Herbert BR (2003) Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins. J Protein Res 2:303–311
Rabilloud T, Blisnick T, Heller M, Luche S, Aebersold R, Lunardi J, Braun-Breton C (1999) Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts. Electrophoresis 20:3603–3610
Righetti PG, Castagna A, Herbert B (2000) Prefractionation techniques in proteome analysis. Anal Chem 73:320A–326A
Samson PR, Milner RJ, Bullard GK (1999) Development of Metarhizium based biopesticides for sugar cane pest management—current progress and future prospects. Proc Aust Sugar Cane Technol 21:156–163
Schirmbock M, Lorito M, Wang YL, Hayes CK, Arisan-Atac I, Scala F, Harman GE, Kubicek CP (1994) Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Appl Environ Microbiol 60:4364–4370
Wilkins MR, Williams KL (1997) Cross-species protein identification using amino acid composition, peptide mass fingerprinting, isoelectric point and molecular mass: a theoretical evaluation. J Theor Biol 186:7–15
Yan JX, Harry RA, Spibey C, Dunn MJ (2000) Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electrophoresis 21:3657–3665
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Grinyer, J., McKay, M., Nevalainen, H. et al. Fungal proteomics: initial mapping of biological control strain Trichoderma harzianum . Curr Genet 45, 163–169 (2004). https://doi.org/10.1007/s00294-003-0474-4
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DOI: https://doi.org/10.1007/s00294-003-0474-4