Oct 2, 2006 - Jacobs/ W. Ardrtes,' Silvia B. Vila/ Marc Van. Montagu^* and Alfredo Herrera-Estrella^. 'Laboratorium voor Genetica, Universiteit Gent, K.L.
Molecular Microbiology (1993) 8(3), 603-613
Molecular characterization of the proteinase-encoding gene, prbi, related to mycoparasitism by Trichoderma harzianum Roberto A. Geremia,^ Gustavo H. Goldman,'^ Dirk Jacobs/ W. Ardrtes,' Silvia B. Vila/ Marc Van Montagu^* and Alfredo Herrera-Estrella^ 'Laboratorium voor Genetica, Universiteit Gent, K.L Ledeganckstraat 35, B-9000Gent, Belgium. ^Centro de Investigacidn y Estudios Avanzados, Apartado Postal 629, 26500 Irapuato, Gto, fvfexico.
Summary The soil fungus Trichoderma harzianum is a mycoptarasitic fungus known for its use as a biocontrol agent of phytopathogenic fungi. Among other factors, Trichoderma produces a series of antibiotics and fungal cell wall-degrading enzymes. These enzymes are believed to play an important role in mycoparasitism. Among the hydrolytic enzymes, we have identified a basic proteinase (Prb1) which is induced by either autoclaved mycelia, fungal cell wall preparation or chitin; however, the induction does not occur in the presence of glucose. The proteinase was purified and biochemically characterized as a serine proteinase of 31 kDa and pi 9.2. Based on the sequence of three intemai peptides, synthetic oligonudeotide probes were designed. These probes allowed subsequent isolation of a cDNA and its corresponding genomic clone. The deduced amino acid sequence indicates that the proteinase is synthesized as a pre-proenzyme and aliows its classification as a serine proteinase. Northem analysis shows that the induction of this enzyme is due to an increase in the corresponding mRNA level.
Introduction Many fungi are the causative agents of a series of plant diseases with an important negative impact on yield, quality, and conservation of many important crops. Despite extensive efforts to find sources of resistance Received 2 October. 1992: revised and accepted 4 January, 1993. 'Present address: Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 0S6S4, USA. 'For con-Mpondence. Tel, (91) 645170; Fax (91) 645349; BITNET: mamon(n'gengenp.njg,ac.be.
which could be inciuded in plant breeding programmes, no solution has been found for many of these diseases. An alternative strategy for limiting fungal disease in crops is the use of naturally occurring fungicides. However, more knowledge is required about how they work before they can be used efficiently. Trichoderma harzianum is a soil fungus that is active as a mycoparasite and has been extensively used as a biocontrol agent because it attacks a large variety of phytopathogenic fungi responsible for major crop diseases. Mycoparasitism has been described as a complex process including several steps. Initially, the mycoparasite hyphae grow towards the host hyphae (Chet e( ai, 1981), attach to the target hyphae and coil around them, presumably recognizing host lectins (Barak et ai, 1985; Elad et at., 1983a,b). Then, apressoria-like structuresareformed(Chete^a/., 1981; Elade? a/., 1983b), after which the mycoparasite penetrates into the host mycelium, apparently by partially degrading its cell walis at localized places (Elad ef ai, 1983b,c). The skeleton of most filamentous fungal cell walls is composed of chitin and p(1.3)-glucans, with the exception of Oomycetes, which contain cellulose (Peberdy, 1990). Proteins and lipids are also important in the celt wall structure (Hunsley and Burnet, 1970). Production of extracellular 3(1.3)-glucanases and chitinases has been observed when Trichoderma is grown on cell walls of several phytopathogens (Chet et ai, 1979; Hadar et ai, 1979; Chet and Baker, 1981). Also, the presence of proteinases in Trichoderma cultures containing Rhizoctonia solani cell walls as sole carbon source was reported by Ridout et ai (1988). However, no further studies were carried out to characterize the hydrolytic enzymes required for cell wall degradation and penetration of the host. Studies on hydrolytic enzymes released by T. harzianum are necessary to understand the mycoparasitic process. Identification of specifically induced enzymes, and isolation of the corresponding genes wili provide tools for studying mycoparasitism at the molecular level. This will allow us, for example, to fuse promoters with reporter genes and to study the spatial and temporal expression. In this paper, we report the identification of a basic proteinase whose secretion is induced by growing T. harzianum in either autoclaved mycelium or cell walls of a phytopathogen (simulated mycoparasitism) (Goldman e(
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LeuTyrLeuG]y*laLeuLeuProilaVaILe-iO2 Ml
1 proenivue CGCTJCTCCTGCAGCICTTCACAAG**GCCTGUGCCGTACCCAACAAGTTC*TCGTCACTCTTiAAG*GGGTGCTTCTATTGArACCGACTCTC*rCTCGCCTGGGT*ACGGAC*TCCA G l l S n T f A S H L A l l V J T n A n H 62
B01 CACCCGTrCGTrGACCAAGCGTAGCACTGCTGGTGTTGAAAAGACr!ATAACATTCArACITGGAArGCTTATGCGGGCGAGrTTGACGAGGAGACAATTGA6CAGArCAAGTCCAACCC srnrArgSerLeuTtirLysArgSerTnrAlaGlYValGluLysTnrTyrAsnIleHiaTrirTrpAsnAlaTyrAlaGlyGluPheAspGluGluTnrlleGJuGlnlleLysSerAsnPp -2 721
CGArgtacgtgtccgctgtattgtgaagataaaagaacaaaaagasgctaatcacactatagGITGCBTCTGTTGAGCCAGACTACAlCATGCATCIGTCGGACATTGTTGAAGACAAGC oAsp valAlaServalGluProAspIyrlleMETHisLeoSerAsprieValGSuAspLysA - 1 i w t u r e enzyiw S41 GTGCTCrGACCACATAATCTGGTGCTCCTTGGGGTCTCGGCACTGTGTCCCATCGCACATCTGGAICCACAAGCTACArrTATGATAGCTCAGCCGGCGCCGGCACCTTTGCCTAIGTGG r A l a L e i j r r r r n r G l n 5 e r G l y A l a P r o r r p G l t i e u G l y T n r V a ] 5 c r t t i s A r g T h r S e r G l y S e r T n p S e r T y r n e T y r A s p S e r S c r A i a G ] y A l a G l y r n r R i ( ^ i a T y ^ ^ JO 961
TTGACICTGGUTCUCACncrCArCiGCAaTTCGGCGGGCGTGCCAGCCTTGGCTACAATGCTGCAGGAGGACAGCATGTCGATACTCTTGGCCATGGTACTCATGITTCCGGAACA* alASDSErGiylleAsriTnrSerHisGlnGlnPfijilYGlyArgAJaSerLeuGlvTyrAsnAJaAlaGlyGlyGlnHisValAsoThrLeuGiyHisGlyTtirHisValSerGlyTnrl ao ^t)pi9 1061 TTGGTGGArcrACATACGGlGTrGCTAAGCAGgtgagctacccuigtcgactttttgttgtgaatacatgaatgcattcaagactaactcagtaetgctcatagGCGAGCCiAATClCl leGiyGiySerTwryrGlyValAlaLysGln AlaSerLeuIleSer 95 1201
GTCAAGGTCTTTGCrGGTGAAAGTGCTTCCACCTCTGTTATCCTTSACGGCTATAACTGGGCTGTAAACGACATCGTCTCGAAAAGCCGTGCTAGCAAGTCTGCTATIAACArGTCGCTT ValLysVBlPtie*laGlyGluSerAlaSerInrSerValIleLeu*3pGlyTyrAsnrrpAla¥a]AsnAspIIei/8lSerLysSerArgAlaSerLysSerAlaI]eAsn«TSerLeu 135
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GGAGGACCTGCCrCATCrACCTGGACGACCGCCATTUCGCAGCCITTAACCAGGGCGTGCrTACCAnGTCGCCGCrGGIAArGGCGACTCICTGGGAAACCCCCAGCCAGTCrCCSGC GlyGlyProAlaSerSerthrTf-pThrTrirAlalleAsnAIaAlaPrieAsnGlnGlyValLeuTrtrllevaUlaAlaGlyAsnGlyAspSerLeuGlvAsnProGlnProValSerGly 175
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irTTrTrrirjriarKrTCCTAACGCCATAACCGTTGCCGCATTGGACATCAACTGGCGCACrGCTTCCnCACCAACGGGCGG rn^SerP^oAJaA3nValP^DA^nAlalleTn^Val*laAlaLeuAap]leAanT^pA^(frnrAlaSerP^eTn^AsnTyf^Gly^l^GlyVal*spvalPne«lyroGlyValAsnIle 215 tip4B 1561 CTGTCGrCirGGATrGGAICCAACACTGCTACCAACACAATCAGCGGTACITCCATGGCrACACCTCACGTTGTTGGCCTEGCCCTCTATCTTCAATCTCTCGAGGGTCTCACCTCTCCG LeuSerSerrroneGlySerAsrtTtirAlaTnrAsnrnrlleSefGlyTriPSerMETAlarrtrProHisValValGlyLeuAlaLeuTyrLeuGlnSenLeuGluGJyLeuinrSerPro 255 leai
JICCGCTGTCACIAACCGAATTAAAGCTCTGGCTACATCTGGTCGTGTAACCGGAAGCCTCAACGGCAGCCCCAACGTTATCArCTTCAACGGAAACAGTTCTTAAATCGTTGATGGAPCG ThrAlayairr>rAsnArglleLy3AlaLeu*laTnrStrGlyArg|?iTT"npGlySerLeuAsnGly5erProAsr
