Curr Genet (1997) 31: 3037

Springer-Verlag 1997

O OR RI IG GI IN NA AL L PA PA P PE ER R

Alberto Flores Ilan Chet Alfredo Herrera-Estrella

Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1

Received: 6 January 1995 / 5 August 1996

Abstract Transformation systems developed for Trichoderma spp. were utilized to improve the biocontrol efficiency of the mycoparasitic fungus Trichoderma harzianum by increasing the copy number of the basic proteinase gene prb1. The transformants were stable and carried from two to ten copies of prb1. High levels of expression of prb1 during fungus-fungus interaction were detected when T. harzianum and Rhizoctonia solani were confronted in vitro. In liquid cultures the proteinase was induced by cell walls of R. solani. Under greenhouse conditions, incorporation of T. harzianum transformants into pathogen-infested 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 antagonistic microorganisms is a potential non-chemical means of plant disease control. Trichoderma harzianum is an active mycoparasite and can be used as a biocontrol agent. It attacks a large variety of phytopathogenic fungi responsible for major crop diseases (Elad et al. 1981). Tricho-

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 de Quimica, Universidad de Guanajuato, Apartado Postal 187, Guanajuato, Gto. 36050, Mexico 2 Otto Warburg Center for Agricultural Biotechnology, Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel Communicated by O. C. Yoder

derma spp. interact with plant pathogens in a variety of ways. The initial detectable interaction shows that the hyphae of the mycoparasite grow directly toward the host by a chemotropic reaction (Chet and Baker 1981; Chet and Elad 1983). When the mycoparasite reaches the host, their hyphae coil around it (Elad et al. 1983). Following these interactions, the mycoparasite penetrates into the host mycelium, by partial degradation of its cell wall (Elad et al. 1983; Benhamou and Chet 1993). It appears that the main mechanism involved in the antagonism to pathogenic fungi by T. harzianum is the release of lytic enzymes. The production of extracellular -1, 3 glucanases, chitinases (Elad et al. 1982 1984) and a proteinase (Geremia et al. 1993) increase significantly when Trichoderma is grown in a medium supplemented with either autoclaved mycelium or fungal cell walls. These enzymes play an important role in the destruction of the plant pathogens (Chet and Baker 1981; Chet et al. 1979; Hadar et al. 1979). In many pathogen-host interactions, proteinases may be used either for penetration into the host tissue or for the utilization of host proteins for nutrition. A correlation between pathogenicity and proteinase activity has been reported for plant pathogens (Ries and Albersheim 1973; Pladys 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; Rchel 1983). In mycoparasitism, fungal proteases may play a significant role in cell-wall lysis, since fungal cell walls contain chitin and/or -glucan fibrils embedded in a protein matrix (Wessels 1986). Cell wall-degrading enzyme preparations are a mixture of several enzymes, but virtually all of them contain some proteases. This activity is necessary for the lysis of whole fungal cells (Scott and Schekman 1980; Andrews and Asenjo 1987). Among the hydrolytic enzymes produced by T. harzianum, a basic proteinase (Prb1) has been identified. The gene (prb1) coding for this proteinase has been cloned and characterized (Geremia et al. 1993). This gene was shown to be active only when the fungus was grown in media containing Rhizoctonia so-

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lani cell walls or chitin as the sole carbon source, and was repressed by glucose. In the present paper we report the relatively rapid induction of prb1 by R. solanicell walls. Using direct confrontation assays, gene transcription was clearly detected even before hyphae from both fungi began to interweave. We report for the first time an improvement in biocontrol efficacy by the introduction of multiple copies of prb1 into Trichoderma.

Protease activity. Protease activity was measured in 500-l reaction mixtures 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 stopped by the addition of 500 l of ice-cold water. Absorbance at 405 nm was measured immediately. Activity was expressed as nmol of p-nitroaniline released in 1 min. Specific activity was referred to 1 mg of protein. The protein content of the culture supernatants was determined using the BioRad microassay kit. Direct confrontation experiments. Confrontation assays in vitro between T. harzianum and R. solani were carried out as follows: agar plugs cut from growing colonies of each fungus were placed, on opposite sides, in 9-cm plates containing MM-2% agar plus 0.3% glucose and covered with sterile cellophane sheets. Control plates were inoculated with R. solani or T. harzianum alone. Fungi were allowed to grow at 28C, and mycelia were collected before they touched each other (day 3), and when the interaction zone was about 1 cm wide (day 6). At day 3, only Trichoderma mycelia 0.5 cm away from the original inoculum and without spores were collected; at day 6, all mycelia in the area of interaction were recovered. Equivalent zones were harvested from control plates. All plates were manipulated and grown in the dark (Carsolio et al. 1994). Greenhouse experiments. Experiments were carried out in a sandy loam 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 provided. T. harzianum was added to the soil in a wheat bran/peat preparation mixture (0.5%, w/w). Chopped potato soil of R. solani was prepared according to Ko and Hora (1971) and used for soil infestation. T. harzianum and R. solani were added to the soil at the same time. Cotton seedlings were used in the experiments which were performed in six replicates, using plastic pots, each containing 0.5 kg of soil (ten plants/replicate). Each experiment was repeated twice.

Materials and methods

Strains and plasmids. T. harzianum (IMI 206040) was used in all experiments. Plasmid pPrBg78 (Fig. 2) is a derivative of pT3T7Lac (Boehringer-Mannheim) carrying a 5.5-kb fragment containing the basic proteinase genomic clone (Geremia et al. 1993); plasmid pHAT (Herrera-Estrella et al. 1990) is a derivative of pAN7-1 (Punt et al. 1987) and carries the E.coli hygromycin phosphotransferase gene as a dominant selectable marker. Preparation of protoplasts and transformation protocol. Protoplast preparation and transformation were carried out essentially as previously described (Laurila et al. 1985; Herrera-Estrella et al. 1990). Proteinase expression. To study the expression of prb1 in submerged cultures, mineral medium (MM) with 2% glucose was inoculated with 1 106 conidia/ml and incubated in an orbital shaker at 200 rpm for 48 h at 28C. Mycelium was collected, washed with distilled water and transferred to fresh MM containing either 2% glucose or 0.2% purified R. solani cell walls as the sole carbon source. Aliquots were removed from each flask at different times. The mycelium from each sample was immediately harvested, washed with STE buffer (10 mM Tris, pH 8.0; 100 mM NaCl and 5 mM EDTA), frozen in liquid nitrogen and used for RNA extraction. DNA techniques. Fungal chromosomal DNA was isolated essentially according to the protocol of Raeder and Broda (1985). DNA fragments were labeled with [-32P]dCTP using a random primer DNA labeling kit (Boehringer). DNA hybridization experiments were carried out under highly stringent conditions (Sambrook et al. 1989). RNA. Fungal RNA was isolated essentially according to the largescale isolation protocol described by Jones et al. (1985). RNA-blot analysis was performed by standard techniques using 20 g of total RNA per sample (Sambrook et al. 1989). SDS-PAGE. Protein samples from T. harzianum were obtained by the inoculation of 10 ml of MM (Chet et al. 1967) containing 0.5% glucose with 5 15 conidia/ml, and cultivated for 48 h at 28C. The mycelium was filtered, transferred to fresh MM containing 0.2% R. solani cell walls as the sole carbon source and grown under agitation for 72 h at 28C. The culture filtrate was collected by filtration through Whatman paper No. 1. Culture filtrates were freezedried, re-suspended in small volumes of 10 mM Tris-HCl, pH 7.0, and dialyzed against the same buffer. The concentrated protein samples were subjected to electrophoresis following standard techniques (Laemmli 1970) in 5% and 10% stacking and separating acrylamide gels, respectively. Each lane was loaded with 22 g of protein. Proteins were stained according to the silver-nitrate procedure (Wray et al. 1981). Protein-blot analysis. Proteins were subjected to electrophoresis in polyacrylamide-SDS gels, and transferred onto Immobilon membranes (Towbin et al. 1979). Blots were sequentially treated with rabbit anti-Prb1 and alkaline phosphatase-conjugated goat anti-rabbit IgG. Phosphatase color development was performed by incubation with BCIP and NBT.

Results

Expression of prb1 in submerged cultures In order to determine the actual time at which prb1 mRNA begins to accumulate, we carried out a study of prb1 expression. Samples of T. harzianum mycelium grown in the presence of R. solani cell walls or glucose were collected at different times of incubation and RNA was extracted for Northern analysis. Figure 1 shows that the prb1 message is detectable as early as 4 h after transfer to cell wall-con-

Fig. 1 Expression of prb1 during growth of T. harzianum in the presence of R. solani cell walls. Total RNA (20 g) extracted from cultures grown in glucose (G)- or cell wall (C)-containing media, were subjected to electrophoresis in an agarose gel under denaturing conditions. RNA was transferred onto a nylon membrane and hybridized against a 535-bp 32P-labeled PstI fragment containing part of the coding sequence of prb1, or a human 28s rDNA clone. The results are representative of three experiments

32 Fig. 3 Southern-blot analysis of transformants. Total DNA from transformants P1, P2, P3, P4, P5, P6, P7 and P8 (lanes 18) and the wild-type (lane 9) was extracted, digested with EcoRI, and equal amounts of DNA were subjected to electrophoresis in a 0.8% agarose gel. Samples were blotted onto a nylon membrane and hybridized with A a 32P-labeled PstI fragment from pPrBg78 containing part of the prb1 coding region or B a 32P-labeled cDNA fragment of the ech42 gene. Numbers on the left indicate the molecular size (kb) of PstI standards

Fig. 2 Map of plasmid pPrBg78. The size of the complete plasmid is indicated in base pairs. Relevant restriction sites are indicated and numbers in brackets indicate the relative position of the site. prb1 basic proteinase gene; T3 phage T3 promoter; T7 phage T7 promoter; AmpR bacterial ampicilin resistance cassette; ori bacterial origin of replication

taining media, and accumulates until 48 h, after which time the message decreases. Densitometric analysis of the Northern indicated that accumulation of the message occurs in a linear fashion. No expression was found at any time in the glucose-supplemented cultures. In control experiments the same blot was hybridized against a human 28s rDNA clone which showed no significant variation throughout the experimental period. Transformation of T. harzianum Because of the correlation between the expression of prb1 and the presence of cell walls in the media, the relatively rapid accumulation of its mRNA upon transfer to cell wallcontaining media, and the potential role of a proteinase such as Prb1 in mycoparasitism, we set up a series of experiments to test the relevance of this enzyme in the Trichoderma-Rhizoctonia interaction. An obvious way to analyze this was to increase the gene dosage in a given strain, which could result in the generation of an improved biocontrol agent. Two different plasmids, pPrBg78 (Fig. 2), carrying the basic proteinase genomic clone including 2.5 kb of the promoter region, and pHAT, carrying the hygromycin phosphotransferase (hph) gene, were introduced into T. harzianum protoplasts by co-transformation. Protoplasts transformed with the plasmids were selected for hygromycin resistance. Monosporic cultures from eight primary transformants (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 of prb1. DNA from transformants (P1P8) and the wild-type strain was isolated, digested with BamHI or EcoRI, and subjected to agarose-gel electrophoresis. When DNA was digested with BamHI, a strongly hybridizing band of approximately 8 kb corresponding to the complete pPrBg78 plasmid was observed in all transformants (data not shown). These results demonstrate that all the selected strains originated by co-transformation and that integration of pPrBg78 had not occurred by gene replacement. As shown in Fig. 3 A (lanes 18), following digestion with EcoRI all transformants gave the expected hybridizing band at 5.5 kb corresponding to prb1. As judged from both the intensity of the bands observed upon hybridization and the pattern of BamHI-digested DNA (data not shown), integration most probably occurred in multiple copies arranged in tandem, as has previously been described in T. 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 rearrangements. As a control (Fig. 3 B) the same blot was hybridized against the endochitinase gene ech42, a single-copy gene (Carsolio et al. 1994). Densitometric analysis of both autoradiograms allowed us to standardize and estimate the number of copies in the different transformants. The copy

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Fig. 4 RNA-blot analysis of proteinase-transformants. Total RNA was extracted from mycelium grown in the presence of R. solani cell walls. RNA (20 g) was subjected to electrophoresis, blotted and hybridized using a 535-bp PstI fragment of pPrBg78 containing the prb1 gene, or a human 28 rDNA clone, as a probe. Lanes 18 transformants P1-P8; lane 9 wild-type. The results are representative of two different experiments

number for transformants P1P8 was 7, 6, 10, 9, 4, 3, 5 and 4, respectively. Comparative expression analysis Steady state mRNA RNA-blot analysis of total RNA extracted from the wildtype and transformant strains after a 3-day period of growth in media containing R. solani cell walls as the sole carbon source (Materials and methods) showed that prb1 messenger RNA accumulated at different levels (Fig. 4). These results demonstrate a significantly higher level of prb1 mRNA for most of the transformants analyzed compared to the wild-type strain (Fig. 4, lane 9), except in the case of transformant P3 which showed an equivalent level. Densitometric analysis of the autoradiography indicated that the levels of prb1 mRNA accumulated by transformants P1P8 was 8, 4, 1, 7, 7, 4, 4, and 5-fold that of the wildtype, respectively. The estimated values for the level of mRNA were standardized according to the hybridization signal obtained in the control experiment using the human 28s rDNA clone as a probe. Analysis of the proteins secreted during inducing conditions Culture filtrates of T. harzianum were obtained as described (see Materials and methods), dialyzed, and freezedried (see SDS-PAGE). The protein concentrate obtained in this way was subjected to SDS-PAGE, transferred onto a Immobilon PVDF membrane, and probed using polyclonal antibodies raised against Prb1. Extracts from the different transformants analyzed showed very similar patterns of protein bands among them. However, several differences between the protein pattern of the transformants and the wild-type were observed. The most evident difference corresponded to an approximately 31-kDa protein band (Fig. 5 A). Protein-blot analysis showed that the 31-kDa protein reacted with Prb1 antibodies (Fig. 5 B). The cul-

Fig. 5 A, B Polyacrylamide-gel electrophoresis of extra-cellular proteins. Equal amounts (22 g) of freeze-dried extra-cellular proteins from cultures grown with cell walls of R. solani were subjected to SDS-PAGE. Lanes 18 transformants P1P8; lane 9 wild-type; lane 10 low-molecular-weight markers (BioRad). The results are representative of three different experiments. A silver nitrate-stained gel. Migration of molecular-weight standards is indicated at the right. B protein blot, probed with anti-Prb1 antibodies (1:2000), and visualized with a phosphatase-conjugated goat anti-rabbit second antibody

ture filtrate of transformant P8 showed four bands that were recognized by the antiserum (Fig. 5 B, lane 8). Two of these bands could be degradation products of Prb1. However, the origin of the third cross-reacting band, also present in the culture 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 different transformants as compared to the wild-type. Densitometric analysis of the protein-blot indicated that the levels of proteinase secreted by the transformants P1P8 was 1.5-, 3.4-, 1.4-, 2.0-, 7.7-, 10.2-, 7.0- and 24.8-fold that of the wild-type strain, respectively. Furthermore, measurements of protease activity using the most specific available substrate for the basic proteinase (Geremia et al. 1993) indicated that all transformants had a higher activity than the wild-type (Table 1). Experiments carried out with lessspecific substrates, such as azocasein, also indicated higher protease activity levels in the transformants, although with

34 Table 1 Protease activity in Trichoderma harzianum cultures. The table shows the results of a representative experiment using Suc-AlaAla-Pro-Phe-pNA as substrate (see Materials and methods) Strain P1 P2 P3 P4 P5 P6 P7 P8 WT WT-glucosec
a b c

Specific activitya 36.36 76.25 50.27 19.01 81.67 75.20 137.34 39.04 6.52 0.18

Relative activityb 5.57 11.69 7.71 2.91 12.52 11.53 21.06 5.98 1.00 0.02

mmoles of pNA liberated min1 mg1 Ratio specific activity of the strain/specific activity of wild type 0.5% glucose as carbon source instead of R. solani cell walls

less marked differences, when compared to the wild-type (data not shown). Expression of prb1 during the T. harzianum-R. solani interaction To determine whether the expression of enzyme activity observed in cell wall-containing media correlated with the in vivo fungus-fungus interaction, direct confrontation assays were performed as described in Materials and methods. Total RNA was extracted from mycelium corresponding to the area of interaction between T. harzianum (wildtype or transformant P6) and R. solani (Fig. 6 A, shaded area) and subjected to RNA-blot analysis. Hybridization with the prb1 gene showed a clear induction of proteinase mRNA in the presence of R. solani in both transformant and wild-type (Fig. 6 B, lanes 2,4,7 and 9). Under these conditions, the amount of mRNA was up to 25-fold higher in the transformant than in the wild-type. The mycelia of the wild-type and the transformant grown in the same conditions but without R. solani showed detectable basal levels of prb1 mRNA, which were always higher in the transformant (Fig. 6 B, lanes 3 and 8) than in the wild-type (Fig. 6 B, lanes 1 and 6). No hybridization was detected with RNA from pure R. solani cultures grown in the same conditions (Fig. 6 B, lanes 5 and 10). As expected, there was no cross-hybridization of prb1 with DNA from R. solani, even at low stringency (data not shown). Note that in the 6-day-old samples, the amount of prb1 RNA was probably underestimated, because the total RNA sample was a mixture of T. harzianum and R. solani RNAs (Fig. 5 B, lane 7). Control experiments, carried out using a human 28s rDNA clone, showed no significant variation in the levels of the corresponding messenger RNA. Greenhouse experiments The successful over-expression of prb1 driven by its own promoter prompted us to test the hypothesis that a strain
Fig. 6 RNA-blot analysis from direct confrontation assays. A schematic representation of the confrontation assays. Th denotes T. harzianum and Rs R. solani. Total RNA was extracted from cells in the shaded area and subjected to RNA-blot analysis using 20 g per lane. B autoradiography of the RNA blot after hybridization against a PstI fragment of the prb1 gene or a human 28s rDNA clone. Lanes 1, 2, 6 and 7 wild-type; lanes 3, 4, 8 and 9 transformant P6; lanes 1, 3, 6 and 8 no interaction controls; lanes 5 and 10 R. solani alone. The results are representative of two independent experiments

over-expressing a gene encoding a lytic enzyme involved in mycoparasitism should be a more effective mycoparasite. For this purpose, greenhouse experiments were carried out to compare the biocontrol efficacy of the wild-type strain and four out of the eight prb1 transformants derived from it. The four transformants used, which were selected on the basis of gene dosage, carried from two to six extracopies of the gene. A wheat bran/peat inoculum preparation of T. harzianum (wild-type and proteinase transformants) was added to R. solani-infested soil. Disease incidence in cotton seedlings attacked by R. solani in the control and the Trichoderma-treated soils were 43.5 and 27.5%, respectively. The corresponding data for plants attacked by R. solani in the P1, P2, P5, and P6 treated soils were 16.6, 5.3, 6.8, and 10.5%, respectively (Fig. 7). When analyzed by Duncans multiple range test (P = 0.05), treatments fell into three significantly different groups. The control experiment where soil was inoculated with the pathogen alone corresponds to group a. Groups b and c encompass treatments with transformants P2, P5 and P6 and with the wild-type, respectively. According to this analysis, treatments with

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Discussion

Fig. 7 Plant disease control by T. harzianum transformants. Six replicates of ten cotton plants were used for each treatment 16. Columns 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

transformant P1 could belong to either group b or c. However, following a comparison of the results by Dunnetts test (based on variance analysis), treatments with all transformants, except P1, are significantly different from those with the wild-.type. All experiments including Trichoderma as antagonist were significantly different from the control with R. solani alone. These data indicate that up to a 5-fold increase in the biocontrol efficiency of Trichoderma strains was achieved by over-expressing a single gene. Experiments using other genes coding for mycolytic enzymes resulted in no significant difference in terms of plant protection when compared to the wild-type strain (unpublished results). Such experiments were carried out using at least five independent transformants in each case. Thus, although the greenhouse experiments were made by comparing different prb1 transformants with the wild-type strain, and not with a transformant carrying the vector alone, no significant difference between the wild-type strain and such a control should be expected.

Despite the well known production of extra-cellular proteinases by hyper-parasitic fungi, little is known about their physiological role in the interaction with their host. Prb1 production in Trichoderma is controlled by two mechanisms: it is induced by the presence of a phytopathogenic fungus, or its cell walls, and repressed by glucose (Geremia et al. 1993). Similarly, proteinase production by the entomopathogenic fungus Metarhizium anisopliae is induced both by its host and by starvation (St Leger et al. 1988). The involvement of the proteinase Prb1 in mycoparasitism has been suggested by Geremia et al. (1993) who observed proteinase induction after growing Trichoderma in media containing purified cell walls of R. solani. Their data indicated that prb1 mRNA accumulated after 24 h but failed to do so when 2% glucose was present in the media, most probably due to glucose repression. However, it could be expected that prb1 expression was switched on earlier if it was actually induced by cell walls, since these contain chitin and -1,3-glucan fibrils embedded in a protein matrix. In T. harzianum, induction of the proteinase gene (prb1) occurs in a relatively short time (4 h) after contact with the phytopathogen cell walls or even before physical contact takes place as shown by direct confrontation assays (Fig. 6). In terms of prey/predator relationship this represents an advantage for Trichoderma because it can very rapidly stop growth of its prey. Increasing the copy number of a gene does not always result in an additive effect. Furthermore, several mechanisms have been described to inactivate gene duplications in fungi (Selker 1991; Pandit and Russo 1992). In our case, multiple copies of the plasmid pPrBg78 integrated in tandem, as shown by Southern analysis, resulted in a higher level of both prb1 messenger and protein, although no direct correlation was observed between the levels of mRNA and protein. Transformants containing multicopies of prb1 showed a much higher level of basal expression than the wild-type. This can be explained if the gene is being transcribed by a promoter sequence nearby its site of integration, or alternatively by an additive effect of the basal expression of each copy of prb1 integrated into the transformant genome. The fact that gene expression maintained its inducibility supports the second hypothesis. It is noteworthy that the levels of prb1 mRNA in the 6-day-old samples without R. solani was higher than that of the 3-day-old culture for both transformant and wild-type strains (Fig. 6 B, compare lane 1 with lane 6 and lane 3 with lane 8). This increase may result from the induction of prb1 by products of an autolytic process or by a dual control of gene expression involving starvation and actual induction by the presence of the host. However, it seems unlikely that starvation alone could induce proteinase production since growth of the wild-type strain in media with low glucose as the sole carbon source (0.5% glucose only at the start of the culture) resulted in much lower activity levels that when the strain was grown in R. solani cell walls as the carbon source (Table 1).

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A comparative analysis of the prb1 mRNA, Prb1 protein levels, and protease activity of the four transformants used in the greenhouse experiments revealed that strains P1 and P5 produced twice the amount of messenger as compared to transformants P2 and P6 (Fig. 4). Transformant P1, on the other hand, produced less enzyme protein and exhibited lower activity levels than the rest of them. In contrast, transformant P2 which produced lower levels of mRNA, showed activity levels comparable to those of transformants P5 and P6. These data led to the conclusion that a high prb1 mRNA production does not always result in high protein levels and/or activity (see Figs. 4, 5 B and Table 1). Strains tested in greenhouse experiments showed a gradient of enzyme protein production in the order P6>P5>P2>P1 and of proteinase activity in the order P5>P6>P2>P1. However, the results of this test indicated that a transformant, such as P2, with intermediate levels of proteinase production and protease activity, confers more protection than others with higher levels of enzyme protein and activity, such as P5 and P6. This might be due to the fact that the growth rate of all the transformants was lower than that of the wild-type, with transformant P2 being the least affected, followed by P5 and P6 which grew at comparable rates and P1 which grew at less than half the rate of the wild-type strain, as determined by the measurement of both protein content and dry weight (data not shown). However, we can not rule out the effect of partial or full degradation of other proteins important in the mycoparasitic process under conditions of high production of Prb1. This possibility is in line with the different protein patterns observed for the wild-type and the transformants. The lower growth rates of the transformants may result from mutations caused by the insertion of transformant DNA in the fungus genome or by a slightly toxic effect of proteinase over-expression. The strategy used in this work for the improvement of fungal mycoparasitic potential, by increasing the copy number of a gene with highly specific expression encoding a lytic enzyme, was substantiated by a significant increase in the protection of plants against the attack of an agrobiologically important pathogen such as R. solani. This increase was up to 5-fold when compared with the original strain which was isolated for its effectiveness as a biocontrol agent. Our results demonstrate that the proteinase Prb1 plays an important role in biological control by T. harzianum. This represents a major step towards the production of improved strains, because those which are good colonizers, or more efficient antibiotic producers, could be easily modified in their ability to produce lytic enzymes. However, a fine physiological analysis of such strains should be run before they are used in field experiments to verify that the production of antibiotics or lytic enzymes other than proteinase, as well as growth rate and other colonizing properties, have not been affected.
Acknowledgements We thank Drs. Jos Ruz-Herrera, June Simpson, Luis Herrera-Estrella and Everardo Lpez Romero for critical reading of the manuscript, Mrs. M. Abramski for excellent assistance in the greenhouse experiments and Ana Gutirrez-Moraga for help in the kinetic experiments. This work was supported by the EEC contract TS3-CT92-0140 and the Chais Family Charity Foundation.

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