INTRODUCTION

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Among the wide array of cell wall-degrading enzymes (CWDEs) are polygalacturonases (PGs), which are involved in the degradation of pectin, a complex polysaccharide that is primarily found in the middle lamella and primary cell wall of higher plants (33). Endopolygalacturonases [endoPGs; poly(1,4--D-galacturonide) galacturonohydrolase; EC 3.2.1.15], which are produced by many organisms and have long been studied with regard to their role in many aspects of pathogenicity, are responsible for depolymerization of homogalacturonan, a major component of plant cell walls (10). The genes encoding a number of fungal endoPGs, including those of Aspergillus flavus, Aspergillus niger, Botrytis cinerea, Cochliobolus carbonum, Colletotrichum lindemuthianum, Cryphonectria parasitica, Fusarium moniliforme, Fusarium oxysporum, and Sclerotinia sclerotiorum, have been cloned and characterized (3-6, 14, 17, 23, 27, 28, 34). However, with a few notable exceptions (28, 30), conclusive evidence for the role of fungal CWDEs in pathogenesis by targeted gene disruption is lacking (17, 26, 27). Besides acting as virulence factors, endoPGs may also function as avirulence determinants through release of oligogalacturonide inducers of plant defense (11) and interactions with plant proteins that modulate activities of PG-inhibiting proteins (PGIPs) (7). Regulation of endoPG gene expression is generally subject to the carbon source available, with the exception of a constitutively expressed gene in B. cinerea (31). These genes are mainly induced by pectin and subject to glucose repression (14, 27, 35), although complete regulation of endoPG gene expression is not completely understood.

EndoPGs are among the first CWDEs secreted by F. oxysporum f. sp. lycopersici upon contact with the host tissue (21). Like other pectinolytic enzymes, endoPGs have been suggested to be of prime importance in a number of key steps during infection, such as root penetration, colonization of the vascular tissue, and perforation of xylem vessel plates (2). To investigate this hypothesis, we have previously isolated an endoPG gene and an exopolygalacturonase (exoPG) gene from F. oxysporum. Both genes are expressed by the fungus during infection, but inactivation of either gene had no effect on virulence (14, 19).

The soilborne plant pathogen F. oxysporum Schlecht causes vascular wilt disease on a wide variety of crops (2). This fungus produces a wide variety of extracellular CWDEs, including xylanases, cellulases, proteases, pectate lyases, and exo- and endoPGs, (9, 12, 13, 16, 18, 20, 24), that may contribute to the degradation of the structural barriers constituted by plant cell walls. The gene encoding the major endoPG secreted by F. oxysporum, PG1, has been cloned, and mutants lacking an active copy of pg1 were shown to retain full virulence (14). These mutants still exhibited extracellular PG activity, although it was strongly reduced in comparison with the wild type, suggesting the presence of additional PG genes in F. oxysporum. The present report describes the isolation of pg5, encoding a novel endoPG, from F. oxysporum. We show that pg5 is expressed during saprophytic growth on citrus pectin and in planta during the initial stages of infection. Targeted inactivation of pg5 suggests that the enzyme is not essential for pathogenesis on tomato plants.

	MATERIALS AND METHODS

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Fungal isolates and culture conditions. F. oxysporum f. sp. lycopersici strain 4287 (race 2) and F. oxysporum f. sp. melonis strain 18 M (race 1) were obtained from J. Tello (Universidad de Almería, Almería, Spain) and stored as microconidial suspensions in 30% glycerol at 80°C. The pathotypes of the isolates were periodically confirmed by plant infection assays in a growth chamber. The detailed origins of the other F. oxysporum isolates used for Southern analysis are described elsewhere (15).

Nucleic acid manipulations. pg5 was isolated from a lambda EMBL3 genomic library of F. oxysporum f. sp. lycopersici isolate 42-87 by using the Cryphonectria parasitica enpg1 clone as a probe (17). Library screening, subcloning, and other routine procedures were performed as described in standard protocols (25). Sequencing of both DNA strands was performed at the Servicio de Secuenciación Automática de DNA, CIB, Madrid, Spain, using a Dyedeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer, Foster City, Calif.) on an ABI Prism 377 Genetic Analyzer apparatus (Applied Biosystems, Foster City, Calif.). Analyses of sequencing data were carried out with the Lasergene programs (DNAStar Inc., Madison, Wis.). DNA and protein sequence databases were searched by using the BLAST algorithm (1) at the National Center for Biotechnology Information (Bethesda, Md.).

Assays of pathogenicity on tomato plants and fruits. Infection of tomato plants was performed as reported elsewhere (14). Briefly, tomato seedlings of cultivar Vemar were inoculated with F. oxysporum f. sp. lycopersici strains by dipping the roots in a microconidial suspension, planting seedlings in minipots with vermiculite, and maintaining them in a growth chamber at 25°C with 14 h of light and 10 h of darkness per day. Plants immersed in sterile water were used as controls. For pathogenicity assays, the severity of disease symptoms was recorded at different times after inoculation, using a scale ranging from 1 (healthy plant) to 5 (dead plant) (14). Twenty plants were used for each treatment group.

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RT-PCR. Five plants from each treatment group, inoculated as described above, were sampled after different time periods; total RNA was isolated from roots and lower parts of stems, and reverse transcription (RT)-PCR was performed as previously reported (14). Total RNA was treated with RNase-free DNase (Roche Molecular Biochemicals) and reverse transcribed into cDNA with murine leukemia virus reverse transcriptase (Gibco BRL, Paisley, United Kingdom), using the specific antisense primer 5'-AAGTTGGTGACGCTGTTGATG-3'. A volume of the RT reaction product was used for PCR amplification with the sense primer 5'-CCGATGCTGCTACTGCTATT-3' and the antisense primer described above, both flanking the intron of pg5. PCR conditions were as follows: 40 cycles with denaturation at 94°C for 35 s, annealing at 50°C for 35 s, and extension at 72°C for 90 s. An initial denaturation step of 2 min at 94°C and a final elongation step at 72°C for 6 min were performed. Total genomic DNA of F. oxysporum was used as a template for PCR to compare the sizes of the amplified fragments with and without the intron. Aliquots of the PCR products were separated on 2% agarose gels, transferred to positively charged nylon membranes, and subjected to Southern hybridization analysis with the labeled pg5 probe.

Expression, purification, and characterization of the pg5 product in a heterologous bacterial system. Total RNA from F. oxysporum strain 4287 grown on citrus pectin was retrotranscribed to cDNA by RT-PCR using the oligonucleotides 5'-CCATCCCTCATATGCGTGCCGGCAGCTGC-3' (sense) and 5'-CTACCGCTCGAGAATAGTATTAACTGATAG-3' (antisense). The amplified cDNA lacked the putative signal peptide and had an NdeI site and a XhoI site introduced at its 5' and 3' ends, respectively. To add a 10-histidine tag at the amino-terminal end of the PG5 protein, the amplified fragment was inserted into the pET-16b vector by digestion with NdeI and XhoI restriction enzymes and ligation. Escherichia coli expression strain BL21(DE3) (29) was transformed with the ligation mixture, and a single transformant colony was isolated. Bacterial cultures were grown on a large scale at 28°C, and the recombinant protein, extracted from cell lysates of 500-ml cultures, was purified under denaturing conditions by using nickel-nitrolotriacetic acid (Ni²⁺-NTA) resin according to the manufacturer's instructions (Qiagen Ltd., Dorking, Surrey, United Kingdom). Aliquots of each eluted fraction (500 µl) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12% (wt/vol) acrylamide resolving gels followed by staining with Coomassie brilliant blue. Fractions containing a single band of the overexpressed protein were pooled and renatured by using a gradient of decreasing concentrations of urea (8 to 0 M). EndoPG activity was measured by determining the release of reducing groups from PGA and expressed in nanokatals as previously described (12). Analysis of degradation products by thin-layer chromatography was carried out as reported elsewhere (18).

Transformation-mediated gene replacement and analysis of transformants. The gene replacement vector pPg5::pANBlue3 was constructed as follows. A 0.6-kb HindIII-ApaI fragment containing 5'-flanking sequence and the first 312 bp of the pg5 coding region and a 0.5-kb BamHI-HindIII fragment containing the final 404 bp of the pg5 coding region and 3'-flanking sequence were cloned, in opposite directions, into the hygromycin resistance vector pAN7Blue3 (14). After linearization with HindIII, the final replacement vector contained 0.6 and 0.5 kb of colinear genomic DNA, with pAN7Blue3 replacing approximately 350 bp of the pg5 coding region. The linearized fragment was used to transform protoplasts of F. oxysporum strain 42-87 by a protocol described previously (14). To obtain protoplasts induced for pg5 expression, microconidia were germinated for 14 h in SM with 1% citrus pectin prior to protoplast formation (19). Transformants appeared after 5 days and were transferred to hygromycin plates and subjected to two consecutive rounds of single-spore isolation before being stored as microconidial suspensions at 80°C.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Isolation and characterization of pg5 In the present study, a novel endoPG gene from F. oxysporum, pg5, was identified using a heterologous endoPG clone (enpg1) from Cryphonectria parasitica as the probe (17). The screening of an F. oxysporum f. sp. lycopersici isolate 4287 genomic library yielded three recombinant phage clones that were characterized by restriction and Southern hybridization analyses. Results indicated that the three clones encompassed different portions of the same genomic region. A 1.6-kb HindIII fragment that hybridized to the probe was subcloned in pBluescript KS(+) and sequenced on both strands. The pg5 coding region consists of an open reading frame of 1,083 bp, encoding a 361-amino-acid polypeptide with five potential N-glycosylation sites, interrupted by one intron of 49 bp whose position was confirmed by sequencing of the cDNA clone. The 5'-flanking region contains two TATA boxes, at the 50 and 64 positions. The N-terminal amino acid sequence of the predicted protein has the characteristic features of a signal peptide with a predicted cleavage site between residues 23 and 24 (32).

View larger version (109K): [in this window] [in a new window]   FIG. 1.   Multiple alignment of the deduced amino acid sequences of F. oxysporum f. sp. lycopersici (F.o.) strains PG5 (GenBank accession no. AF078156) and PG1 (U96456), Cryphonectria parasitica ENPG-1 (U49710), Colletotrichum lindemuthianum CLPG1 (X89370), Sclerotinia sclerotiorum (S.s.) PG1 (L12023), and Cochliobolus carbonum PGN1 (M37819). Identical amino acids are shaded.

Expression of pg5 in E. coli and characterization of the gene product. A polyhistidine (His₁₀) affinity tag was attached to the N terminus of the mature PG5 protein by subcloning the cDNA without the signal peptide coding sequence into the pET16b expression vector. High levels of a 35-kDa protein were produced in E. coli BL21(DE3) transformed with the recombinant vector and grown in the presence of isopropyl--D-thiogalactopyranoside (IPTG) (Fig. 2A). This protein band was absent from E. coli transformants grown in medium lacking IPTG and from a strain transformed with the pET16b vector alone. No PG activity was detected in the recombinant protein samples, indicating that most of the protein was insoluble and found in inclusion bodies (results not shown). Therefore, Ni²⁺-NTA affinity chromatography of the polyhistidine-tagged PG5 protein was carried out under denaturing conditions, in 8 M urea, after which the protein was renatured in vitro under a gradient of decreasing urea concentration. This protocol produced an apparently homogeneous 35-kDa protein band, as determined by SDS-PAGE (Fig. 2A), that had PG activity. This fraction showed a total activity of 0.75 nkat, corresponding to a specific activity of 0.5 nkat/µg of protein (Fig. 2B). To determine the mode of action of PG5, the end products of enzymatic hydrolysis of PGA were analyzed by thin-layer chromatography. The presence of oligogalacturonides with intermediate degrees of polymerization together with di- and monogalacturonic acids was consistent with a classical endo mode of cleaving activity of PG5 (results not shown).

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Purification of the recombinant pg5 gene product. (A) pg5 was overexpressed in E. coli grown in the presence of IPTG, and proteins were separated on an SDS-12% polyacrylamide gel and stained with Coomassie blue. Lane 1, molecular size markers; lanes 2 and 3, lysates of BL21(DE3) cells containing pET16b vector without (lane 2) or with (lane 3) the insert; lane 4, purified PG5 protein eluted from the Ni2+-NTA column. The molecular mass of the recombinant PG5 protein (in kilodaltons) is indicated on the left. (B) PG activities (specific activities) in bacterial lysates and in pure protein extract, expressed in nanokatals per microgram of protein. Column numbers correspond to lane numbers in panel A. One nanokatal was defined as the amount of enzyme that produced 1 nmol of galacturonic acid.

Expression of pg5 during saprophytic growth and during infection of tomato plants. Expression of pg5 was determined by Northern hybridization analysis of total RNA obtained from mycelia of F. oxysporum f. sp. lycopersici grown for different periods of time in SM supplemented with different carbon sources. A single 1-kb transcript was detected in mycelia grown on citrus pectin, with maximum expression at 12 h of growth, whereas no transcript was detected in mycelia grown on glucose, PGA, or TVT (Fig. 3). Moreover, 1% D-galacturonic acid also induced pg5 expression, whereas 1% rhamnose did not (data not shown). The highly reduced accumulation of transcript levels in mycelia grown on 1% pectin plus 1% glucose suggested that glucose acts as a partial repressor of pg5 expression (data not shown). The two endoPGs of F. oxysporum, PG1 and PG5, show similar patterns of regulation during growth in axenic culture. Both are strongly induced by pectin and repressed by glucose.

View larger version (50K): [in this window] [in a new window]   FIG. 3.   Northern hybridization analysis of pg5 transcript accumulation in F. oxysporum f. sp. lycopersici mycelium grown for the indicated time periods (in hours) in synthetic medium containing 1% citrus pectin, 1% glucose, 0.5% PGA, or 2.5% TVT as the carbon source. (Lower panel) Total RNA blotted onto a nylon membrane and stained with 0.02% methylene blue. (Upper panel) Same filter, destained and hybridized with the digoxigenin-dUTP-labeled pg5 probe. The size of the transcript is indicated in kilobases.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Southern hybridization analysis of RT-PCR products, showing the expression pattern of pg5 during infection of tomato plants by F. oxysporum f. sp. lycopersici. First-strand cDNAs were generated from total RNA isolated at the indicated time points (in hours) from roots and stems of uninfected or infected plants and used as templates for PCR with primers specific for pg5 (see Materials and Methods). Aliquots of the PCR products were electrophoresed on a 2% agarose gel, blotted onto a nylon membrane, and hybridized with the labeled pg5 probe. The position and size (in base pairs) of the pg5 fragment are indicated. The numbers represent days after inoculation. gDNA, genomic DNA.

Targeted replacement of pg5 Mutants carrying a disrupted copy of the pg5 gene were generated by a one-step gene replacement protocol. Vector pPg5::pANBlue3 was constructed, as shown in Fig. 5A and B, by replacing a 0.3-kb ApaI-BamHI fragment within the pg5 coding region with the hygromycin B resistance plasmid pAN7Blue3. A linear HindIII restriction fragment containing the disrupted pg5 gene was used to transform F. oxysporum f. sp. lycopersici strain 4287, which is highly virulent to tomato plants. Eight hygromycin-resistant transformants were selected, and their genomic DNAs were isolated, digested with HindIII, and subjected to Southern analysis with the pg5 probe. As shown in Fig. 5C, in two of the transformants, Dpg5-1 and Dpg5-2, the 1.6-kb HindIII fragment corresponding to the wild-type pg5 allele was replaced by a larger, 6.0-kb fragment. This indicates that both transformants contain a single copy of the replacement vector integrated by double homologous recombination, thereby generating a disrupted copy of pg5. Consistent with gene replacement, the same single fragment hybridized with a probe for the hygromycin resistance gene (data not shown). The rest of the transformants, exemplified by NDpg5-3, contained the wild-type pg5 allele together with an additional hybridizing fragment, indicative of ectopic insertion of the replacement vector. Northern analysis of pectin-grown mycelia of the mutant strains Dpg5-1 and Dpg5-2, as well as the wild-type strain and the ectopic transformant NDpg5-3, confirmed the absence of the pg5 transcript in the two mutants, while this transcript was readily detected in the other strains (Fig. 6). Transcript levels of the exo- and endoPG pgx4 and pg1 genes, respectively, were comparable in pg5-mutants and in the control strains.

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Targeted replacement of the F. oxysporum pg5 gene. (A) Physical map of the pg5 genomic region. The pg5 coding region is shown as a black arrow orientated with the open reading frame. Restriction enzymes are abbreviated as follows: A, ApaI; B, BamHI; and H, HindIII. (B) Gene replacement vector pPg5::pANBlue3. (C) Analysis of transformants by Southern blotting. Genomic DNAs from the transformants Dpg5-1, Dpg5-2, and NDpg5-3 (lanes 1 to 3, respectively) and wild-type strain 4287 (lane 4) were digested with HindIII, separated in a 0.7% agarose gel, blotted onto a nylon membrane, and hybridized with a pg5 probe. Sizes of hybridizing bands are indicated in kilobases.

View larger version (72K): [in this window] [in a new window]   FIG. 6.   Northern hybridization analysis of F. oxysporum f. sp. lycopersici wild-type strain 4287 (lane 5), replacement mutants Dpg5-1 and Dpg5-2 (lanes 1 and 2, respectively), and ectopic transformants NDpg5-3 and NDpg5-4 (lanes 3 and 4, respectively). (Upper panels) Total RNA from mycelia grown in synthetic medium containing 0.5% PGA (sodium salt) was hybridized with probes of the endoPG pg5 and pg1 genes and of the exoPG pgx4 gene. (Lower panel) Total RNA was blotted onto a nylon membrane and stained with 0.02% methylene blue. *, sample was not included in the hybridization with the pg1 probe.