FEMS Microbiology Letters 194 (2001) 27^32
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aspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis Nathalie Poussereau, Ste¨phanie Gente 1 , Christine Rascle, Genevie©ve Billon-Grand, Michel Fe©vre * Laboratoire de Biologie Cellulaire Fongique [Bat. 405], ERS 2009 CNRS INSA LYON1, Microbiologie et Ge¨ne¨tique, 43 bd 11 novembre 1918, 69622 Villeurbanne Cedex, France Received 21 September 2000; received in revised form 24 October 2000; accepted 26 October 2000
Abstract The gene aspS encoding an aspartyl protease has been cloned from Sclerotinia sclerotiorum by screening a genomic library with a PCRamplified fragment of the gene. The open reading frame of 1368 bp interrupted by one intron would encode a preproprotein of 435 amino acids. The catalytic aspartyl residues characteristic of aspartyl proteases are conserved; however, the active-site motif (DSG) in the N-terminal lobe is unusual in that Ser replaced Thr used in the active-site motif (DTG) of the C-terminal lobe and in all other fungal aspartyl proteases. RT-PCR revealed that aspS expression in axenic culture is not subjected to catabolite repression and demonstrated that aspS is expressed from the beginning of infection of sunflower cotyledons. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Sclerotinia sclerotiorum; Aspartyl protease gene; Gene expression ; Plant pathogenesis
1. Introduction Sclerotinia sclerotiorum is a ubiquitous necrotrophic fungus infecting more than 400 plant species including many economically important crops [1]. Tissue maceration and water soaking are part of the symptoms associated with diseases caused by S. sclerotiorum [1]. Degradation of the plant cell wall components and maceration of the host tissues is possible by the concerted action of a number of extracellular lytic enzymes such as cellulases, hemicellulases and pectinases secreted by the fungus [2]. Among the cell wall polysaccharide-degrading enzymes produced by necrotrophic fungi such as S. sclerotiorum, pectic enzymes are thought to be responsible for the ex-
* Corresponding author. Tel. : +33 (4) 72 44 83 78; Fax: +33 (4) 72 43 11 81; E-mail :
[email protected] 1 Present address: Laboratoire de Microbiologie Alimentaire, Universite¨ de Caen Basse Normandie, Esplanade de la Paix, F-14032 Caen Cedex, France.
tensive maceration and cell death. It has been shown that these fungi produce a set of pectinolytic enzymes and polygalacturonase isozymes which are encoded by a gene family [3,4]. Among other enzymes produced by necrotrophic fungi, proteolytic enzymes are of interest because of their potential ability to degrade the host cell wall and plasma membrane proteins. Proteases may also counter host defense-related proteins such as antifungal enzymes. Proteases are synthesized by many phytopathogenic fungi including the biotroph Uromyces viciae-fabae [5], the hemibiotroph Glomerella cingulata [6] and the necrotroph Botrytis cinerea [7]. However, there have been very few studies related to the protease expression by plant pathogenic fungi. A limited number of genes such as those encoding subtilisin-like proteinases from Magnaporthe poae [8] and Cochliobolus carbonum [9], a trypsin-like protease from Stagonospora nodorum [10] and aspartyl proteases from Cryphonectria parasitica [11] and G. cingulata [6] have been cloned. In the closely related species Sclerotinia sclerotiorum and Botrytis cinerea the proteolytic activity seems to be correlated with the development of symptoms [12,13]. Aspartic proteases are ¢rstly produced by these necrotrophic fungi during infection of various hosts and they are able to cause death of the host cells [12,14]. Since this type of
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 5 0 0 - 0
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enzyme may be an important virulence factor [15], we have isolated and characterized an aspartyl protease gene of S. sclerotiorum and analyzed its expression in vitro and in planta during the interaction of the phytopathogen with sun£ower cotyledon leaves. 2. Materials and methods 2.1. Strains and culture conditions S. sclerotiorum S5 was grown and maintained on potato-dextrose agar. It was also grown in minimal liquid medium containing (per liter) 1 g KH2 PO4 , 0.1 g MgSO4 , 0.05 mg biotin, 250 mg citric acid, 250 mg ZnSO4 , 50 mg Fe(SO4 )2 , 15 mg Cu(SO4 )2 , 25 mg each of MnSO4 , H3 BO3 , NaMoO4 , supplemented with 1% sun£ower cell wall extracts or 2% glucose and 100 mM NH4 Cl. Sun£ower cell wall extracts were prepared from cotyledons of 1-week-old germlings. Cotyledon leaves were ground in a blade blender then the homogenate was centrifuged at 10 000Ug for 10 min to pellet cell walls which were frozen until use. The culture medium was bu¡ered at different pH in 0.15 M MacIlvaine bu¡er (citrate/phosphate bu¡er). The cultures were inoculated with mycelial disks cut from 4-day-old colonies and incubated at 24³C under constant agitation. Escherichia coli strain Sure R was the host for recombinant plasmids and was grown in LB broth medium supplemented with 50 Wg ml31 of ampicillin. E. coli P2392 was used for bacteriophage lambda EMBL3 screening and was grown in NZY medium [16]. Plasmid pUC 18 was used for cloning experiments. 2.2. Pathogenicity tests Phytopathogenicity assays were performed on sun£ower cotyledons as hosts. Sun£ower seeds were sown in a peat^ pouzzolane mix. Germlings were maintained at 25³C (95% humidity) with a 14/10-h photoperiodic (day/night) illumination. Cotyledon leaves from 1-week-old germlings were infected by depositing a 4-mm mycelial disk on the upper face of the cotyledons. At various intervals after inoculation (corresponding to di¡erent stages of disease development), infected cotyledons were harvested and frozen at 380³C. Controls were performed using mycelial disks previously heated for 30 min at 65³C. 2.3. DNA and RNA isolation DNA was prepared from freeze-dried mycelium grown on potato-dextrose agar [17]. Total RNA was isolated from freeze-dried mycelium or infected cotyledons after lysis in a bu¡er containing 50% guanidinium thiocyanate, followed by centrifugation in cesium chloride solutions [16]. RNA concentration was measured spectrophotometrically.
2.4. Preparation of a probe by PCR For ampli¢cation of an aspS-speci¢c fragment, S. sclerotiorum genomic DNA was used as the template in a PCR reaction with two mixed degenerate oligonucleotide primers, synthesized as follows : primer mixture A: 5PCTGAAGCTTATNWSNTAWGGNGAYGG-3P primer mixture B: 5P-CTGTCTAGAGTNGTNCCNGTRTC-3P where N is an equal mixture of all four nucleotides, R one of A and G, and Y one of T and C. The ampli¢cation was initiated with a 5-min denaturation at 94³C, followed by 35 cycles: denaturation, 94³C for 1 min; annealing, 56³C for 1 min; primer extension, 72³C for 3 min; the ¢nal elongation step was 7 min at 72³C. The ampli¢ed products were analyzed by agarose gel electrophoresis, isolated by adsorption to glass silica beads (Geneclean II, Bio 101), digested and cloned into the HindIII/XbaI-digested pUC 18 vector. The cloned PCR fragment was sequenced and then used as a probe to screen a genomic library. 2.5. RT-PCR RT-PCR was performed according to the manufacturer's instructions. One microgram of total RNA was treated with RNase-free DNase (Boehringer Mannheim) and reverse-transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Promega) with the speci¢c antisense primer 5P-CGTTGAATACGACAAATTGA-3P. The cDNA products were ampli¢ed by PCR with the sense primer 5P-ATTGCTGACACCGGAACC3P and the antisense primer described above. Competitor DNA was serial dilution of the genomic clone. The PCR conditions (temperature, time, number of cycles) were as follows : 95³C, 1 min, 55³C, 1 min, 72³C, 1 min, 35 cycles. Aliquots of the PCR products were analyzed on 2% agarose gels. For ampli¢cation of S. sclerotiorum gpd transcripts, the forward primer was 5P-TGGCTCCTACTAAAGTTG-3P and the reverse was 5P-CAAGCAGTTGGTTGTGCAAG-3P. 3. Results and discussion 3.1. Isolation and characterization of aspS To clone an aspartyl proteinase gene from S. sclerotiorum by PCR, primers, ISYGDG and ADTGTT, synthesized on the basis of the homology among the proteinases [18], allowed ampli¢cation of a single fragment of 750 bp. Its identity was established by sequencing and comparison to those of known acid proteinases. The PCR fragment cloned in pUC 18 was used as a probe to screen a genomic library of S. sclerotiorum constructed in lambda phage EMBL3. Among 104 individual recombinant bacteriophage plaques, four hybridizing clones were selected. A band of 3.6 kb present in these phages and hybridizing
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Fig. 1. Nucleotide sequence of the S. sclerotiorum aspS gene encoding aspartyl protease and the deduced amino acid sequence. The intron is indicated as lowercase within the coding sequence. Double underlined regions represent the catalytic domains. Putative N-glycosylation sites are boxed.
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Fig. 2. Comparison of the deduced amino acid sequence of the aspartyl proteases from the phytopathogenic fungi C. parasitica [11], G. cingulata [6] and S. sclerotiorum. Identical residues are in bold. Asterisks indicate the catalytic motif. N-Glycosylation sites are underlined. Dashes indicate gaps introduced to optimize the sequence alignment.
to the probe was cloned. The nucleotide sequence of the cloned fragment determined on both strands revealed that it contained the entire nucleotide sequence of the gene aspS (GenBank accession number : AF271387). Southern blot analysis of genomic DNA of S. sclerotiorum showed that the PCR fragment hybridized to a single band indicating the presence of one copy of the gene. The sequence revealed an open reading frame of 1368 bp interrupted by an intron of 61 bp (Fig. 1). The open reading frame encodes a polypeptide of 435 amino acids. By analogy with the N-terminal region of secreted aspartyl proteinases of C. parasitica [11] and G. cingulata [6], it may be assumed that aspS is synthesized as a zymogen and contains a Nterminal preproregion of 97 amino acids. The mature form of the enzyme would be a 338-residue protein with a calculated molecular mass of 36.2 kDa and a calculated pI of 5.14. In aspartyl proteases, the catalytic Asp residues occur within the motif Asp-Xaa-Gly in which Xaa can be Ser or Thr [19]. In fungal aspartyl proteases the catalytic Asp residues are contained in a DTGS and a DTGT motif in the N- and C-terminal lobes of the enzyme, respectively. In ASPS, the region around the active-site residue Asp322 corresponds to the conserved motif DTGT while the active-site residue Asp137 occurs within the motif DSGS, which is unusual for fungal enzymes [19]. The amino acid sequences of aspartyl proteases of phytopathogenic fungi are strongly conserved (Fig. 2) and the degree of amino acid sequence identity between ASPS and
the other fungal aspartyl proteases varies from 41 to 47%. Compared to the aspartyl protease from the other phytopathogenic fungi, ASPS presents several speci¢c features. The polypeptide chain is longer due to the presence of a supplementary sequence of 18 residues in the N-terminal lobe. The active-site motif in the N-terminal lobe is unusual in that Ser is present instead of the Thr used in all other fungal aspartyl proteases. ASPS contains four putative N-linked glycosylation consensus sites NXS/T while this motif is not found in other proteases (Fig. 2). 3.2. aspS expression during saprophytic growth of S. sclerotiorum. E¡ects of ambient pH, carbon and nitrogen sources on aspS expression In order to determine the pattern of aspS expression, S. sclerotiorum grown in a minimal glucose/NH4 medium was transferred to culture media containing sun£ower cotyledon extracts. No hybridization signal was detected by Northern blotting of total RNA with the aspS probe while clear signals were observed when S. sclerotiorum gpd was used (not shown). This indicates that aspS was too weakly expressed to be detected by RNA gel blots. The more sensitive RT-PCR technique was employed using speci¢c oligoprimers £anking the intron and genomic DNA as a template for competitive PCR reaction (Fig. 3). The intron within the competitive template allowed the target cDNA and genomic product to be size-separated on agarose gels and identi¢ed according to the size of the ampli¢ed frag-
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ments (367 and 306 bp with and without the intron, respectively). Electrophoresis analysis of the RT-PCR products showed a fragment of the expected size (306 bp). Addition of competitor DNA revealed two bands, one corresponding to the competitive genomic fragment (367 bp) and the other to the cDNA-derived fragment (307 bp), con¢rming that aspS is expressed in vitro. The e¡ect of carbon and nitrogen sources on aspS expression was determined by transferring mycelia cultivated in a minimal glucose/NH4 medium to unbu¡ered media containing sun£ower extracts and glucose, glycerol and/ or NH4 as easily metabolizable carbon and nitrogen sources (Fig. 3). aspS expressed during the preculture in the glucose/NH4 medium was also expressed after transfer to the sun£ower extract medium. When glucose, glycerol or glycerol and NH4 were added to this medium aspS was still expressed at an apparent constant level indicating that aspS expression is not controlled by glucose and ammonium catabolite repression. During the course of these cultures in unbu¡ered media the pH became very acidic because of oxalic acid secretion and NH4 consumption. As ambient pH controls acid protease gene expression [18,20] and may a¡ect aspS expression, mycelia obtained in glucose/NH4 media were transferred to bu¡ered sun£ower media at pH 4 and pH 6. Constant levels of aspS mRNA were observed regardless of the ambient pH con¢rming that in vitro expression of aspS is constitutive and not pH-regulated (not shown). 3.3. aspS expression during sun£ower infection In planta expression of the aspS gene was analyzed in a time course experiment. Sun£ower cotyledons were in-
Fig. 3. Ethidium bromide-stained RT-PCR products showing expression pattern of aspS from S. sclerotiorum grown in di¡erent culture conditions. Lane 1: ladder marker. Lane 2: PCR ampli¢cation of the aspS genomic fragment. Mycelia were grown for 48 h in a glucose/NH4 medium (lane 3) then transferred to a medium containing sun£ower extracts (lane 4) supplemented with glycerol (lane 5), glucose (lane 6), and ammonium and glycerol (lane 7). Eight hours after transfer, the pH of the culture medium was measured and RNA was extracted. The genomic fragment and cDNA-derived fragments of aspS are indicated with arrows as gDNA and cDNA, respectively.
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Fig. 4. Ethidium bromide-stained RT-PCR products showing the expression pattern of aspS (top) and gpd (bottom) during infection of sun£ower cotyledons by S. sclerotiorum. Lanes 2^9 refer to the time of collection of the infected tissues (4, 8, 12, 16, 20, 24, 48, and 56 h after infection). Lane 1 corresponds to uninfected cotyledons. Lane 10: ladder marker. Lane 11: PCR ampli¢cation of the aspS genomic fragment. The genomic fragment and cDNA-derived fragments of aspS are indicated with arrows as gDNA and cDNA, respectively. Gpd was used to evaluate fungal transcripts.
fected with mycelial disks and harvested at di¡erent times after inoculation. The S. sclerotiorum glyceraldehyde 3-phosphate dehydrogenase structural gene gpd was used as a fungal-speci¢c gene. gpd transcripts were detected throughout the infection kinetics, expression of gpd increased from the beginning of infection and remained high 24 h after infection (Fig. 4). No ampli¢cation product was obtained from the RNA samples of non-infected cotyledons proving the fungal speci¢city of the nucleotides (Fig. 4, lane 1). The 306-bp ampli¢cation product of aspS detected 4 h after inoculation (Fig. 4, lane 2) remained low and constant during the ¢rst 12 h, increased rapidly between 16 and 24 h (Fig. 4, lanes 4^6), and then decreased. The increase of the signal coincided with the phase of symptom development in which intensive mycelial colonization of the cotyledon leaves occurred. The decrease of the signal was observed when the cotyledons were completely invaded and degraded. In the present work, the aspS gene of S. sclerotiorum encoding an aspartyl protease was cloned. In pure cultures, the presence and the levels of aspS transcripts were not altered by the ambient pH and the availability of repressive carbon and nitrogen sources indicating that aspS is constitutively expressed. In planta, aspS expression detected from the early stages of infection was activated. The constitutive expression of aspS and the absence of global regulation of its expression give S. sclerotiorum the capacity to produce, from the beginning of infection, an enzyme able to attack structural wall proteins and to degrade host defense proteins. Aspartyl proteases have been considered important virulence factors [15], it will be of importance to know if other acid proteases that use di¡erent catalytic mechanisms participate in and increase the e¤ciency of the attack enzymes of necrotrophic fungi.
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Acknowledgements This research was supported by grants from the CNRS and the Universite¨ Claude Bernard Lyon 1. S.G. was the recipient of a doctoral MESR fellowship. References [1] Purdy, L.H. (1979) Sclerotinia sclerotiorum : history, diseases and symptomatology, host range, geographic distribution and impact. Phytopathology 69, 875^880. [2] Riou, C., Freyssinet, G. and Fe©vre, M. (1991) Production of cell walldegrading enzymes by the phytopathogenic fungus Sclerotinia sclerotiorum. Appl. Environ. Microbiol. 54, 1478^1484. [3] Fraissinet-Tachet, L., Reymond-Cotton, P. and Fe©vre, M. (1995) Characterization of a multigene family encoding an endopolygalacturonase in Sclerotinia sclerotiorum. Curr. Genet. 29, 96^99. [4] Wubben, J.P., Mulder, W., ten Have, A., van Kan, J.A.L. and Visse, J. (1999) Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea. Appl. Environ. Microbiol. 65, 1569^ 1602. [5] Raucher, M., Mendgen, K. and Deising, H. (1995) Extracellular proteases from the rust fungus Uromyces viciae-fabae. Exp. Mycol. 19, 26^34. [6] Clark, S.J., Templeton, M.D. and Sullivan, P.A. (1997) A secreted aspartic proteinase from Glomerella cingulata: puri¢cation of the enzyme and molecular cloning of the cDNA. Microbiology 143, 1395^ 1403. [7] Mohavedi, S. and Heale, J.B. (1990) Puri¢cation and characterization of an aspartic proteinase secreted by Botrytis cinerea Pers ex. Pers in culture and in infected carrots. Physiol. Mol. Plant Pathol. 36, 289^ 302. [8] Sreedhar, L., Kobayashi, D.Y., Bunting, T.E., Hillman, B.I. and Belanger, F.C. (1999) Fungal protease expression in the interaction of the plant pathogen Magnaporthe poae with its host. Gene 235, 121^129.
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