INTRODUCTION

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Many bacteria and fungi synthesize extracellular enzymes that can degrade pectin or its demethylesterified form, polygalacturonic acid, and a number of such enzymes and their encoding genes have been isolated from plant pathogenic microorganisms and studied for their role in the disease process. Extracellular pectin-degrading enzymes clearly contribute to symptom development in soft rot diseases and can induce defense responses in a number of plants (32), but their exact roles in the process of pathogenesis, especially in diseases not characterized by soft rotting, are largely unknown. A major barrier to a more precise definition of the role of pectin degradation in the disease process is the presence in most microorganisms of multiple pectin-degrading enzymes and -encoding genes (14, 26, 32).

Cochliobolus carbonum, the causal agent of Northern leaf spot of corn, attacks mainly foliar tissue but also the stalks and ears of susceptible maize. This filamentous fungus produces a number of extracellular cell wall-degrading enzymes including xylanases, cellulases, proteases, mixed-linked glucanases, exo-1,3-glucanases, -arabinosidase, and -xylosidase (32). It produces at least three pectin-degrading enzymes, i.e., endo-1,4-polygalacturonase (endoPG), exo-1,4-polygalacturonase (exoPG), and pectin methylesterase (PME) (25, 26, 33). Strains of C. carbonum mutated in the gene encoding endoPG, PGN1, grow normally on pectin and are still pathogenic. Total PG activity is reduced by ca. 60%, and residual activity is due to one or more exo-acting activities that appear as a set of peaks of variable sizes when fractionated by cation-exchange chromatography (26). Although these earlier results demonstrated that PGN1 by itself is not required for pathogenicity, they leave unanswered the more general question of the requirement for any pectin-degrading ability in pathogenesis by C. carbonum. In order to address this, it is necessary to create a strain of the fungus that completely lacks all pectin-degrading activity. As a step in this direction, we report here the purification of the exoPG activity of C. carbonum and the cloning of its gene, PGX1. exoPGs have previously been identified and purified from plant pathogenic fungi (8, 22), and exoPG-encoding genes have been isolated from the bacterial pathogens Erwinia chrysanthemi and Ralstonia (Pseudomonas) solanacearum (9, 11). exoPG contributes to growth of E. chrysanthemi on polygalacturonic acid but not to tissue maceration (9), whereas the exoPG of R. solanacearum is a virulence factor (11). To the best of our knowledge, no exoPG-encoding gene has previously been isolated from a plant pathogenic fungus.

	MATERIALS AND METHODS

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Fungal cultures and enzyme purification. C. carbonum was grown and maintained as previously described (34). PG activity was determining the production of reducing sugars from sodium polygalacturonic acid (Sigma) at 30°C. Reducing sugars were detected with p-hydroxybenzoic acid hydrazide (18, 33). endoPG activity was distinguished from exoPG activity by comparing activity as measured by viscometry and activity as measured by the release of reducing sugars (26, 33).

Nucleic acid manipulations. Genomic DNA and RNA isolation, DNA and RNA analysis, and library screening were performed as described elsewhere (24, 26). PCR was carried out in a Perkin-Elmer thermocycler under the following conditions: 3 min of denaturation at 94°C; 35 cycles of 1 min of denaturation at 94°C, 2 min of annealing at 54°C, and 3 min of primer extension at 72°C; and 10 min of primer extension at 72°C. The PCR primers used were CARTAYCCNGGNGARGT and CCNGGCCANACYTTHAT, corresponding to the exoPG amino acid sequences QYPGEV and IKVWPG, respectively (R represents A or G, Y represents C or T, H represents A, T, or C, and N represents any base). A third oligonucleotide, TTYTCNACHATRTC, corresponding to the sequence DIVEN, was end labelled with polynucleotide kinase and used as a probe to select the correct PCR product. A 99-bp DNA fragment was identified, cloned, sequenced, and used to screen genomic and cDNA libraries as described elsewhere (17, 23). cDNA and genomic clones were sequenced by using nested deletions (26). Sequencing was done by automated fluorescent sequencing at the MSU-Department of Energy Plant Research Laboratory Plant Biochemistry Facility. PGN1 and PGX1 were chromosome mapped by DNA hybridization to a DNA blot of C. carbonum chromosomes separated by pulsed-field gel electrophoresis (1).

Transformation-mediated gene disruptions and replacements. Gene disruption mutants were obtained with vectors containing internal fragments from the coding region of each gene. For PGN1, an internal 655-bp BamHI fragment and a 4-kb KpnI/SalI fragment containing the amdS gene for acetamide utilization from Aspergillus nidulans (12) were cloned into vector pSP72 (Promega) to make plasmid pPGNK1. For PGX1, an internal 1.5-kb HindIII/BamHI fragment was cloned into the corresponding sites in pHyg1, which contains the hph1 gene for resistance to hygromycin driven by the P1 promoter of C. heterostrophus (28) to make plasmid pPGXK1. Plasmids were linearized by cutting at unique restriction sites in the cloned segments of C. carbonum DNA prior to transformation (26). Transformants were purified by seven rounds of single-spore isolation.

Nucleotide sequence accession number. The sequence of PGX1 was deposited in GenBank in November 1995 under accession number L48982.

	RESULTS

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Isolation of exoPG. pgn1 mutants of C. carbonum have residual PG activity due to one or more exo-acting enzymes (26). Similarly to endoPG activity, production of exoPG activity is stimulated by the presence of pectin and is suppressed by 2% sucrose (33). In contrast to endoPG activity, which reaches a maximum after 7 days of growth on pectin and then declines to almost undetectable levels by 14 days (33), exoPG activity continues to increase until at least day 14 (25). After concentration and passage through a column of DEAE-cellulose to remove pigments and anionic proteins, culture filtrates of the C. carbonum pgn1 mutant strain grown for 14 days on pectin were analyzed by cation-exchange HPLC. exoPG activity appears as multiple peaks of activity of variable sizes and numbers, with a major broad peak of activity eluting just before Pgn1p, the product of PGN1 (26). These multiple exoPG activity peaks could be due to multiple exoPG-encoding genes and/or to variable posttranslational modifications of a single gene product.

View larger version (68K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of purified exoPG before (lane 2) and after (lane 3) chemical deglycosylation. Molecular mass markers (lane 1) are in kilodaltons. Lane 2 was loaded with 2 µg of protein, and lane 3 was loaded with what was left of 2 µg of protein after it had been deglycosylated.

Cloning of PGX1. Oligonucleotides based on portions of peptide sequences 1 (IKVWPG) and 2 (QYPGEV) were synthesized, and a 99-bp fragment of PGX1 was amplified by PCR. This fragment hybridized with an oligonucleotide based on DIVEN in peptide sequence 2. The PCR product was used as a probe to isolate cDNA and genomic copies of the encoding gene, PGX1, and both were sequenced on both strands. The DNA sequence of PGX1 contains an open reading frame of 1,341 bases that is interrupted by three introns of 51, 63, and 70 bp (Fig. 2). The predicted protein product of PGX1 contains 446 amino acids with a molecular mass of 47.8 kDa, which is in good agreement with the molecular mass (45 kDa) of the deglycosylated gene product determined by SDS-PAGE (Fig. 1). The product of PGX1 (called Pgx1p) has 12 potential N-glycosylation sites (Fig. 2). The N terminus of the mature protein could not be experimentally determined, but the SignalP program predicted a signal peptide cleavage site between amino acids 19 and 20 (21). The dipeptide KR at amino acid 33 could be a second processing site for a Kex-2-like protease (6). Pgn1p is also likely to undergo a second proteolytic processing event based on comparison of its predicted signal peptide cleavage site and the known N terminus of the mature protein (SwissProt P26215) (26).

View larger version (72K): [in this window] [in a new window]   FIG. 2.   DNA and deduced amino acid sequences of PGX1. Amino acids are shown below the corresponding codons. The 12 potential N-glycosylation sites (NXS/T) are underlined. The two peptides used for the design of PCR primers are doubly underlined. The three introns are indicated by lowercase letters. *, stop codon; +, polyadenylation site.

Expression of PGX1 in culture and in planta. RNA was extracted from mycelial mats grown with sucrose, pectin, or purified maize cell walls as the carbon source. PGX1 mRNA was abundant in 7-day-old fungal mycelium grown on pectin, was present in mycelium grown on maize cell walls, and was absent in mycelium grown on sucrose (Fig. 3). During the course of infection of maize leaves, PGX1 mRNA could be detected starting 3 days after inoculation (Fig. 4). At this point, infection was well developed, with large, expanding lesions. PGN1 was also expressed during infection and could be detected before either PGX1 or GPD1 (Fig. 4).

View larger version (30K): [in this window] [in a new window]   FIG. 3.   RNA blot of total RNA (10 µg) extracted from wild-type strain 367-2 and probed with the PGX1 cDNA. The fungus was grown in still culture for 7 days on modified Fries' medium salts with 2% sucrose (lane 1), 1% pectin (lane 2), or 1% maize cell walls (lane 3). Equal loading of each lane was confirmed by staining of the blot with methylene blue (10). The size of the PGX1 mRNA is 1.4 kb.

View larger version (100K): [in this window] [in a new window]   FIG. 4.   RNA blot of expression of PGN1, PGX1, and GPD1 during infection of maize plants. Insofar as the expression of GPD1 is constitutive, it serves as an indicator of fungal mass. Numbers indicate days after inoculation (day 0 is immediately after inoculation). Fifteen micrograms of poly(A)+ RNA per lane was loaded. The blot was first probed with PGN1, stripped, probed with PGX1, stripped, and probed with GPD1. Approximately equal loading of mRNA in each lane, including days 0 and 1, was confirmed by staining of the gel with ethidium bromide.

Gene disruptions and replacements of pgx1 and pgn1. Initially, pgx1 and pgn1 were mutated by single-crossover gene disruption (26). In the pgx1 mutant, the major peak as well as the minor peaks of exoPG activity eluting from the cation-exchange HPLC column disappeared (Fig. 5). One explanation for this result is that the multiple peaks of exoPG activity are all encoded by PGX1 and represent glycosylation isoforms of Pgx1p. An alternative explanation is that the other peaks are the products of other genes whose expression is dependent on expression of PGX1; for example, the action of Pgx1p on pectin may release essential inducers of these genes.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Cation-exchange HPLC analysis of PG activity of wild-type, pgn1, and pgx1 single mutants and a pgn1/pgx double mutant made by gene disruption. Culture filtrates were harvested after 7 days of growth on 1% pectin as the carbon source. Approximately equal amounts of protein were loaded onto the HPLC column in each case. One-milliliter fractions were collected. Of each fraction, 25 µl was assayed in a total volume of 300 µl, and, at the end of a 30-min incubation at 30°C, 25 µl was sampled for total reducing sugars. Enzyme activity is expressed as change in the optical density at 410 nm (OD410); an OD410 of 1.0 represents 7.5 µg of galacturonic acid.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Genomic restriction maps and strategies for gene replacement mutation of PGN1 and PGX1. Dashed lines, DNA that was deleted and the DNA that was inserted. (Upper diagram) PGN1 replacement vector pPGNGR1; (lower diagram) PGX1 replacement vector pPGXGR1.

View larger version (91K): [in this window] [in a new window]   FIG. 7.   DNA blot analysis of progenies from a cross between PGN1 and PGX1 replacement mutants made as diagrammed in Fig. 6. DNA was cut with HindIII in all lanes. (Top panel) DNA blot probed with hph1 encoding hygromycin phosphotransferase; (bottom panel) the same blot after stripping and reprobing with PGN1 and PGX1 simultaneously (the faint bands are residual hph1 hybridization). In each lane of the bottom panel, the upper band, when present, is PGN1, and the bottom band, when present, is PGX1. Lanes: 1, DNA from 367-2 (wild type); 2 through 13: strains 655-1 through 655-12, respectively, which are progenies from a cross between 639-2 (pgn1) and 640-11 (pgx1).

View larger version (26K): [in this window] [in a new window]   FIG. 8.   Cation-exchange chromatography analysis of wild type (SB111) and the double mutant pgn1/pgx1 (655-5) made by gene replacement. Cultures were harvested after 7 days of growth on 1% pectin. One-milliliter fraction were collected. (Top panel) Short-term assay (25 µl of each fraction assayed in a total volume of 300 µl for 30 min at 30°C, followed by 25 µl of each reaction removed for measurement of released reducing sugars); (bottom panel) extended duration assay (50 µl of each fraction assayed in a total volume of 300 µl for 25 h at 30°C, followed by 50 µl of each reaction removed for measurement of released reducing sugars). Enzyme activity is expressed as change in the OD410; an OD410 of 1.0 represents 8 µg of galacturonic acid.

	DISCUSSION

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A strain of C. carbonum mutated both in PGN1, which encodes endoPG, and in PGX1, which encodes exoPG, had less than 0.8% of wild-type levels of total PG activity but was still pathogenic and grew almost as well as the wild-type strain in culture on pectin.

The simplest explanation for the unaltered pathogenicity of the double mutant is that Pgx1 and Pgn1 have no (or only a minor) role in pathogenesis. However, the importance of pectin degradation in pathogenesis is still an open question, because in vitro studies indicate that the double mutant is still able to utilize pectin for growth.

There are several possible explanations for why C. carbonum without its two major extracellular PGs can still grow on pectin. Residual heterokaryosis in the transformants was excluded by multiple rounds of single-spore isolation and by putting the mutant strains through a cross (17). Heterokaryosis due to reversion of a small percentage of the mutant genes in strains created by gene disruption was excluded by the construction of gene replacements in which reversion cannot occur.

Utilization of low-molecular-weight contaminants in the commercial pectin source is unlikely, because the fungus grows just as well on pectin that has been extensively washed with acidified ethanol. Utilization of nonpectinaceous high-molecular-weight contaminants also seems unlikely, because the same pectin was used for growth and for enzyme assays, and, therefore, any additional pectin-degrading enzymes contributing to growth would have been detected in the enzyme assays.

The double pgn1/pgx1 mutant probably grows on pectin by degrading pectin itself, and, if so, C. carbonum must have additional pectin-degrading enzymes. On the one hand, these enzymes could be ones that have avoided identification because they are not detectable by any of the assays that we have used (including different pHs and addition of divalent cations), are expressed more transiently than Pgn1p and Pgx1p, or are not stable during culture and purification. On the other hand, C. carbonum has three pectin-degrading activities that, alone or together, could account for the growth of the double mutant on pectin. The first of these is pectin methylesterase, which in addition to its role in converting pectin to polygalacturonic acid, the preferred substrate for Pgn1p and Pgx1p, releases methanol. C. carbonum can grow to a slight extent on methanol as the sole carbon source if it is added to cultures periodically in small amounts (25). However, even if the pectin used in these experiments were 100% methoxylated, and even if 100% of the released methanol were incorporated into fungal dry weight, this could account for less than half of the growth of the double mutant on pectin (Table 1). The role of pectin methylesterase in supporting growth of C. carbonum on pectin is currently being investigated by isolation and disruption of its encoding gene (25).

The second pectin-degrading activity in the double pgn1/pgx1 mutant is the small residual peak that is coeluted from a cation-exchange column with Pgn1p (Fig. 5). Based on its continued presence in gene replacement mutants that have been genetically purified by multiple rounds of single-spore isolation and crossing, it might be the product of a new PG-encoding gene. If so, this gene is not closely related to PGN1 or PGX1, because low-stringency hybridization to DNA blots of total genomic DNA with these genes as probes does not detect any related genes (25). We are purifying this residual peak of PG activity in order to characterize it further and to obtain amino acid sequence data for comparison with Pgn1p.

A third pectin-degrading activity that might support growth of the double mutant is an enzyme(s) that remains bound to the mycelium of C. carbonum. Wall-bound xylanase, cellulase, and pectinase have previously been reported from prokaryotes (19, 27). The major PG of the filamentous fungus Venturia inaequalis, an apple pathogen, is present in the mycelium, and, based on its extractability with high salt, is probably present in fungal cell walls (30). Mycelial extracts of the double mutant of C. carbonum do, in fact, contain pectin-degrading activity that is distinct from that of either Pgn1 or Pgx1 because it is optimal at pH 6, prefers methoxylated pectin over polygalacturonic acid, is stimulated by calcium ions, and is anionic in character (25). At this point, the presence of the mycelial pectinase seems the most likely of the three possible explanations for the continued ability of the double mutant to grow on pectin.

Although the two residual activities in the double mutant are weak compared with those due to Pgn1p and Pgx1p, they might be sufficient to support near-normal growth. If so, then C. carbonum in culture must normally produce approximately 100-fold more PG activity than is necessary. Strains of Saccharomyces cerevisiae with as little as 0.6% of their wild-type levels of invertase can still grow on sucrose (13, 16).