articulo afm trichoderma[1]

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ORIGINAL PAPER

Abstract Transformation systems developed for Tricho-derma spp. were utilized to improve the biocontrol effi-ciency of the mycoparasitic fungus Trichoderma harzia-

num by increasing the copy number of the basic proteinasegeneprb1. The transformants were stable and carried fromtwo to ten copies ofprb1. High levels of expression ofprb1during fungus-fungus interaction were detected whenT. harzianum and Rhizoctonia solani were confronted invitro. In liquid cultures the proteinase was induced by cellwalls ofR. solani . Under greenhouse conditions, incorpo-ration of T. harzianum transformants into pathogen-in-fested soil significantly reduced the disease caused by

R. solani in cotton plants.

Key words Biocontrol Transformation Proteinase Mycoparasitism

Introduction

Biological control of soilborne plant pathogens by antag-onistic microorganisms is a potential non-chemical meansof plant disease control. Trichoderma harzianum is an ac-tive mycoparasite and can be used as a biocontrol agent. Itattacks a large variety of phytopathogenic fungi respon-sible for major crop diseases (Elad et al. 1981). Tricho-

derma spp. interact with plant pathogens in a variety ofways. The initial detectable interaction shows that the hy-phae of the mycoparasite grow directly toward the host by

a chemotropic reaction (Chet and Baker 1981; Chet andElad 1983). When the mycoparasite reaches the host, theirhyphae coil around it (Elad et al. 1983). Following theseinteractions, the mycoparasite penetrates into the host my-celium, by partial degradation of its cell wall (Elad et al.1983; Benhamou and Chet 1993).

It appears that the main mechanism involved in the an-tagonism to pathogenic fungi by T. harzianum is the re-lease of lytic enzymes. The production of extracellular-1, 3 glucanases, chitinases (Elad et al. 1982 1984) and aproteinase (Geremia et al. 1993) increase significantlywhen Trichoderma is grown in a medium supplementedwith either autoclaved mycelium or fungal cell walls.

These enzymes play an important role in the destruction ofthe plant pathogens (Chet and Baker 1981; Chet et al. 1979;Hadar et al. 1979).

In many pathogen-host interactions, proteinases may beused either for penetration into the host tissue or for theutilization of host proteins for nutrition. A correlation be-tween pathogenicity and proteinase activity has been re-ported for plant pathogens (Ries and Albersheim 1973; Pla-dys and Esquerr-Tugaye 1974; Khare and Bompeix 1976),entomopathogenic fungi (Kucera 1980; St Leger et al.1987; Goettel et al. 1989), and fungi pathogenic to humans(Minocha et al. 1972; MacDonald and Odds 1980; Rchel1983). In mycoparasitism, fungal proteases may play a sig-

nificant role in cell-wall lysis, since fungal cell walls con-tain chitin and/or -glucan fibrils embedded in a proteinmatrix (Wessels 1986). Cell wall-degrading enzyme prep-arations are a mixture of several enzymes, but virtually allof them contain some proteases. This activity is necessaryfor the lysis of whole fungal cells (Scott and Schekman1980; Andrews and Asenjo 1987). Among the hydrolyticenzymes produced by T. harzianum, a basic proteinase(Prb1) has been identified. The gene (prb1) coding for thisproteinase has been cloned and characterized (Geremiaet al. 1993). This gene was shown to be active only whenthe fungus was grown in media containingRhizoctonia so-

Curr Genet (1997) 31: 3037 Springer-Verlag 1997

Received: 6 January 1995 / 5 August 1996

Alberto Flores Ilan Chet Alfredo Herrera-Estrella

Improved biocontrol activity of Trichoderma harzianum

by over-expression of the proteinase-encoding gene prb1

ORIGINAL PAPER

A. Flores1 I. Chet2 A. Herrera-Estrella ()Centro de Investigacin y Estudios Avanzados, Unidad Irapuato,Km. 9.6 del Libramiento Norte de la Carretera Irapuato/Len,Apartado Postal 629, Irapuato, Gto., Mexico

Present addresses:1 Instituto de Investigacin en Biologa Experimental, Facultad deQuimica, Universidad de Guanajuato, Apartado Postal 187, Guana-

juato, Gto. 36050, Mexico2 Otto Warburg Center for Agricultural Biotechnology, Faculty ofAgriculture, The Hebrew University of Jerusalem, P.O. Box 12, Re-hovot 76100, Israel

Communicated by O. C. Yoder


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lani cell walls or chitin as the sole carbon source, and wasrepressed by glucose.

In the present paper we report the relatively rapid in-duction ofprb1 byR. solanicell walls. Using direct con-frontation assays, gene transcription was clearly detectedeven before hyphae from both fungi began to interweave.We report for the first time an improvement in biocontrolefficacy by the introduction of multiple copies ofprb1 into

Trichoderma.

Materials and methods

Strains and plasmids. T. harzianum (IMI 206040) was used in all ex-periments. Plasmid pPrBg78 (Fig. 2) is a derivative of pT3T7Lac(Boehringer-Mannheim) carrying a 5.5-kb fragment containing thebasic proteinase genomic clone (Geremia et al. 1993); plasmidpHAT (Herrera-Estrella et al. 1990) is a derivative of pAN7-1 (Puntet al. 1987) and carries the E.coli hygromycin phosphotransferasegene as a dominant selectable marker.

Preparation of protoplasts and transformation protocol. Protoplastpreparation and transformation were carried out essentially as pre-viously described (Laurila et al. 1985; Herrera-Estrella et al. 1990).

Proteinase expression. To study the expression ofprb1 in submergedcultures, mineral medium (MM) with 2% glucose was inoculatedwith 1106 conidia/ml and incubated in an orbital shaker at 200 rpmfor 48 h at 28C. Mycelium was collected, washed with distilled wa-ter and transferred to fresh MM containing either 2% glucose or 0.2%purifiedR. solani cell walls as the sole carbon source. Aliquots wereremoved from each flask at different times. The mycelium from eachsample was immediately harvested, washed with STE buffer (10 mMTris, pH 8.0; 100 mM NaCl and 5 mM EDTA), frozen in liquid ni-trogen and used for RNA extraction.

DNA techniques. Fungal chromosomal DNA was isolated essential-

ly according to the protocol of Raeder and Broda (1985). DNA frag-ments were labeled with [-32P]dCTP using a random primer DNAlabeling kit (Boehringer). DNA hybridization experiments were car-ried out under highly stringent conditions (Sambrook et al. 1989).

RNA. Fungal RNA was isolated essentially according to the large-scale isolation protocol described by Jones et al. (1985). RNA-blotanalysis was performed by standard techniques using 20 g of totalRNA per sample (Sambrook et al. 1989).

SDS-PAGE.Protein samples from T. harzianum were obtained by theinoculation of 10 ml of MM (Chet et al. 1967) containing 0.5% glu-cose with 5 15 conidia/ml, and cultivated for 48 h at 28C. Themycelium was filtered, transferred to fresh MM containing 0.2%

R. solani cell walls as the sole carbon source and grown under agi-tation for 72 h at 28C. The culture filtrate was collected by filtra-

tion through Whatman paper No. 1. Culture filtrates were freeze-dried, re-suspended in small volumes of 10 mM Tris-HCl, pH 7.0,and dialyzed against the same buffer. The concentrated protein sam-ples were subjected to electrophoresis following standard techniques(Laemmli 1970) in 5% and 10% stacking and separating acrylamidegels, respectively. Each lane was loaded with 22 g of protein. Pro-teins were stained according to the silver-nitrate procedure (Wrayet al. 1981).

Protein-blot analysis. Proteins were subjected to electrophoresis inpolyacrylamide-SDS gels, and transferred onto Immobilon mem-branes (Towbin et al. 1979). Blots were sequentially treated with rab-bit anti-Prb1 and alkaline phosphatase-conjugated goat anti-rabbitIgG. Phosphatase color development was performed by incubationwith BCIP and NBT.

Protease activity. Protease activity was measured in 500-l reactionmixtures containing 520 l of induced culture filtrates (see above)and 0.5 mM Suc-Ala-Ala-Pro-Phe-pNA in 50 mM MOPS, pH 7.0.The reaction mixture was incubated at 37C for 20 min and stoppedby the addition of 500 l of ice-cold water. Absorbance at 405 nmwas measured immediately. Activity was expressed as nmol ofp-nitroaniline released in 1 min. Specific activity was referred to1 mg of protein. The protein content of the culture supernatants wasdetermined using the BioRad microassay kit.

Direct confrontation experiments. Confrontation assays in vitro be-tween T. harzianum andR. solani were carried out as follows: agarplugs cut from growing colonies of each fungus were placed, on op-posite sides, in 9-cm plates containing MM-2% agar plus 0.3% glu-cose and covered with sterile cellophane sheets. Control plates wereinoculated withR. solani or T. harzianum alone. Fungi were allowedto grow at 28C, and mycelia were collected before they touchedeach other (day 3), and when the interaction zone was about 1 cmwide (day 6). At day 3, only Trichoderma mycelia 0.5 cm away fromthe original inoculum and without spores were collected; at day 6,all mycelia in the area of interaction were recovered. Equivalentzones were harvested from control plates. All plates were manipu-lated and grown in the dark (Carsolio et al. 1994).

Greenhouse experiments. Experiments were carried out in a sandyloam soil consisting of 82.3% sand, 2.3% silt, 15% clay and 0.4%organic matter, pH 7.4, with a moisture holding capacity of 12.2%.The temperature ranged from 27 to 30C. Daily irrigation was pro-vided. T. harzianum was added to the soil in a wheat bran/peat prep-aration mixture (0.5%, w/w). Chopped potato soil of R. solani wasprepared according to Ko and Hora (1971) and used for soil infesta-tion. T. harzianum andR. solani were added to the soil at the sametime. Cotton seedlings were used in the experiments which were per-formed in six replicates, using plastic pots, each containing 0.5 kgof soil (ten plants/replicate). Each experiment was repeated twice.

Results

Expression ofprb1 in submerged cultures

In order to determine the actual time at whichprb1 mRNAbegins to accumulate, we carried out a study of prb1 ex-pression. Samples of T. harzianum mycelium grown in thepresence ofR. solani cell walls or glucose were collectedat different times of incubation and RNA was extracted forNorthern analysis. Figure 1 shows that the prb1 messageis detectable as early as 4 h after transfer to cell wall-con-

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Fig. 1 Expression ofprb1 during growth of T. harzianum in the pre-sence ofR. solani cell walls. Total RNA (20 g) extracted from cul-tures grown in glucose (G)- or cell wall (C)-containing media, weresubjected to electrophoresis in an agarose gel under denaturing con-ditions. RNA was transferred onto a nylon membrane and hybridi-zed against a 535-bp 32P-labeled PstI fragment containing part of thecoding sequence ofprb1, or a human 28s rDNA clone. The resultsare representative of three experiments


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taining media, and accumulates until 48 h, after which timethe message decreases. Densitometric analysis of theNorthern indicated that accumulation of the message oc-curs in a linear fashion. No expression was found at anytime in the glucose-supplemented cultures. In control ex-

periments the same blot was hybridized against a human28s rDNA clone which showed no significant variationthroughout the experimental period.

Transformation of T. harzianum

Because of the correlation between the expression ofprb1and the presence of cell walls in the media, the relativelyrapid accumulation of its mRNA upon transfer to cell wall-containing media, and the potential role of a proteinasesuch as Prb1 in mycoparasitism, we set up a series of ex-periments to test the relevance of this enzyme in the Tri-

choderma-Rhizoctonia interaction. An obvious way to an-alyze this was to increase the gene dosage in a given strain,which could result in the generation of an improved bio-control agent.

Two different plasmids, pPrBg78 (Fig. 2), carrying thebasic proteinase genomic clone including 2.5 kb of the pro-moter region, and pHAT, carrying the hygromycin phos-photransferase (hph) gene, were introduced into T. harzi-anum protoplasts by co-transformation. Protoplasts trans-formed with the plasmids were selected for hygromycinresistance. Monosporic cultures from eight primary trans-formants (P1P8) were selected for further analysis.

Southern analysis of transformants.

Monosporic cultures of the selected transformants were

subjected to Southern analysis to test for the presence ofprb1. DNA from transformants (P1P8) and the wild-typestrain was isolated, digested with BamHI or EcoRI, andsubjected to agarose-gel electrophoresis. When DNA wasdigested withBamHI, a strongly hybridizing band of ap-proximately 8 kb corresponding to the complete pPrBg78plasmid was observed in all transformants (data notshown). These results demonstrate that all the selectedstrains originated by co-transformation and that integra-tion of pPrBg78 had not occurred by gene replacement. Asshown in Fig. 3 A (lanes 18), following digestion with

EcoRI all transformants gave the expected hybridizingband at 5.5 kb corresponding toprb1. As judged from both

the intensity of the bands observed upon hybridization andthe pattern ofBamHI-digested DNA (data not shown), in-tegration most probably occurred in multiple copies ar-ranged in tandem, as has previously been described inT. harzianum (Goldman et al. 1990; Herrera-Estrella et al.1990). Transformant P1 showed two extra bands (Fig. 3 A,lane 1) which were probably generated by DNA rearrange-ments. As a control (Fig. 3 B) the same blot was hybridizedagainst the endochitinase gene ech42, a single-copy gene(Carsolio et al. 1994). Densitometric analysis of bothautoradiograms allowed us to standardize and estimate thenumber of copies in the different transformants. The copy

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Fig. 2 Map of plasmid pPrBg78. The size of the complete plasmidis indicated in base pairs. Relevant restriction sites are indicated andnumbers in brackets indicate the relative position of the site.prb1basic proteinase gene; T3 phage T3 promoter; T7phage T7 promo-ter;AmpR bacterial ampicilin resistance cassette; ori bacterial ori-gin of replication

Fig. 3 Southern-blot analysisof transformants. Total DNAfrom transformants P1, P2, P3,P4, P5, P6, P7 and P8 ( lanes18) and the wild-type (lane 9)was extracted, digested with

EcoRI, and equal amounts ofDNA were subjected to electro-phoresis in a 0.8% agarose gel.Samples were blotted onto anylon membrane and hybridi-zed with A a 32P-labeled PstIfragment from pPrBg78 contain-ing part of theprb1 coding re-gion or B a 32P-labeled cDNAfragment of the ech42 gene.

Numbers on the left indicate themolecular size (kb) of PstIstandards


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number for transformants P1P8 was 7, 6, 10, 9, 4, 3, 5 and4, respectively.

Comparative expression analysis

Steady state mRNA

RNA-blot analysis of total RNA extracted from the wild-type and transformant strains after a 3-day period of growthin media containingR. solani cell walls as the sole carbonsource (Materials and methods) showed that prb1 messen-ger RNA accumulated at different levels (Fig. 4). These re-sults demonstrate a significantly higher level of prb1mRNA for most of the transformants analyzed comparedto the wild-type strain (Fig. 4, lane 9), except in the caseof transformant P3 which showed an equivalent level. Den-

sitometric analysis of the autoradiography indicated thatthe levels of prb1 mRNA accumulated by transformantsP1P8 was 8, 4, 1, 7, 7, 4, 4, and 5-fold that of the wild-type, respectively. The estimated values for the level ofmRNA were standardized according to the hybridizationsignal obtained in the control experiment using the human28s rDNA clone as a probe.

Analysis of the proteins secreted during inducingconditions

Culture filtrates of T. harzianum were obtained as de-

scribed (see Materials and methods), dialyzed, and freeze-dried (see SDS-PAGE). The protein concentrate obtainedin this way was subjected to SDS-PAGE, transferred ontoa Immobilon PVDF membrane, and probed using polyclo-nal antibodies raised against Prb1. Extracts from the dif-ferent transformants analyzed showed very similar patternsof protein bands among them. However, several differ-ences between the protein pattern of the transformants andthe wild-type were observed. The most evident differencecorresponded to an approximately 31-kDa protein band(Fig. 5 A). Protein-blot analysis showed that the 31-kDaprotein reacted with Prb1 antibodies (Fig. 5 B). The cul-

ture filtrate of transformant P8 showed four bands that wererecognized by the antiserum (Fig. 5 B, lane 8). Two of thesebands could be degradation products of Prb1. However, theorigin of the third cross-reacting band, also present in theculture filtrates of transformants P5, P6 and P7 (Fig. 5 B,lanes 57), is still unclear. These results demonstrate that

larger quantities of the proteinase were secreted by the dif-ferent transformants as compared to the wild-type. Densit-ometric analysis of the protein-blot indicated that the lev-els of proteinase secreted by the transformants P1P8 was1.5-, 3.4-, 1.4-, 2.0-, 7.7-, 10.2-, 7.0- and 24.8-fold that ofthe wild-type strain, respectively. Furthermore, measure-ments of protease activity using the most specific avail-able substrate for the basic proteinase (Geremia et al. 1993)indicated that all transformants had a higher activity thanthe wild-type (Table 1). Experiments carried out with less-specific substrates, such as azocasein, also indicated higherprotease activity levels in the transformants, although with

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Fig. 4 RNA-blot analysis of proteinase-transformants. Total RNAwas extracted from mycelium grown in the presence ofR. solani cellwalls. RNA (20 g) was subjected to electrophoresis, blotted and hy-bridized using a 535-bp PstI fragment of pPrBg78 containing the

prb1 gene, or a human 28 rDNA clone, as a probe.Lanes 18trans-formants P1-P8; lane 9 wild-type. The results are representative oftwo different experiments

Fig. 5A, B Polyacrylamide-gel electrophoresis of extra-cellularproteins. Equal amounts (22 g) of freeze-dried extra-cellular pro-teins from cultures grown with cell walls of R. solani were subjec-ted to SDS-PAGE.Lanes 18transformants P1P8; lane 9 wild-ty-pe; lane 10 low-molecular-weight markers (BioRad). The results are

representative of three different experiments. A silver nitrate-stai-ned gel. Migration of molecular-weight standards is indicated at theright. B protein blot, probed with anti-Prb1 antibodies (1:2000), andvisualized with a phosphatase-conjugated goat anti-rabbit second an-tibody


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less marked differences, when compared to the wild-type(data not shown).

Expression of prb1 during the T. harzianum-R. solaniinteraction

To determine whether the expression of enzyme activityobserved in cell wall-containing media correlated with thein vivo fungus-fungus interaction, direct confrontation as-says were performed as described in Materials and meth-ods. Total RNA was extracted from mycelium correspond-ing to the area of interaction between T. harzianum (wild-type or transformant P6) and R. solani (Fig. 6 A, shadedarea) and subjected to RNA-blot analysis. Hybridizationwith theprb1 gene showed a clear induction of proteinasemRNA in the presence of R. solani in both transformantand wild-type (Fig. 6 B, lanes 2,4,7 and 9). Under theseconditions, the amount of mRNA was up to 25-fold higherin the transformant than in the wild-type. The mycelia ofthe wild-type and the transformant grown in the same con-ditions but withoutR. solani showed detectable basal lev-els ofprb1 mRNA, which were always higher in the trans-formant (Fig. 6 B, lanes 3 and 8) than in the wild-type(Fig. 6 B, lanes 1 and 6). No hybridization was detectedwith RNA from pureR. solani cultures grown in the sameconditions (Fig. 6 B, lanes 5 and 10). As expected, therewas no cross-hybridization ofprb1 with DNA fromR. so-lani, even at low stringency (data not shown). Note that inthe 6-day-old samples, the amount ofprb1 RNA was prob-ably underestimated, because the total RNA sample wasa mixture of T. harzianum andR. solani RNAs (Fig. 5 B,lane 7). Control experiments, carried out using a human28s rDNA clone, showed no significant variation in thelevels of the corresponding messenger RNA.

Greenhouse experiments

The successful over-expression ofprb1 driven by its ownpromoter prompted us to test the hypothesis that a strain

over-expressing a gene encoding a lytic enzyme involvedin mycoparasitism should be a more effective mycopara-site. For this purpose, greenhouse experiments were car-ried out to compare the biocontrol efficacy of the wild-typestrain and four out of the eightprb1 transformants derivedfrom it. The four transformants used, which were selectedon the basis of gene dosage, carried from two to six extra-copies of the gene.

A wheat bran/peat inoculum preparation of T. harzia-num (wild-type and proteinase transformants) was added

toR. solani-infested soil. Disease incidence in cotton seed-lings attacked byR. solani in the control and the Tricho-derma-treated soils were 43.5 and 27.5%, respectively. Thecorresponding data for plants attacked byR. solani in theP1, P2, P5, and P6 treated soils were 16.6, 5.3, 6.8, and10.5%, respectively (Fig. 7). When analyzed by Duncansmultiple range test (P = 0.05), treatments fell into threesignificantly different groups. The control experimentwhere soil was inoculated with the pathogen alone corre-sponds to group a. Groups b and c encompass treatmentswith transformants P2, P5 and P6 and with the wild-type,respectively. According to this analysis, treatments with

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Table 1 Protease activity in Trichoderma harzianum cultures. Thetable shows the results of a representative experiment using Suc-Ala-Ala-Pro-Phe-pNA as substrate (see Materials and methods)

Strain Specific activitya Relative activityb

P1 36.36 5.57P2 76.25 11.69P3 50.27 7.71P4 19.01 2.91P5 81.67 12.52P6 75.20 11.53P7 137.34 21.06P8 39.04 5.98WT 6.52 1.00WT-glucosec 0.18 0.02

a mmoles of pNA liberated min1 mg1b Ratio specific activity of the strain/specific activity of wild typec 0.5% glucose as carbon source instead ofR. solani cell walls

Fig. 6 RNA-blot analysis from direct confrontation assays. A sche-matic representation of the confrontation assays. Th denotesT. harzianum and RsR. solani. Total RNA was extracted from cellsin the shaded area and subjected to RNA-blot analysis using 20 gper lane. B autoradiography of the RNA blot after hybridizationagainst a PstI fragment of the prb1 gene or a human 28s rDNAclone.Lanes 1, 2, 6 and 7wild-type; lanes 3, 4, 8 and 9 transformantP6; lanes 1, 3, 6 and 8 no interaction controls; lanes 5 and 10

R. solani alone. The results are representative of two independentexperiments


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transformant P1 could belong to either group b or c. How-ever, following a comparison of the results by Dunnettstest (based on variance analysis), treatments with all trans-formants, except P1, are significantly different from thosewith the wild-.type. All experiments including Tricho-derma as antagonist were significantly different from thecontrol withR. solani alone. These data indicate that up toa 5-fold increase in the biocontrol efficiency of Tricho-

derma strains was achieved by over-expressing a singlegene.

Experiments using other genes coding for mycolytic en-zymes resulted in no significant difference in terms of plantprotection when compared to the wild-type strain (unpub-lished results). Such experiments were carried out using atleast five independent transformants in each case. Thus,although the greenhouse experiments were made by com-paring different prb1 transformants with the wild-typestrain, and not with a transformant carrying the vectoralone, no significant difference between the wild-typestrain and such a control should be expected.

Discussion

Despite the well known production of extra-cellular pro-teinases by hyper-parasitic fungi, little is known about theirphysiological role in the interaction with their host. Prb1production in Trichoderma is controlled by two mecha-nisms: it is induced by the presence of a phytopathogenic

fungus, or its cell walls, and repressed by glucose (Gere-mia et al. 1993). Similarly, proteinase production by theentomopathogenic fungus Metarhizium anisopliae is in-duced both by its host and by starvation (St Leger et al.1988). The involvement of the proteinase Prb1 in mycop-arasitism has been suggested by Geremia et al. (1993) whoobserved proteinase induction after growing Trichodermain media containing purified cell walls ofR. solani. Theirdata indicated thatprb1 mRNA accumulated after 24 h butfailed to do so when 2% glucose was present in the media,most probably due to glucose repression. However, it couldbe expected thatprb1 expression was switched on earlierif it was actually induced by cell walls, since these contain

chitin and -1,3-glucan fibrils embedded in a protein ma-trix. In T. harzianum, induction of the proteinase gene(prb1) occurs in a relatively short time (4 h) after contactwith the phytopathogen cell walls or even before physicalcontact takes place as shown by direct confrontation as-says (Fig. 6). In terms of prey/predator relationship thisrepresents an advantage for Trichoderma because it canvery rapidly stop growth of its prey.

Increasing the copy number of a gene does not alwaysresult in an additive effect. Furthermore, several mecha-nisms have been described to inactivate gene duplicationsin fungi (Selker 1991; Pandit and Russo 1992). In our case,multiple copies of the plasmid pPrBg78 integrated in tan-

dem, as shown by Southern analysis, resulted in a higherlevel of bothprb1 messenger and protein, although no di-rect correlation was observed between the levels of mRNAand protein.

Transformants containing multicopies ofprb1 showed amuch higher level of basal expression than the wild-type.This can be explained if the gene is being transcribed by apromoter sequence nearby its site of integration, or alterna-tively by an additive effect of the basal expression of eachcopy ofprb1 integrated into the transformant genome. Thefact that gene expression maintained its inducibility sup-ports the second hypothesis. It is noteworthy that the levelsofprb1 mRNA in the 6-day-old samples withoutR. solani

was higher than that of the 3-day-old culture for both trans-formant and wild-type strains (Fig. 6 B, compare lane 1 withlane 6 and lane 3 with lane 8). This increase may result fromthe induction ofprb1 by products of an autolytic process orby a dual control of gene expression involving starvationand actual induction by the presence of the host. However,it seems unlikely that starvation alone could induce pro-teinase production since growth of the wild-type strain inmedia with low glucose as the sole carbon source (0.5% glu-cose only at the start of the culture) resulted in much loweractivity levels that when the strain was grown inR. solanicell walls as the carbon source (Table 1).

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Fig. 7 Plant disease control by T. harzianum transformants. Six re-plicates of ten cotton plants were used for each treatment 16. Co-lumns headed with different letters (ac) are significantly different(P = 0.05) according to Duncans multiple range test. (1) Control,R.solani in soil, (2)R. solani + wild-type T. harzianum, (3)R. solani+ P1 transformant, (4)R. solani + P2 transformant, (5)R. solani +P5 transformant, (6)R. solani + P6 transformant


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A comparative analysis of theprb1 mRNA, Prb1 proteinlevels, and protease activity of the four transformants usedin the greenhouse experiments revealed that strains P1 andP5 produced twice the amount of messenger as comparedto transformants P2 and P6 (Fig. 4). Transformant P1, onthe other hand, produced less enzyme protein and exhibitedlower activity levels than the rest of them. In contrast, trans-formant P2 which produced lower levels of mRNA, showed

activity levels comparable to those of transformants P5 andP6. These data led to the conclusion that a highprb1 mRNAproduction does not always result in high protein levelsand/or activity (see Figs. 4, 5 B and Table 1). Strains testedin greenhouse experiments showed a gradient of enzymeprotein production in the order P6>P5>P2>P1 and of pro-teinase activity in the order P5>P6>P2>P1. However, theresults of this test indicated that a transformant, such as P2,with intermediate levels of proteinase production and pro-tease activity, confers more protection than others withhigher levels of enzyme protein and activity, such as P5 andP6. This might be due to the fact that the growth rate of allthe transformants was lower than that of the wild-type, with

transformant P2 being the least affected, followed by P5 andP6 which grew at comparable rates and P1 which grew atless than half the rate of the wild-type strain, as determinedby the measurement of both protein content and dry weight(data not shown). However, we can not rule out the effectof partial or full degradation of other proteins important inthe mycoparasitic process under conditions of high produc-tion of Prb1. This possibility is in line with the different pro-tein patterns observed for the wild-type and the transfor-mants. The lower growth rates of the transformants may re-sult from mutations caused by the insertion of transformantDNA in the fungus genome or by a slightly toxic effect ofproteinase over-expression. The strategy used in this work

for the improvement of fungal mycoparasitic potential, byincreasing the copy number of a gene with highly specificexpression encoding a lytic enzyme, was substantiated bya significant increase in the protection of plants against theattack of an agrobiologically important pathogen such as

R. solani. This increase was up to 5-fold when comparedwith the original strain which was isolated for its effective-ness as a biocontrol agent.

Our results demonstrate that the proteinase Prb1 playsan important role in biological control by T. harzianum.This represents a major step towards the production of im-proved strains, because those which are good colonizers,or more efficient antibiotic producers, could be easily mod-

ified in their ability to produce lytic enzymes. However, afine physiological analysis of such strains should be runbefore they are used in field experiments to verify that theproduction of antibiotics or lytic enzymes other than pro-teinase, as well as growth rate and other colonizing prop-erties, have not been affected.

Acknowledgements We thank Drs. Jos Ruz-Herrera, June Simp-son, Luis Herrera-Estrella and Everardo Lpez Romero for criticalreading of the manuscript, Mrs. M. Abramski for excellent assistancein the greenhouse experiments and Ana Gutirrez-Moraga for helpin the kinetic experiments. This work was supported by the EEC con-tract TS3-CT92-0140 and the Chais Family Charity Foundation.

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