Mol Genet Genomics (2001) 265: 922±929 DOI 10.1007/s004380100491
O R I GI N A L P A P E R
A. PeÂrez-Espinosa á T. RoldaÂn-Arjona á M. Ruiz-Rubio
Pantothenate synthetase from Fusarium oxysporum f. sp. lycopersici is induced by a-tomatine
Received: 6 November 2000 / Accepted: 16 March 2001 / Published online: 5 May 2001 Ó Springer-Verlag 2001
Abstract The steroidal glycoalkaloid a-tomatine which is present in tomato (Lycopersicum sculentum) is assumed to protect the plant against phytopathogenic fungi. We have isolated a gene from the fungal pathogen Fusarium oxysporum f. sp. lycopersici that is induced by this glycoalkaloid. This gene, designated panC, encodes a predicted protein with a molecular mass of 41 kDa that shows a high degree of sequence similarity to pantothenate synthetases from yeast, plants and bacteria. Recombinant PanC protein from F. oxysporum has been over-expressed in Escherichia coli and puri®ed to homogeneity. It shows pantothenate synthetase activity in the presence of D-pantoate, b-alanine and ATP. The panC gene from F. oxysporum functionally complements an E. coli panC mutant, demonstrating that the PanC protein functions in vivo as a pantothenate synthetase. Southern analysis of F. oxysporum genomic DNA from other formae speciales indicates that there is a single copy of the pantothenate syntethase gene in this fungus. The presence of a STRE consensus sequence (CCCCT) in the promoter region of the gene suggests that the induction of panC may be part of a cellular stress response triggered by a-tomatine. Keywords Lycopersicum esculentum á Phytopathogenic fungi á Pantothenate synthetase á a-Tomatine
Introduction Saponins are secondary metabolites produced constitutively by many plants, including many major food crops Communicated by C. A. M. J. J. van den Hondel A. PeÂrez-Espinosa á T. RoldaÂn-Arjona á M. Ruiz-Rubio (&) Departamento de GeneÂtica, Facultad de Ciencias, Universidad de CoÂrdoba, Avda. San Alberto Magno s/n, 14071 CoÂrdoba, Spain E-mail:
[email protected] Tel.: +34-957-761153 Fax: +34-957-761297
(Price et al. 1987). They consist of a triterpenoid, steroid or steroidal glycoalkaloid bearing one or more sugar chains (Price et al. 1987; Hostettmann and Marston 1995; Osbourn 1996). Due to their antifungal eects, saponins have been implicated as preformed determinants of plant resistance to fungal attack (Fenwick et al. 1992; Bowyer et al. 1995). The term phytoanticipin has been proposed to describe saponins and other low-molecular-weight antimicrobial compounds that are produced as part of normal plant metabolism and that may function to protect plants from disease (Van Etten et al. 1994). The saponin a-tomatine (Fig. 1) is a steroidal glycoalkaloid found in the leaf, stem, root, ¯ower and green fruit of the tomato plant (Lycopersicon esculentum) (Friedman and Levin 1995, 1998). It consists of an aglycone moiety (tomatidine) and a tetrasaccharide moiety (b-lycotetraose) which is composed of two molecules of glucose and one each of galactose and xylose (Roddick 1974). Tomatine is known for its inhibitory eect on fungal growth (Fontaine et al. 1948; Arneson and Durbin 1968; Jadhav et al. 1981; Lacey and Mercadier 1998; Sandrock and Van Etten 1998). The toxic eect of tomatine is attributed to its interaction with cell membrane sterols containing free 3b-hydroxyl groups, the formation of such complexes resulting in loss of membrane integrity, increased permeability and leakage of cell contents (Roddick and Drysdale 1984; Price et al. 1987; Steel and Drysdale 1988; Fenwick et al. 1992; Keukens et al. 1995). Tomato pathogens are generally more tolerant to a-tomatine than most non-pathogenic fungi (Arneson and Durbin 1968; Sandrock and Van Etten 1998). This suggests that tomato pathogens have evolved speci®c mechanisms to counteract the toxic eect of this chemical. Tolerance to tomatine is based on two main mechanisms: some fungi are resistant to the compound because they have a modi®ed membrane composition, while others produce speci®c tomatine-detoxifying enzymes, known as tomatinases. These enzymes carry out a number of reactions that cleave tomatine, removing either all four sugars or a single sugar from the steroidal
923 Fig. 1 The structure of a-tomatine
glycoalkaloid. In all these cases, deglycosylation appears to be sucient to destroy the ability of tomatine to form complexes with membrane sterols and therefore to eliminate or reduce its toxic eect. Detoxi®cation of tomatine may therefore allow these tomato pathogens to avoid the glycoalkaloid barrier (Osbourn 1999). Fusarium oxysporum is a widespread and economically important soil-borne plant pathogen that causes vascular wilt diseases on a wide variety of crops. It exists in many forms, which are grouped into formae speciales on the basis of their ability to evoke disease in a particular host (Armstrong and Armstrong 1981). Most of the formae speciales seem to be limited to one host species, although sometimes their host range is wider. In the case of F. oxysporum f. sp. lycopersici the only host known is the tomato plant (Beckman 1987). Despite its agronomic importance and the recent increase in biochemical and genetic studies, the mechanisms of pathogenicity used by this fungus remain poorly understood. It is known that production of tomatinase by F. oxysporum f. sp. lycopersici is induced by atomatine (Ford et al. 1977; Lairini et al. 1996; RoldanArjona et al. 1999), and this may be part of a fungal defence mechanism against the compound. The identi®cation of other genes whose expression is activated in the presence of the glycoalkaloid may provide new insights into alternative mechanisms of resistance and further our knowledge of the metabolism of tomatine. In this work we applied mRNA dierential display analysis to mycelium grown on minimal medium in the presence and absence of a-tomatine, and we found that this saponin induces the F. oxysporum f. sp. lycopersici gene for pantothenase synthetase. The signi®cance and role of this induction is unknown, but it may represent an adaptive metabolic change in the fungus induced by the presence of a-tomatine and other signals which are generated as a plant defence response. The study of their physiological role may contribute to our knowledge of the interaction between the tomato plant and phytopathogenic fungi, and to the development of new
strategies to restrict pathogen propagation inside the plant.
Materials and methods Culture methods for F. oxysporum For dierential display analysis, cultures inoculated with microconidia from isolate 42-87 of F. oxysporum f. sp. lycopersici (Lairini and Ruiz-Rubio 1997) were grown with shaking for 72 h at 28°C in CA medium (10 g/l casamino acids, 10 mM ammonium sulphate and 0.5 g/l Yeast Nitrogen Base). The culture was ®ltered through nylon cloth (Monodur; mesh size 10 lm) to separate mycelium from spores, and the mycelium was re-inoculated into 25 ml of fresh CA medium. After 30 min of incubation at 28°C, a-tomatine (Sigma), dissolved in 50 mM potassium citrate buer (pH 4.0), was added aseptically to one ¯ask to a ®nal concentration of 40 lg/ml, and incubation was continued for 6 h. The mycelium was then collected by ®ltration through nylon cloth, frozen in liquid nitrogen and stored at ±80°C. A ¯ask inoculated with tomatine-free buer was incubated in parallel and used as a control. For gene expression analysis, cultures inoculated with microconidia from the same isolate, 42-87 (Lairini and Ruiz-Rubio 1997), were grown with shaking for 36 h at 28°C in CA medium. The culture was ®ltered through nylon cloth and 0.3 g of mycelium was re-inoculated into 100 ml of fresh CA media. After 2 h of incubation at 28°C, a-tomatine dissolved in 50 mM potassium citrate buer (pH 4.0) was added aseptically to the fungal growth medium to a ®nal concentration of 20 lg/ml, and incubation was continued for various periods up to 48 h. The mycelia were then collected by ®ltration through nylon cloth, frozen in liquid nitrogen and stored at ±80°C. Flasks inoculated with tomatine-free buer were incubated in parallel and sampled at the same time points. For DNA extraction, mycelium from dierent formae speciales (Lairini and Ruiz-Rubio 1997) was obtained from cultures grown for 4 days at 28°C in potato dextrose broth (PDB, Difco). Dierential display analysis of mRNA, and cloning of dierential cDNA fragments The Hieroglyph mRNA Pro®le kit (Genomix) was used for differential display analysis of mRNA according to the manufacturer's instructions. Total RNA (200 ng) was reverse transcribed and then used in PCRs containing 32P-labeled dATP and the combinations of primers recommended by the kit manufacturers. Aliquots of duplicate PCRs were electrophoresed in a 6%
924 polyacrylamide sequencing gel to separate the ampli®ed cDNAs, and bands that diered between the various samples were chosen for further analysis. After elution from the display gel, the dierentially expressed cDNAs were reampli®ed, cloned into the pGEM-T vector (Stratagene) and used as a probe for Northern analysis. Clones that gave a positive result when used as probes for Northern analysis were sequenced; available databases were subsequently searched for related sequences.
Nucleic acid isolation and analysis Genomic DNA was extracted from mycelium as described previously (Aljanabi and Martinez 1997). DNA was digested with SacI, and subjected to Southern hybridization analysis by standard procedures (Sambrook et al. 1989) using a non-isotopic digoxigenin labelling kit (Boehringer Mannheim) according to the instructions of the manufacturer. Total RNA was prepared from mycelium of F. oxysporum grown for periods between 30 min and 48 h in the absence or the presence of a-tomatine (®nal concentration 20 lg/ml) as described (Farrell 1993). Aliquots (5 lg) of total RNA were fractionated on a formaldehyde-1% agarose gel and transferred to positively charged nylon membranes (Boehringer Mannheim) by capillary transfer at 4°C. For quanti®cation, transferred RNA was stained for 5 min with 0.02% methylene blue in 0.3 M sodium acetate, pH 5.2. After destaining in 20% ethanol, ®lters were subjected to Northern hybridization analysis using the protocol supplied with the nonisotopic digoxigenin labelling kit. The probe used for Southern analysis corresponds to the 651-bp partial cDNA obtained in the dierential display analysis. For Northern analysis a single-stranded antisense DNA probe of 651 bp corresponding to the cDNA band obtained from the dierential display was generated using a standard protocol (Konat et al. 1994).
for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 3 min were followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 3 min. An initial denaturation for 1 min at 94°C and a ®nal elongation at 72°C for 2 min were included. In order to add a His10 tag at the N-terminal end of the PanC protein, the ampli®ed fragment was inserted into the pET-16b vector after digestion with NdeI and BamHI. The resulting construct, called pFoPanC, was used to transform the E. coli strain BL21(DE3) (Studier et al. 1990), and a single transformant colony was inoculated into 10 ml of LB medium containing carbenicillin (50 lg/ml) and incubated overnight at 37°C with shaking. A 1-ml aliquot of the overnight culture was inoculated into 1 l of fresh medium and incubated on a shaker at 37°C for 24 h, the cells were then collected by centrifugation at 4000´g for 30 min and the pellet frozen at ±80°C. The stored cell pellet was thawed and resuspended in 15 ml of sonication buer (TSB: 20 mM TRIS-HCl pH 8.0, 0.5 M NaCl, 5 mM imidazole). Cells were disrupted by sonication and the lysate was clari®ed by centrifugation. The supernatant was mixed with 0.7 ml of Ni2+-NTA resin (Qiagen) pre-equilibrated with TSB buer, and stirred gently for 1 h. The resin was then packed into a column and washed with 12 ml of TSB, followed by washing with 16 ml of TSB supplemented with 60 mM imidazole. The inclusion of 5 mM imidazole in the sonication buer (TSB) reduced nonspeci®c binding. Washing of the Ni2+-NTA column with TSB and TSB containing 60 mM imidazole removed non-speci®cally bound proteins. Histidine-tagged protein was eluted with 5 ml of TSB supplemented with 500 mM imidazole, and collected in 0.5-ml fractions. An aliquot of each fraction was analysed by SDS-PAGE, and those fractions containing a single band of the overexpressed protein were pooled. The protein preparation was divided into aliquots, frozen and stored at ±80°C. All steps were carried out at 4°C or on ice. Protein concentrations were determined by the Bradford assay. Denatured proteins were analysed on SDS-PA (12%) gels using Broad-Range molecular weight standards (Bio-Rad).
Screening of cDNA and genomic libraries A cDNA library was constructed in lambda ZAP Express (Stratagene) using RNA isolated from strain 42-87 of F. oxysporum f. sp. lycopersici grown in the presence of a-tomatine (Roldan-Arjona et al. 1999). This library and a lambda EMBL3 genomic library of strain 42-87 were screened using the a NcoI-SalI fragment of the pantothenate synthetase gene as a probe. Library screening and subcloning was performed as described in standard protocols (Sambrook et al. 1989). The autoexcision protocol supplied with the kZAP Express cDNA Synthesis kit (Stratagene) was followed. DNA sequence analysis was performed using the Dyedeoxy Terminator Cycle Sequencing kit (Perkin Elmer) on an ABI Prism 310 Genetic Analyser (Applied Biosystems). DNA and predicted amino acid sequence comparisons were carried out using the BLAST (Altschul et al. 1990) network service at the National Centre for Biotechnology Information (NCBI, Bethesda, Md.). Contiguous peptide sequences in the nonredundant protein database were scored against the query sequence using the BLOSUM62 homology matrix (Heniko and Heniko 1992). Sequences retrieved by the BLAST search were analysed using multiple sequence alignments (Feng and Doolittle 1987). Overexpression and puri®cation of pantothenate synthetase Plasmid DNA containing the panC cDNA was puri®ed using a mini-plasmid puri®cation kit (Qiagen) and used as a template for PCR. The oligonucleotide primers 5¢-CAGTACTCCATATGCTTCCTCTACGCTTC-3¢ and 5¢-ATAGGATCCAGCGTCTATGAATCC-3¢ were used to engineer NdeI and BamHI restriction sites (underlined) at the beginning and the end of the panC cDNA, respectively. The PCR was carried out in two stages, using Pfu DNA polymerase (Stratagene). Five cycles of denaturation at 94°C
Pantothenate synthetase assay Pantothenate synthetase activity was measured as described (Genschel et al. 1999). The reaction mixture (1 ml) contained 100 mM TRIS-HCl pH 7.8, 10 mM MgSO4, 5 mM ATP, 1 mM potassium phosphoenolpyruvate, 0.36 lmol NADH, myokinase (4 U), pyruvate kinase (6 U) lactate dehydrogenase (4 U), 10 mM b-alanine, 5 mM potassium D-pantoate and an adequate amount of enzyme. The assay components, except pantoate, were ®rst assembled in a 1-ml UV quartz cuvette, various amounts of the puri®ed protein were added and the volume was adjusted to 950 ll with distilled water. The absorption change at 340 nm was monitored immediately for about 2 min in order to determine background activity. The reaction was then initiated by addition of 50 ll of potassium D-pantoate solution and the absorbance at 340 nm was monitored until no further decrease in OD was observed.
Complementation assay The full-length panC cDNA in the vector pBK-CVM was used to transform the E. coli panC mutant AT1371 [panC4, D(gpt-proA)62, lacY1, tsx-29, glnV44(AS), galK2, LAM-, rac-0, hisG4(Oc), rfbD1, xylA5, mtl-1, argE(Oc), thi; Cronan et al. 1982], which was obtained form the E. coli Genetic Stock Center at Yale University (New Haven, Conn.). Four independent transformants were inoculated into 5 ml of LB medium containing kanamycin (30 lg/ml) and incubated at 37°C until the OD600 reached 0.7. Cells were then centrifuged, resuspended in the same volume of 10 mM MgSO4, and 10-ll drops were placed on M9 minimal medium plates supplemented, or not, with potassium D-pantoate. The plates were incubated overnight at 37°C.
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Results Isolation of a clone encoding an mRNA that is dierentially expressed in the presence of a-tomatine The dierential display technique (Liang and Pardee 1992) was used to compare the mRNA population of F. oxysporum f. sp lycopersici mycelium grown in the presence of a-tomatine with that of mycelium grown in the absence of the glyoalkaloid. Several dierential bands were revealed and one band of 651 bp was chosen for further characterisation. Northern analysis (Fig. 2) showed that it hybridised preferentially to a 1.3-kb mRNA which was induced during growth of the fungus in the presence of a-tomatine. This mRNA was not detectable when the cells were grown in the absence of the glyoalkaloid. A genomic library of the F. oxysporum f. sp. lycopersici isolate 42-87, constructed in kEMBL3, was screened with the 651-bp partial cDNA as a probe, and one positive clone was isolated and further characterised by restriction digestion and Southern hybridisation analysis. A 3.5-kb SacI fragment was subcloned in pBluescript KS±, and sequenced on both strands. The genomic clone contains an ORF of 1128 bp that encodes a predicted protein of 375 amino acid residues with a calculated molecular mass of 41 kDa. The 5¢ noncoding region shows the characteristic features of a lower eukaryotic promoter, including a TATA box (Gannon et al. 1979) at position ±74 relative to the start codon and two putative CAAT boxes (positions ±239, ±309). A cDNA expression library was prepared from mycelium of isolate 42-87 grown in the presence of 40 lg/ml a-tomatine, in the vector kZAP Express (Roldan-Arjona et al. 1999). This cDNA library was screened using a NcoI-SalI fragment of the genomic clone as a probe. Three positive clones were isolated and the phagemids were excised. One of the positive
Fig. 2 Northern hybridization analysis of total RNA from the F. oxysporum f. sp lycopersici isolate 42-87. Total RNA was obtained from fungi grown in the absence (lane 1) or the presence (lane 2) of a-tomatine (40 lg/ml), subjected to electrophoresis in a denaturing 1% agarose gel and blotted onto a nylon ®lter. The ®lter was probed with a digoxigenin-dUTP-labeled probe of 651 bp corresponding to a partial panC cDNA sequence obtained by dierential display analysis. The panel on the left shows the ®lter after staining of RNA with methylene blue
clones contained a full-length cDNA for pantothenate synthase, and the plasmid was designated pBKPanC. Comparison of the genomic and cDNA sequences con®rmed that the coding region contains no introns (Fig. 3). A database search with the deduced amino acid sequence of the tomatine-induced gene revealed a high degree of similarity to pantothenate synthetases from various organisms, including bacteria, yeast and plants (Fig. 4), with identity scores ranging from 42.8% (Sacharomyces cerevisiae) to 35.6% (Oryza sativa) (Fig. 4). In view of the functional characterization described below we have designated the gene panC. Southern hybridisation analysis of genomic DNA digested with SacI from six dierent formae speciales of F. oxysporum (strains 42-87, 18 M, G-60301, 2871, 77r and 699) using the 651-bp fragment from the partial cDNA as a probe, detected a single 3.5-kb hybridising band in all cases (data not shown). This result suggests that the genome of F. oxysporum f. sp. lycopersici contains a single copy of the panC gene, which is also present as a single copy in other formae speciales of the species.
Fig. 3 Nucleotide and deduced amino acid sequence of the pantothenate synthetase gene (EMBL Accession No. AJ298881). The nucleotide sequence is numbered relative to the A of the predicted start codon. The putative TATA and STRE sequences are marked by the black and grey boxes, respectively. The two putative CAAT boxes are underlined. The stop codon is indicated by an asterisk. Non-coding sequences are shown in lower-case letters. A putative polyadenylation consensus sequence is marked by an open box
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Fig. 4 Comparison of the amino acid sequence of F. oxysporum f. sp lycopersici PanC (EMBL Accession No. AJ298881) with pantothenate synthetases from various other organisms. Amino acids that are identical in at least four of the sequences are boxed. GenBank Accession Nos. for the sequences are as follows: Escherichia coli, P31663; Mycobacterium tuberculosis, O06280; Thermotoga neapolitana, O86953; Schizosaccharomyces pombe, Q09673; Oryza sativa, O24210; Lotus japonicus, O24035; Saccharomyces cerevisiae, P40459
Overexpression and puri®cation of PanC A polyhistidine (His10) anity tag was attached to the N-terminus of the PanC protein by subcloning the cDNA into the pET16b expression vector to obtain the recombinant plasmid pFoPanC. F. oxysporum PanC was expressed at a high level by growing the E. coli strain BL21 (DE3), transformed with the plasmid pFoPanC, at 37°C in the presence of IPTG, but most of the protein was insoluble and found in inclusion bodies. However, when cells were grown at 37°C in the absence of IPTG, leaky expression was still observed and soluble PanC protein was produced. The histidine-tagged protein was puri®ed to apparent homogeneity by Ni2+-NTA anity chromatography. Elution with 500 mM imidazole revealed a major protein band, as assessed by SDS-PAGE, which migrated with an apparent molecular mass of 43 kDa (Fig. 5). To con®rm that PanC is indeed a pantothenate synthetase, its enzymatic activity was measured by coupling AMP production to the activities of myokinase, pyruvate kinase and lactate dehydrogenase (Genschel et al. 1999). In this assay the rate of pantothenate formation is proportional to the rate of NADH oxidation. The recombinant PanC protein was active in the presence of dierent concentrations of D-pantoate ranging from 0.5 mM to 5 mM (Fig. 6). Protein fractions obtained from cells transformed with the vector (pET16b) alone were used as a negative control and showed no detectable pantothenate synthetase activity.
Functional complementation of an E. coli panC mutant The panC gene from F. oxysporum was checked for its ability to complement the panC phenotype of E. coli strain AT1371. The E. coli mutant was transformed with the pBKpanC vector and drops of cell suspension was placed on M9 plates with or without pantothenate as described in Materials and methods. The panC cDNA from F. oxysporum complemented the PS de®ciency in E. coli AT1371, whereas the pBK-CMV vector did not (Fig. 7). This result demonstrates that PanC protein from F. oxysporum functions in vivo as a pantothenate synthetase. Expression of the panC gene of Fusarium Northern analysis showed that the panC gene encodes a mRNA of approximately 1.3 kb that is inducibly expressed in the presence of a-tomatine (Fig. 8). In the absence of the glycoalkaloid a low level of panC transcript was observed, but transcript levels increased in its presence, reaching a maximum between 2 and 4 hours after addition of tomatine to the culture, although a slow decrease was observed afterwards.
Discussion Pathogenic attack is a stressful process for infected plants because the pathogen activates multiple mechanisms which allow it not only to penetrate, colonise and invade the host plant but also to counter all the defence mechanisms that the plant deploys to avoid fungal infection. The saponin a-tomatine is a preformed compound with antifungal properties that is present in the tomato plant. In order to avoid the toxic eects of tomatine F. oxysporum f. sp lycopersici produces a spe-
927
Fig. 5 Puri®cation of recombinant F. oxysporum f. sp lycopersici pantothenate synthetase. Pantothenate synthetase was overexpressed in E. coli, and proteins were visualised on a 12% SDS/ polyacrylamide gel by staining with Coomassie blue. Lanes 1 and 2 show lysates of BL21(DE3) cells containing the pET16b vector and the vector carrying panC cDNA, respectively. Lane 3 shows the puri®ed His-tagged pantothenate synthetase eluted from the nickelchelate column in the presence of 0.5 M imidazole. Positions of protein size markers (Bio-Rad) are indicated (in kDa) on the left
Fig. 6 Activity of the recombinant pantothenate synthetase. Enzyme activity was measured by coupling AMP production to the activities of myokinase, pyruvate kinase and lactate dehydrogenase. In this assay the rate of pantothenate formation is proportional to the rate of NADH oxidation. The decrease in absorbance at 340 nm was monitored in the presence of 0.5 mM (squares), 1 mM (diamonds) and 5 mM (circles) pantoate. The assay was repeated on two independent occasions with similar results, and the result of one of these experiments is shown
ci®c tomatine-detoxifying enzyme called tomatinase. A dierential display analysis of mRNA F. oxysporum f. sp. lycopersici shows that, besides the gene encoding tomatinase, several other genes are induced in the presence of the glycoalkaloid. Here we report the cloning of a gene encoding a pantothenate synthetase activity from F. oxysporum f. sp. lycopersici that is
Fig. 7 Functional complementation of the E. coli panC mutant. E. coli AT1371 (panC) cells were transformed with plasmid pBKCMV (left panels) or the plasmid pBKPanC containing the full-length panC cDNA (right panels). Cells of independent transformants were grown on LB medium containing kanamycin, harvested by centrifugation and resuspended in 10 mM MgSO4. A drop (10 ll) from this suspension was plated in M9 minimal medium plates supplemented (+) or not (±) with potassium D-pantoate. The plates were incubated overnight at 37°C
Fig. 8 Induction of panC in the presence of a-tomatine. Northern hybridization analysis of panC transcript accumulation in F. oxysporum f. sp lycopersici mycelium grown in medium containing a-tomatine. Total RNA was puri®ed from mycelium grown in the absence (±) or the presence (+) of a-tomatine (20 lg/ ml), for 30 min (lanes 1 and 2), 1 h (lanes 3 and 4), 2 h (lanes 5 and 6), 4 h (lanes 7 and 8), 8 h (lanes 9 and 10), 12 h (lanes 11 and 12), 24 h (lanes 13 and 14) and 48 h (lanes 15 and 16) after addition of the glycoalkaloid. The RNA was blotted onto a nylon membrane and stained with 0.02% methylene blue (upper panel). The ®lter was then destained and hybridised with a digoxigenin-dUTP-labelled, single-stranded antisense DNA probe corresponding to the 651-bp partial panC cDNA identi®ed by dierential display analysis (lower panel)
selectively induced in the presence of a-tomatine. Pantothenate synthetase from F. oxysporum f. sp. lycopersici is encoded by a single gene which is also present as a single copy in other formae speciales of F. oxyxporum, including melonis (18 M), niveum (G60301 and
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2871), radicis lycopersici (77r) and conglutinans (699). The recombinant PanC protein overexpresed in E. coli and puri®ed by anity chromatography is active as a pantothenate synthetase and is able to complement the PS de®ciency of the E. coli strain AT1371, which lacks a functional panC gene. The signalling pathway leading to tomatine-dependent induction of panC is unknown. Sequence analysis of the promoter region revealed the presence of a putative STRE consensus motif (CCCCT) which has been shown in yeast to be related to gene activation in response to a variety of stress conditions, especially heat and osmotic stress, low pH and nutrient starvation, although the primary cellular events triggering this response are not well understood at present (Siderius and Mager 1997). The fungitoxic eect of tomatine is attributed to its interaction with membrane sterols, which results in an increase in fungal membrane permeability, pore formation and leakage of cell contents. Therefore, the possibility exists that damage caused by tomatine at the membrane level represents a stress signal that evokes a cellular response which in turn activates STREdependent panC transcription. This hypothesis is supported by the ®ndings of Moskvina et al. (1999) in Saccharomyces cerevisiae. These authors showed that dierent stress factors acting at the level of the plasma membrane, including tomatine and other antifungal agents, induce transcription via the STRE motif. An important question arising from our results is the role played by pantothenate synthetase during the infection process. Panthotenic acid is an essential precursor of CoA and acyl carrier protein, which are cofactors required in many energy-yielding reactions and in a large number of metabolic processes (Abiko 1975). Therefore, the induction of panC in the presence of a-tomatine seems important to ensure a continuous supply of such a cofactor to deal with the presence of the glycoalkaloid. In light of the role of pantothenate in ATP generation and fatty acid biosynthesis, it could be important for the fungus to induce pantothenate synthetase during plant infection for at least two reasons. First, tomatine stimulates sporulation in vitro (Smith and MacHardy 1982) which implies a high energy demand. Secondly, the toxic eects of tomatine, as mentioned above, are attributed to its ability to form complexes with membrane sterols (Safe et al. 1977; Dow and Callow 1978; Roddick 1979; Roddick and Drysdale 1984; Steel and Drysdale 1988; Keukens et al. 1995). Thus, it is reasonable to envisage a scenario in which the sterol components of the membrane are depleted and biosynthesis of fatty acids is increased in order to repair the damage to the cell membrane. Acknowledgements The authors would like to thank Dr. A.G. Smith and Dr. J. Ashurst for help with the pantothenate synthetase assay and Dr. R. R. Ariza for helpful discussion and careful reading of the manuscript. A. P.-E. was supported by a predoctoral fellowship from the University of CoÂrdoba and T. R.-A. by Marie Curie Fellowship from the European Union. This work was ®nanced by a grant from the European Union (BIOTECH, contract no. BIO2-CT94±3001).
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