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Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
Correspondence
Prasun K. Mukherjee
prasunmukherjee1{at}gmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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tac1 mutants; of these 11 clones, nine sequences were homologous to secondary metabolism-related gene sequences. Therefore, cAMP signalling positively regulates secondary metabolism in T. virens. This is believed to be the first direct genetic study on the role of cAMP signalling in a Trichoderma sp. Tac1 is also believed to be the first regulatory protein to be identified in T. virens that is involved in growth, germination, mycoparasitism and secondary metabolism.
The GenBank/EMBL/DDBJ accession no. for the sequence reported in this paper is EF189190.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Robinson & Sutherland, 1971). Although cAMP is an important regulator of growth, development and pathogenicity in filamentous fungi, genes encoding adenylate cyclase have been studied in a few species only (Adachi & Hamer, 1998; Choi & Dean, 1997; Fillinger et al., 2002; Ivey et al., 2002; Klimpel et al., 2002; Liebmann et al., 2003). The crisp mutants of Neurospora crassa which lack a functional copy of the adenylate cyclase are characterized by a colonial growth habit, with impairment in elongation of aerial hyphae, and a premature accumulation of conidia (Rosenberg & Pall, 1979; Terenzi et al., 1974, 1976). Evidence from the analysis of Aspergillus nidulans strains defective for adenylate cyclase (CyaA) or PkaA has shown the involvement of cAMP signalling in conidial germination in response to carbon-source sensing (Fillinger et al., 2002). In PkaA-defective strains, cAMP levels have been shown to increase dramatically by 250-fold, indicating that PkaA regulates intracellular cAMP levels (Fillinger et al., 2002). In the human pathogen Aspergillus fumigatus, disruption of the adenylate-cyclase-encoding gene acyA results in reduced colony diameter and conidiation; the mutants are less virulent as a result of reduced defence against killing by macrophages (Liebmann et al., 2003). In the rice pathogen Magnaporthe grisea, mac1 disruption results in abolition of appressorium formation, and reduction in vegetative growth, conidiation and conidial germination (Adachi & Hamer, 1998; Choi & Dean, 1997). magB (G-protein) and mac1 (adenylate cyclase) mutants share several phenotypes, indicating that adenylate cyclase is downstream of MagB in M. grisea (Liu & Dean, 1997). The Gβ subunit MGB1 is involved in cAMP signalling for regulating conidiation, surface recognition and appressorium formation in M. grisea (Nishimura et al., 2003). In Botrytis cinerea, the adenylate cyclase (BAC) is required for full pathogenicity (Klimpel et al., 2002).
Trichoderma spp. are economically important as sources of many industrial enzymes, and as commercial biofungicides (Harman & Bjorkmann, 1998; Hjeljord & Tronsmo, 1998; Mach & Zeilinger, 2003). These fungi inhabit soil and the rhizosphere, and inhibit other fungi through antibiosis and mycoparasitism (Chet et al., 1998; Sivasithamparam & Ghisalberti, 1998). They are also good plant growth promoters, and they induce resistance in plants against invading pathogens through limited colonization of plant roots (Harman et al., 2004; Viterbo et al., 2005). Two species (Trichoderma longibrachiatum and Trichoderma citrinoviride) are opportunistic human pathogens (Kuhls et al., 1999). Trichoderma virens IMI 304061 is an aggressive mycoparasite on the plant pathogens Sclerotium rolfsii and Rhizoctonia solani (Mukherjee et al., 1995). To understand the role of signalling pathways in mycoparasitism and development, we previously isolated loss-of-function mutants for two G
proteins (TgaA and TgaB) and an MAPK (TmkA) (Mukherjee et al., 2003b; 2004). However, there has been no direct evidence reported in the literature for a role of cAMP signalling in any Trichoderma species. Antisense-mediated silencing of the G protein Tga1 results in reduced cAMP level in Trichoderma atroviride IMI 206040 (Rocha-Ramirez et al., 2002). The mutants are hypersporulating, and have reduced mycoparasitic coiling. Interestingly, deletion of Tga1 in T. atroviride ATCC 78058 results in elevated internal cAMP levels (Reithner et al., 2005). These mutants also show hypersporulation, a total loss of mycoparasitism, and increased antibiosis against R. solani, even though the production of an antifungal metabolite, 6-pentyl--pyrone, is reduced. The deletion of another G protein, Tga3, results in reduced intracellular cAMP levels in T. atroviride, and is associated with reduced growth rate and germination, light-independent conidiation, and loss of mycoparasitism (Zeilinger et al., 2005). It is interesting that the phenotypes associated with the deletion of Tga1 or Tga3 are similar, although the cAMP level is elevated in Tga1 mutants and reduced in Tga3 mutants. Recently, Casas-Flores et al. (2006) studied the role of a protein kinase A regulatory subunit in blue-light-induced conidiation of T. atroviride. Expression of an antisense copy of this gene results in a non-sporulating phenotype, whereas overexpression results in light-independent conidiation, indicating the involvement of cAMP signalling in conidiation in T. atroviride. In contrast to most of the fungi studied, including the closely related T. atroviride, the deletion of the G protein TgaA has no effect on growth, sporulation or spore germination frequency in T. virens (Mukherjee et al., 2004). This raised the question of whether the functioning of the G-protein–cAMP pathway in T. virens differs from other ascomycetes. Also, what are the physiological roles that this pathway plays in this fungus? Since there has been no direct evidence on the role of cAMP signalling through the deletion of adenylate cyclase in any Trichoderma sp., we cloned the adenylate-cyclase-encoding gene tac1 of T. virens, which is an important biocontrol fungus, and studied the role of the cAMP signalling by gene knockout.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Routinely, the fungi were grown on potato glucose agar (PDA; Difco) at ambient temperature (26–28 °C), and stored as glycerol stocks at –80 °C for long-term storage and maintenance of genetic stability. For growth on dialysis membranes, 106 conidia were spread on PDA plates overlaid with a sterile dialysis membrane (MWCO 12 000–14 000; Spectra/Por), and incubated for 2 days at ambient temperature. The mycelial mat was harvested with a sterile spatula, frozen in liquid nitrogen, and stored at –80 °C until further use. For analysis of the secondary metabolites, the agar was extracted in solvent as described below.
Cloning of the full-length gene.
Using the M. grisea mac1 sequence, two primers, ACFor (CCATATGACTGGAATTGGAA) and ACRev (ACCATGAAGGCGTCACCT), were designed from the region showing high homology with other fungal adenylate cyclase gene sequences, and part of the gene (1.4 kb) was amplified from T. virens genomic DNA by PCR. This fragment was cloned, and used as a probe for screening a T. virens cosmid library described previously (Mukherjee et al., 2003b). The full-length gene was subcloned from the cosmid clone by a three-step inverse PCR using the primer pairs INV1 (GCCGCCTCATGCTGTCTG) and INV2 (GGAGATGTCCTCGACTCTTC), INV2 and INV3 (GAATATGCGAATAAAGTAAGG), and INV4 (GTTCGGGAGGCTGCTCTCG) and INV5 (GCCTGCGATCCACGAATAG). The 3' end of the gene was cloned as a DraI fragment amplified with the gene-specific primer Xin1.2-2GW1 (GATCGGAAGCCTCTTGCGAAGATGAG) by using the Clontech GenomeWalker Universal kit (BD Biosciences). All the clones were sequenced, assembled and translated by using Gene Runner software, and the putative ORF and introns were identified. Homology with adenylate cyclase from other fungi was studied by BLASTP on the NCBI server.
Construction of the gene-deletion construct, protoplast transformation, and selection and purification of transformants.
A double-crossover construct, pACDC, was made by replacing 5087 bp of the ORF with a hygromycin-resistance cassette consisting of hph (hygromycin phosphotransferase), TrpC promoter and TrpC terminator, taken from the plasmid pAT-BS (Mukherjee et al. 2003a). The linear construct (Fig. 1a), consisting of the left flank (2441 bp), the marker (2.2 kb), and the right flank (2010 bp), was amplified by the primer pair AC5'2s (GAGATGCGAGACTGTCAGCG) and ACRFNot (CAATGATATGAGAGCGGCCGCCATTC), and was used for transforming T. virens protoplasts, as described previously (Mukherjee et al., 2003b). The transformants were selected using 200 mg hygromycin B l–1 (Roche), and transferred to PDA plates containing 100 mg hygromycin B l–1. The transformants that showed non-wild-type phenotypes were purified by repeated single-spore isolation, and three putative knockout mutants were selected based on atypical colony growth and morphology.
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tac1 mutants (hereafter referred to as ‘mutants’) was extracted, and subjected to PCR and Southern analysis. PCR was performed by the primer pair INV3 and INV5 for the adenylate cyclase gene, and hphfor (GAGGGCGAAGAATCTCGTGC) and hphrev (CACTGACGGTGTCGTCCATC) for the hph gene. For genomic Southern analysis, the DNA was digested with XbaI, and hybridized with a 1.1 kb fragment amplified from the genomic DNA using the primer pair INV3 and INV5. For analysis of the tac1 transcript level, total RNA was isolated from the mycelial mat of the WT and mutants grown on the dialysis membrane, and first-strand cDNA was synthesized from 1 µg total RNA using the First Strand cDNA Synthesis kit (Roche) in a 20 µl reaction volume. A 5 µl volume of the first-strand cDNA was used for amplification with Taq polymerase (Genei) in a 25 µl volume, using the primer pairs INV3 and INV5 (for tac1), hphfor and hphrev (for hph), and h3for (GCCCGCACCAAGCAGACCG) and h3rev (GGCGGGCGAGCTGGATGTC) (for histone3).
Assay for intracellular cAMP concentration.
For measuring the intracellular cAMP concentration, the WT strain and the mutants were grown on a dialysis membrane, as described above. Two-day-old mycelial mat was harvested, frozen in liquid nitrogen, ground, homogenized in 10 vols 0.1 M HCl, and centrifuged (600 g). The cAMP level was measured by using the Direct cAMP Enzyme Immunoassay kit (Sigma), as per the manufacturer's instructions. Protein concentration was determined by using the Folin phenol reagent (Lowry method). The intracellular cAMP concentration was expressed as pmol (mg protein)–1.
Growth and morphology of the mutants.
Mycelial discs were inoculated on PDA plates, and the linear growth measured. For studying the colony development from single spores, PDA plates were spread with diluted conidial suspension. Dry weight (biomass) was measured after 3 days of shake incubation of 100 ml potato glucose broth (PDB) inoculated with 105 conidia. For the germination assay, conidia were harvested from 10-day-old cultures grown on PDA plates, and washed thrice in double-distilled water. Approximately 5x104 conidia in 50 µl water were incubated on sterile glass slides in a moist chamber. The conidia were observed for germination after 20 h.
Confrontation assay.
The ability of the mutants and the WT to overgrow and lyse the mycelia of the test plant pathogens was assessed using a confrontation assay on PDA plates (Mukherjee et al., 2003b). Observation was recorded for overgrowth of Trichoderma on the test fungi, and for the lysis of the mycelia.
Antibiosis and HPLC analysis.
The ability of the mutants and the WT to inhibit the growth of Pythium sp. through the production of diffusible antibiotics was assessed by bioassay and HPLC. Conidial suspension was spread on dialysis membrane, and grown for 2 days, as described above. The dialysis membrane and the mycelial mat were removed, and the agar was blended in 80 % aqueous acetone (1 g agar in 3 ml acetone), and centrifuged at 10 000 r.p.m. (13 000 g) for 10 min. The supernatant was extracted with an equal volume of chloroform. The chloroform was evaporated under N2 flush, and the metabolites were reconstituted in methanol (1/100 original volume). A 10 µl volume of the reconstituted metabolites was assayed by the agar-well technique, in which the solution was added to a 5 mm well that had been cut at the centre of a PDA plate. After evaporation of the solvent, three mycelial discs of Pythium sp. were inoculated near the edge of the plate, and photographed after 2 days incubation. HPLC was performed as described previously (Mukherjee et al. 2006a).
Suppression subtractive hybridization (SSH), array and sequencing of the differential clones.
Total RNA was isolated from the mycelial mat of the WT and the tac1 mutant ACM1 grown on a dialysis membrane, as described above. First-strand cDNA from the WT and ACM1 total RNA was synthesized using the BD SMART RACE cDNA amplification kit with 3'-RACE CDS Primer A and BD SMART IIA oligonucleotide (BD Biosciences). The first-strand cDNA was purified by selective precipitation (ammonium acetate precipitation), and the second-strand cDNA was amplified using 2.5 µl first-strand cDNA as the template, the Nested Universal Primer A (BD Biosciences) as the oligonucleotide, and Advantage polymerase (BD Biosciences), in a reaction volume of 100 µl. The cycling parameters were as follows: initial denaturation at 95 °C for 1 min, then 17 cycles (optimized initially by taking samples up to 30 cycles, based on the protocol described in BD Super SMART-PCR cDNA synthesis kit) at 95 °C for 15 s, 65 °C for 30 s, and 68 °C for 6 min. The amplified cDNA was treated with RNaseA (Roche), purified using the High Pure PCR product purification kit (Roche), reconstituted in 20 µl nuclease-free water, and the concentration was determined spectrophotometrically. Amplified cDNA (2 µg) from the WT or the tac1 mutant ACM1 was subjected to SSH using the Clontech PCR-Select cDNA subtraction kit (BD Biosciences), according to the manufacturer's instructions. The WT cDNA was used as the tester, and the mutant cDNA was used as the driver. After the second (nested) PCR, the products were cloned in the pTZ57R/T vector (Fermentas). The presence of single inserts was confirmed by colony PCR using the Nested PCR primers 1 and 2R, which were supplied with the kit, and 53 clones showing a single insert were arrayed on a positively charged nylon membrane (Roche). Histone h3 cDNA was included as a control. A 2 µl volume of the heat-denatured PCR product of each clone was blotted on membranes, and hybridized with WT or mutant cDNA, which was labelled with [32P]dCTP (BRIT). After hybridization, the blots were exposed to X-ray films overnight. The clones that were clearly differential were purified, then sequenced with T7 primer using an automated sequencer, and homologous sequences in the database were identified by BLASTX on the NCBI server.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of Tac1 loss-of-function mutants
Using protoplast-mediated transformation of T. virens, with the disruption cassette amplified from the plasmid construct pACDC, several hundred transformants were obtained by selection on 200 mg hygromycin B l–1. One hundred transformants were transferred to fresh PDA containing 100 mg hygromycin B l–1, and three colonies were found to be extremely slow growing, and they did not appear to be similar to the WT colony. These three colonies were purified by repeated single-spore isolations. The mutants (designated ACM1, ACM2 and ACM3) were confirmed for deletion of the adenylate cyclase gene by genomic PCR, Southern hybridization and RT-PCR. Using the primer pair INV3 and INV5, we obtained a 1.1 kb band in the WT, and this was found to be absent in the three mutants (Fig. 1b
). All the mutants were positive for the hph gene, which was not detected in the WT (Fig. 1b). Gene deletion was also confirmed by Southern hybridization (data not presented). RT-PCR data (Fig. 1c) indicated the absence of the tac1 transcript in the mutants, but it was present in the WT. The opposite was true for the hph transcript. The intracellular cAMP level in the 2-day-old WT mycelial mat harvested from the dialysis membrane overlying PDA was 6.227±0.415 pmol (mg protein)–1. The cAMP levels in the mutants were below the detection limit, although the protein level ranged from 1.19 to 1.57 µg (µl extract)–1, compared with 0.59 µg (µl extract)–1 in the WT.
Growth, morphology, and conidial germination
The tac1 mutants, when grown on PDA plates from the disc inoculum, showed a marked reduction in growth rate (1.1±0.3 mm day–1 linear growth, compared with WT growth of 19.2±1.4 mm day–1). The WT fully covered the 85 mm plates in 2 days, while the mutants took more than a month to cover the entire plate (Fig. 2a). The mutants had aerial growth, and sporulated initially towards the centre of the plates, but subsequently they showed submerged growth devoid of aerial hyphae and conidiation. A dark-yellow pigment diffusing from the mutant colonies was seen, and the mutant colony growth was very compact. When PDA plates with the WT or mutants were incubated in the dark, the mutants failed to form conidia, but the colonies responded to light by forming green conidia when incubated under constant illumination for 2 days (Fig. 2b). When the plates were inoculated with a diluted conidial suspension, the mutant colonies were visible 2 days after plating, as opposed to the WT colonies, which were visible after 1 day. The mutant colonies from single spores were very small, discrete and compact, and they produced mainly aerial hyphae, in contrast to the typical spreading growth habit of the WT strain (Fig. 2c). On subsequent incubation, the mutant colonies began to sporulate, produce dark-yellow diffusible pigments, and they showed a submerged growth habit. In 3-day-old liquid shake culture, there was no significant difference between the WT and the mutants with respect to dry matter production (672±21 mg for the WT, compared with 630±49, 640±40 and 657±31 mg for the mutants ACM1, ACM2 and ACM3, respectively). The mutants, when transferred from liquid culture to agar medium, grew typically as slow-growing mutant colonies. This shows that no irreversible modification occurred during growth in liquid medium. When incubated in water for 20 h, the conidia from the mutants failed to germinate, while nearly all the conidia of the WT strain germinated through the production of normal germ tubes (Fig. 2d).
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). The mutants, however, failed to overgrow any of the test pathogens, even after prolonged incubation. In contrast, R. solani and S. rolfsii overgrew the mutant colonies, indicating that the mutants could not offer resistance to overgrowth by these plant pathogens. Against Pythium sp., a clear zone of inhibition was seen when it was inoculated opposite the mutants. In the bioassay of chloroform-extracted metabolites on Pythium sp., the mutants showed reduced inhibition compared with the WT (Fig. 3b). HPLC analysis of the non-polar fractions showed the presence of several unknown metabolites, along with viridiol, which is a reduction product of viridin. The mutants produced lesser concentrations of metabolites in agar, compared with the WT (Fig. 3c).
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); of these, the most abundant was a cytochrome P450 gene (vir2), representing four clones (clone nos 4, 10, 21 and 25). The other clones related to secondary metabolism were cyp1 (a cytochrome P450 gene, clone no. 9), vir4 (a cyclase gene, clone no. 12), vir3 (a cytochrome P450 gene, clone no. 40), omtB-2 (an O-methyl transferase gene, clone no. 41), and a homologue of the GA14-synthase gene of Fusarium proliferatum (a cytochrome P450 gene, clone no. 44). Clone no. 2 was a homologue of the stress-response gene ric-1 of N. crassa, while clone no. 19 had no homology in the database.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Calvo et al., 2002; Xu, 2000). We have previously established the role of a T. virens MAPK TmkA in repression of conidiation in the dark, and identified a gene whose expression is repressed by TmkA (Mukherjee et al., 2003b, 2006b). tmkA mutants have reduced antagonism against mycelia and sclerotia of some plant pathogens. However, since the deletion of the G-protein TgaA or TgaB does not alter the growth, conidiation or conidial germination frequency of T. virens (Mukherjee et al., 2004), the role of cAMP signalling in this fungus has remained elusive. In addition, G-protein-related studies in T. atroviride have not established the role of cAMP signalling, since the results have been inconclusive. There is, in general, no direct evidence of cAMP signalling mechanisms in any Trichoderma sp., as, to the best of our knowledge, the gene encoding adenylate cyclase has not been cloned and characterized in any member of this genus. We cloned the T. virens gene tac1 encoding adenylate cyclase, and studied its cellular functions. The deletion of the gene through homologous recombination resulted in drastic effects on the colony morphology, and hyphal elongation rate. The linear growth rate was reduced by 94–95 %, which is a very strong reduction compared, for example, with M. grisea and A. fumigatus, where the deletion of adenylate cyclase reduced the growth rate to 60 and 68 % of the WT, respectively. Though the growth on PDA plates was drastically reduced in the mutants, the growth/biomass production in shake culture was similar to the WT. The normal biomass production in liquid shake culture might be due to a compensatory mechanism through decreased phosphodiesterase activity in submerged culture, as has been suggested for N. crassa (Ivey et al., 1999). Alternatively, shake culture may provide a richer environment for growth, as has been suggested for CPG-1 (Gi) mutants of Cryphonectria parasitica (Segers & Nuss, 2003). Amendment of the medium with 5 mM cAMP did not restore the phenotypes in the mutants (data not presented). The phenotypes of A. fumigatus cyaA mutants and T. atroviride Tga3 mutants, likewise, are not rescued by the addition of cAMP (Liebmann et al., 2003; Zeilinger et al., 2005).
Unlike the G-protein mutants of T. atroviride, and MAP kinase mutants of T. atroviride and T. virens (Mukherjee et al., 2003b, 2006b; Reithner et al., 2005; Zeilinger, 2004; Zeilinger et al., 2005), the tac1 mutants did not sporulate constitutively in darkness, and responded to light in the same way as the WT. Thus, the regulation of conidiation in these two closely related species appears to be different: cAMP-dependent regulation in T. atroviride, and cAMP-independent regulation in T. virens. Also, with respect to the regulation of conidiation, there could be a cross-talk between the Tmk1 and cAMP pathways in T. atroviride, but not in T. virens.
In the confrontation assay, tac1 mutants were not able to overgrow and lyse the colonies of the test plant pathogens S. rolfsii, R. solani and Pythium sp. (Fig. 3a). The loss of virulence against the host fungi could be associated with extremely slow growth rate of the mutants. Against Pythium sp., however, the mutants produced a prominent zone of inhibition. This visible zone of inhibition could be due to the overproduction of antifungal metabolites that diffuse into agar. However, as measured by both bioassay and HPLC (Fig. 3b, c), secondary metabolite production was decreased in the mutants. This is consistent with the results of SSH, where, out of 11 genes that are underexpressed in the mutants, nine are known to be associated with secondary metabolism in T. virens and other fungi (Mukherjee et al., 2006a). T. virens strain IMI 304061 shows abundant production of the antifungal viridin and its reduction product viridiol in culture (Mukherjee et al., 2006a). HPLC analysis of the non-polar fraction of the metabolites revealed that the concentration of viridiol was lower in the mutants than in the WT. We did not detect viridin in WT or the mutants, probably because of faster conversion of viridin to viridiol in plate culture compared with liquid shake culture. Nevertheless, the presence of viridiol is an indication of early viridin production. Therefore, it seems that the enhanced antifungal properties of the tac1 mutants in the confrontation assay are a result of the slow growth of the mutant colonies, rather than of the enhanced production of secondary metabolites. Reducing the growth rate by adding a sublethal dose of fungicides to visualize the zone of inhibition in Trichoderma–fungal interactions is a standard practice. For example, Howell (1987) amended PDA with 0.4 µg ml–1 benomyl to visualize the zone of inhibition produced as a result of gliotoxin formation by a strain of T. virens against R. solani. The tga1 mutants of T. atroviride produce a lower amount of the antifungal metabolite 6-pentyl--pyrone, although there is a clear zone of inhibition in the confrontation assay against R. solani (Reithner et al., 2005). Those authors attributed this enhanced antibiosis to the possible overproduction of some unidentified low-molecular-mass compounds by the mutants. The relationship between G-protein–cAMP signalling, conidiation and secondary metabolism is well documented in Aspergillus spp., where increased secondary metabolite production is associated with, or the result of, enhanced conidiation (Calvo et al., 2002; Keller et al., 2005). The deletion of the Aspergillus G subunit FadA results in premature conidiation, and enhanced sterigmatocystin production (Hicks et al., 1997). The overexpression of PkaA results in reduced sterigmatocystin biosynthesis, while the deletion of this gene results in hypersporulation and aberrant sterigmatocystin production in A. nidulans (Shimizu & Keller, 2001). T. virens tac1 mutants did not sporulate constitutively, and had reduced secondary metabolite production. This species, therefore, appears to have a novel regulation of conidiation vis-à-vis secondary metabolism that is different from other fungi studied, for example T. atroviride and Aspergillus spp. The present findings clearly establish that cAMP signalling is essential for growth, conidial germination and biocontrol properties, including mycoparasitism and secondary metabolism, in T. virens. We believe that this is the first report on the cloning and functional analysis of a gene encoding adenylate cyclase in any species of Trichoderma. The findings could help to improve the commercial exploitation of Trichoderma spp. for industrial and agricultural applications by manipulating the cAMP-signalling pathway, e.g. by overexpression/constitutive activation of one or more of the components to enhance secondary metabolite production.
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ACKNOWLEDGEMENTS |
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Edited by: H. A. B. Wösten
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Received 3 January 2007;
revised 12 February 2007;
accepted 14 February 2007.
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