Laboratoire de Biologie Cellulaire Fongique (bât 405), ERS CNRS 2009, Microbiologie et Génétique, Université Claude Bernard-Lyon I, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France1
Author for correspondence: Michel Fevre. Tel: +33 4 72 44 83 78. Fax: +33 4 72 43 11 81. e-mail: mfevre{at}biomserv.univ-lyon1.fr
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ABSTRACT |
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Keywords: acid protease, gene expression, pH regulation, carbon and nitrogen repression, plant pathogenesis
a Present address: Département de Biochimie Médicale, Centre Médical Universitaire, 1 rue Michel Servet -1211 Genève 4, Switzerland.
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INTRODUCTION |
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The possible roles of fungal proteases in plant pathogenicity have only been investigated in a few systems and contradictory results have been obtained. An aspartyl protease has been identified as an important virulence factor in infections by Botrytis cinerea and as one of the factors required for establishing fungal growth within the host (Movahedi & Heale, 1990 ). In Pyrenopeziza brassicae, evidence that a cysteine protease may represent a pathogenicity determinant has been presented by complementation of a protease-negative, non-pathogenic mutant to obtain a fully pathogenic protease-positive transformant (Ball et al., 1991
). In contrast, disruption of two alkaline protease encoding genes in Cochliobolus carbonum reduced the proteolytic activity without affecting virulence of the mutants. This indicates that these two enzymes are not required for pathogenicity of C. carbonum (Murphy & Walton, 1996
). The importance of proteases in pathogenicity is also suggested by the fact that plants have evolved mechanisms to counter pathogen-secreted enzymes by producing protease inhibitors (for review, see Ryan, 1990
). These inhibitors may act as regulators of endogenous enzymes. They could also contribute to plant defence and host resistance by inhibiting fungal proteases and by exhibiting a strong antifungal activity (Pernas et al., 1999
; Chen et al., 1999
).
Extracellular proteases are produced by many pathogenic fungi but the profiles of proteases these organisms produce have often not been thoroughly investigated. It has been proposed that the range of proteases produced by a fungus reflects the adaptation of saprophytic or pathogenic fungi to the requirements of their ecological niches (St Leger et al., 1997 ; Bidochka et al., 1999a
, b
). One can imagine that in response to environmental signals, protease production during pathogenesis must be regulated by the structural cell-wall proteins, nitrogen and/or nutrient limitation, and the ambient pH.
Sclerotinia sclerotiorum is an ubiquitous necrotrophic fungus that is able to infect a wide range of cultivated plants resulting in important economic losses. Extracellular proteins secreted by this fungus are able to degrade host cell-wall components and to macerate the plant tissues. They contain the glycoside hydrolase activities that complement polysaccharidase enzymes to release monomers from each plant cell-wall polymer (Riou et al., 1991 ). Each enzymic system contains several endo- and exo- enzymes and, like the pectinolytic system, must be encoded by a multigene family (Martel et al., 1996
; Fraissinet-Tachet et al., 1995
). With respect to their potential contribution to cell-wall degradation and matrix-protein hydrolysis, characterization of proteases and isolation of all the genes for a given enzyme are of importance in order to test the role of proteases in plant pathogenicity. During saprophytic growth and pathogenesis, Scl. sclerotiorum secretes large amounts of oxalic acid which accumulates in infected tissues and decreases rapidly the ambient pH (Magro et al., 1984
). As ambient pH regulates gene expression (Caddick et al., 1986
; Tilburn et al., 1995
), we have focused our attention on acid proteases and we describe here the characterization and regulation of acp1, a gene that encodes a non-aspartyl acid protease expressed during the infection process.
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METHODS |
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Escherichia coli strain Sure R (Stratagene) was the host for recombinant plasmids and was grown in LB broth medium supplemented with ampicillin (50 µg ml-1). E. coli P2392 was used for bacteriophage lambda EMBL3 screening and was grown in NZY medium (Sambrook et al., 1989 ). The plasmid pUC18 was used for cloning experiments.
Pathogenicity tests.
Phytopathogenicity assays were performed on sunflower cotyledons as hosts. Sunflower seeds were sown in a peat/pouzzolane mix. Germlings were grown at 25 °C (95% humidity) with a 14 h light period per day. Cotyledonary leaves from 1-week-old germlings were infected by depositing a 4 mm mycelial disk on the upper face of the cotyledons. At various times after inoculation (corresponding to different stages of disease development), infected cotyledons were harvested and frozen at -80 °C. Each assay was carried out three times on separate plants. Controls were performed using mycelial disks previously heated for 30 min at 65 °C. All experiments were performed twice and the same pattern of symptom development was found.
Acid protease assays.
The culture medium (200 ml) was filtered to eliminate the mycelia, dialysed against distilled water then freeze-dried. Thirty infected sunflower cotyledons, collected at different stages of symptom development, were ground in a mortar and pestle in cold Tris/HCl (0·05 M), pH 7·5. After 10 min centrifugation at 10000 g, supernatants were collected, dialysed against distilled water then freeze-dried. Lyophilized proteins (i.e. proteins secreted in the culture medium or extracted from infected cotyledons) were dissolved in 3 ml water and assayed for protease activity according to Griffen et al. (1997 ). ACP1 was assayed in a reaction mixture containing 200 µl enzyme solution, 900 µl 100 mM KCl/HCl buffer, pH 2, and 100 µl 15% (w/v) azocasein dissolved in the buffer. The following buffers were used to determine the pH profile of the secreted proteases: 100 mM KCl/HCl (pH 1·52·5); 100 mM citrate/phosphate (pH 2·57·5); and 100 mM Tris/HCl (pH 7·59). Following a 2 h incubation at 37 °C, the reaction was stopped by the addition of 300 µl 30% (w/v) TCA. After centrifugation of the assay mixtures for 7 min at 13000 g, 500 µl supernatant was withdrawn and the colour reaction was developed by the addition of 500 µl 1 M NaOH and measured at 450 nm. Appropriate controls without either enzyme or substrate were run simultaneously. One arbitrary unit of protease activity was defined as the amount of enzyme necessary to develop an absorbance of 0·5. Protein determinations were carried out by the method of Bradford (1976
). Specific active-site inhibitors were incubated with the enzymes for 30 min prior to assaying the protease activity. The final concentrations for protease inhibitors (Sigma) used were 0·04 mM E-64 (a cysteine protease inhibitor), 0·08 mM PMSF (a serine protease inhibitor), 0·04 mM phosphoramidon (a metalloprotease inhibitor) and 0·04 mM pepstatin A (an aspartyl protease inhibitor). All protease assays were run in triplicate and data are shown as mean and standard deviations. The results of one representative experiment are presented.
DNA isolation and Southern blot analysis.
DNA was prepared as described by Brownlee et al. (1988 ) from freeze-dried mycelium grown on potato-dextrose agar. DNA was digested to completion, electrophoresed on a 0·8% agarose gel and blotted onto Nytran membranes (Schleicher and Schuell). Membranes were hybridized (50%, w/v, formamide, 5x SSC, 0·2%, w/v, SDS, 1x Denhardts solution, 100 µg denatured salmon sperm DNA ml-1) at 42 °C with a 32P-labelled PCR fragment or acp1 coding region. The probes were radiolabelled using a random-primed DNA labelling kit (Promega). Following overnight hybridization, membranes were washed with 2x SSC, 0·1% (w/v) SDS then with 0·2x SSC, 0·1% (w/v) SDS.
RNA isolation and Northern analysis.
Total RNA was isolated from freeze-dried mycelium or infected cotyledons after lysis in a buffer containing 50% (w/v) guanidinium thiocyanate, followed by centrifugation in caesium chloride solutions (Sambrook et al., 1989 ). For Northern blotting, 15 or 30 µg total RNA was loaded per lane onto 1·5% (w/v) formaldehyde agarose gels, transferred to Nytran membranes after electrophoresis and UV cross-linked. Hybridization and washing of the membranes were done as described above. The membranes were stripped between hybridizations with different probes by washing in 5 mM Tris/HCl, pH 8·0, 0·2 mM EDTA, pH 8·5, 0·05% (w/v) pyrophosphate, 0·1x Denhardts solution for 1 h at 65 °C (Sambrook et al., 1989
).
Preparation of a probe by PCR.
For amplification of an acp1-specific fragment, Scl. sclerotiorum genomic DNA was used as the template in a PCR reaction with two degenerate oligonucleotide primers. They were respectively a 16-mer and a 14-mer of 8- and 96-fold degeneracy and were synthesized as follows: primer mixture A, 5'-TGGTAYGARTGGTAYC-3'; primer mixture B, 5'-ATVMAYTCNGCRTT-3' where N is A/C/G/T, R is G/A, Y is T/C, V is A/C/G and M is A/C. The amplification was initiated with a 5 min denaturation at 94 °C, followed by 35 cycles of denaturation at 94 °C for 1 min; annealing at 56 °C for 1 min; and primer extension at 72 °C for 3 min; the final elongation step was 7 min at 72 °C. The amplified products were analysed by agarose gel electrophoresis, isolated by adsorption to glass silica beads (GeneClean II; Bio101), digested and cloned into the HindIII/XbaI-digested pUC18 vector. The cloned PCR fragment was sequenced and then used as a probe to screen a genomic library.
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RESULTS |
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Isolation and characterization of acp1
Acid proteases have been isolated and characterized for only three filamentous fungi: A. niger (Takahashi et al., 1991 ), Scy. lignicolum (Maïta et al., 1984
) and C. parasitica (Jara et al., 1996
). Comparison of the amino acid sequences of these proteases revealed several conserved regions which could be used for designing primers for PCR amplification. Oligonucleotides corresponding to the conserved regions WYEWY and NAEWI allowed amplification of a single fragment of 204 bp from Scl. sclerotiorum genomic DNA. Its identity was confirmed by sequencing and by comparison with the sequences of known acid proteases.
The PCR fragment, cloned in pUC18, was used as a probe to screen a genomic library of Scl. sclerotiorum constructed in the lambda phage EMBL3 (Riou, 1991 ). Four hybridizing clones were selected out of 104 individual recombinant bacteriophage plaques. The phages were isolated, purified, and their DNA digested with several restriction enzymes and subjected to Southern analysis. A DNA fragment of 1·3 kb, present in all the phages hybridizing to the probe, was cloned into the PstI site of pBluescript, yielding the plasmid pAC1.
Southern blot analysis of genomic DNA of Scl. sclerotiorum was performed using the 204 bp PCR fragment described above as a probe. Genomic DNA was digested with different restriction enzymes for which no internal site was present in the amplified fragment, transferred to a Nylon membrane and hybridized under low stringency. Southern analysis showed that the PCR fragment hybridized to a single band (data not shown), indicating that the acid protease activity is encoded by a single gene, as described for A. niger and Scy. lignicolum (Takahashi et al., 1991 ; Oda et al., 1998
).
The nucleotide sequence of the cloned fragment determined by sequencing both strands revealed that it contains the entire nucleotide sequence of the acp1 gene (GenBank accession no. AF221843). The acp1 gene consists of an intronless ORF of 759 bases, which encodes a polypeptide of 252 aa. By comparison with the N-terminal region of secreted acid proteases of C. parasitica (Jara et al., 1996 ) and A. niger (Takahashi et al., 1991
), it may be assumed that acp1 is synthesized as a zymogen following the general rule for fungal acid proteases (aspartyl or non-aspartyl proteases) and contains an N-terminal preproregion of 52 aa (Fig. 1
). The mature form of the enzyme would be a 200 residue protein with a calculated molecular mass of 20·7 kDa and a calculated pI of 3·9. The deduced primary sequence of the mature protease contains one potential N-linked glycosylation site.
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acp1 expression during saprophytic growth of Scl. sclerotiorum
Transfer experiments were performed to investigate acp1 expression during growth in the presence of plant extracts. Scl. sclerotiorum was grown for 48 h in a medium containing glucose and NH4 then collected, washed with distilled water and transferred to a minimal medium containing sunflower extracts. Total RNA isolated at different times after transfer was hybridized with acp1 then with 16S rDNA (Fig. 2). No acp1 signal was detected at the time of transfer to minimal medium, indicating that the protease gene is not constitutively expressed. Four hours after transfer, a strong signal was observed and 8 h after transfer the hybridization signal decreased and then appeared to remain at a constant level. On the other hand, the enzymic activity measured in the culture medium, which seems likely to be attributable to the acp1 gene product, continued to increase up to 8 h after transfer then remained high. Scl. sclerotiorum secretes oxalic acid during growth (Magro et al., 1984
). Consequently, the pH of the culture medium decreased rapidly and 6 h after transfer stabilized to pH 2·53 (Fig. 2
). Constant levels of pH and of acp1 transcripts were observed at the same time following transfer, suggesting that the ambient pH may affect acp1 expression.
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Analysis of the acp1 promoter sequence
The experiments described above show that acp1 expression is controlled in a complex manner by several environmental factors. It was of interest to examine the 5' non-coding sequence for the presence of motifs involved in the regulation of fungal gene expression (Fig. 5). The sequence contains 19 copies, in each orientation, of the GATA motif which is the recognition sequence of the nitrogen regulatory proteins AreA from A. nidulans (Kulmburg et al., 1993
) and NIT2 from Neurospora crassa (Fu & Marzluf, 1990
). However, only eight of these motifs at the positions -912 and -903, -867 and -858, -522 and -498, -131 and -111 are in an inverted-repeat orientation separated by less than 30 bp, corresponding to the functional organization of the GATA binding sequences in N. crassa (De Bernardis et al., 1998
; Marzluf, 1997
; Caddick et al., 1994). There are three degenerate copies of the recognition sequence 5'-SYGGRG-3' of CREA, a factor involved in glucose-mediated carbon catabolite repression (Kulmburg et al., 1993
). Only one copy of the motif 5'-GCCARG-3', the recognition site for the PACC protein mediating pH regulation in A. nidulans (Tilburn et al., 1995
), is present. The presence of these motifs, together with the data obtained from Northern blot analyses, suggest that acp1 is under the control of homologous wide-domain regulatory genes in Scl. sclerotiorum.
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DISCUSSION |
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acp1 expression was induced when the mycelium was cultivated in the presence of sunflower extracts. The presence of glucose prevented this induction, indicating that production of this protease is regulated by carbon catabolite repression. These data are compatible with the presence, in the Scl. sclerotiorum acp1 promoter, of several binding sites for the glucose repressor CREA, and with the synthesis and nuclear compartmentation of the homologous protein CRE1 in the presence of glucose (Vautard-Mey et al., 1999 ). The addition of ammonium to the plant-cell-extract medium did not repress acp1 expression, indicating that this preferred nitrogen source is, on its own, unable to induce nitrogen repression. However the addition of glycerol, a non-repressive carbon source on its own, to media which contained plant-cell extracts and an increasing concentration of ammonium, reduced markedly acp1 expression leading to full repression at an ammonium concentration of 100 mM. This control, induced by the simultaneous presence of carbon and nitrogen sources, indicates that the sunflower cell-wall preparation did not contain available single carbon sources, as acp1 was expressed in the presence of ammonium and the cell-wall preparation. The products released by cell-wall-protein proteolysis must serve as both carbon and nitrogen sources with nitrogen repression by a convenient nitrogen source (i.e. NH4) only occurring when the requirement for a carbon source is fulfilled i.e. by the addition of glycerol. This situation is reminiscent of the situation for a set of proline-specific genes in A. nidulans: repression of these genes requires the presence of both ammonium and glucose (Gonzalez et al., 1997
). Inducer exclusion appears to be responsible for the carbon and nitrogen repression of the genes of the proline utilization gene cluster of A. nidulans (Marzluf, 1997
; Cubero et al., 2000
). It will be of interest to determine whether protease inducers are released in the presence of nitrogen and carbon sources in Scl. sclerotiorum; their absence would signify that nitrogen and carbon metabolite repression of acp1 expression is indirect by prevention of inducer production or by inducer exclusion.
Expression of acp1 is also tightly regulated by ambient pH. Following transfer of mycelium into inducing medium, acp1 transcripts were only detected within a very narrow range of ambient pH, between pH 3 and 5. Transcription of acp1 requires acidic environmental conditions; the presence of a proteinaceous inducer is not sufficient for expression of this gene. pH control overrides induction. Moreover, synthesis of the acidic ACP1 is prevented at neutral or alkaline pH values at which the enzyme is inactive. pH regulation is an adaptive response for the production of secreted enzymes at their optimal pH of activity (Jarai & Buxton, 1994 ; Gente et al., 1997
). In A. nidulans, the PacC transcription factor has been shown to activate transcription of alkaline genes and to repress transcription of acidic genes (Tilburn et al., 1995
). Identification of a pacC homologue in Scl. sclerotiorum (S. Creton & N. Poussereau, unpublished data) together with the presence of a PacC binding site in the promoter region of acp1 (Fig. 5
), suggest the involvement of a pH regulator in the control of acp1.
We have investigated the pattern of acp1 expression in response to changes in the availability of carbon and/or nitrogen sources, and alteration of the ambient pH demonstrating that pH controls the circuit of acp1 induction and repression prevails over specific induction. acp1 is strongly expressed in planta during the course of symptom development of infected sunflower cotyledons. The ambient conditions which impose a very tight control of acp1 expression in vitro must be encountered in planta to allow acp1 expression during infection. The pH-conditional expression observed in vitro suggests that pH during infection must rapidly become acidic to reach pH 4, the value allowing acp1 expression. In vitro, glucose imposes full repression but carbon catabolite repression must not occur during infection as acp1 is expressed at the early stages of infection. Similarly, nitrogen limitation may also allow gene expression during infection as illustrated in other systems (Talbot et al., 1997 ; Coleman et al., 1997
).
In summary, using acp1 as a reporter system and by comparing in vitro and in planta gene expression it was possible to define conditions which mirror growth conditions in plant tissues. Our data suggest that glucose and nitrogen starvation together with acidification are key factors which control Scl. sclerotiorum gene expression during pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 30 May 2000;
revised 2 November 2000;
accepted 16 November 2000.