Proteinases and exopeptidases from the phytopathogenic fungus ...

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pathogenic fungi in which proteases have been de- scribed include: Botrytis cinerea (Urbanek and Ka- czmare 1985), Fusarium culmorum (Urbanek and Yir-.
Mycologia, 95(2), 2003, pp. 327–339. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897

Proteinases and exopeptidases from the phytopathogenic fungus Ustilago maydis Yuridia Mercado-Flores Ce´sar Herna´ndez-Rodrı´guez

Key words: acid proteinase, dimorphic phytopathogenic fungus, proteases, Ustilago maydis

Departamento de Microbiologı´a. Escuela Nacional de Ciencias Biolo´gicas, IPN. Casco de Santo Toma´s. Me´xico DF

INTRODUCTION

Jose´ Ruiz-Herrera

The heterobasidiomycete Ustilago maydis causes corn smut, a common disease in most parts of the world. In this pathosystem, the infection process is associated with sexual development. In its saprophytic phase, the fungus grows in the form of haploid, budding, yeast-like cells (sporidia). Sporidia of opposite mating types might fuse and give rise to the dikaryotic stage, which is the infective form of the fungus. In the host tissues, U. maydis grows as a septate mycelium, which produces diploid teliospores. These spores fill the galls that are characteristic of the disease. Germination of teliospores involves the formation of the promycelium and meiosis with the formation of basidiospores. Basidiospores reproduce by budding, completing the life cycle of the fungus (for review see Banuett 1995, Ruiz-Herrera and MartinezEspinoza 1998). Ustilago maydis requires the maize plant to complete its sexual life cycle. It has been suggested that the plant produces diffusible compounds needed for completion of the sexual cycle of the fungus (for review see Banuett 1995). By control of pH in synthetic media, it is possible to control dimorphism of haploid cells of U. maydis. At neutral pH the fungus grows as a homogeneous population of sporidia, whereas at acidic pH the mycelial form developes (Ruiz-Herrera et al 1995). Proteolysis plays an important role in different physiological functions—protein digestion, hormone maturation, immune response, inflammation, coagulation, fertilization, germination and other morphological processes (Holzer and Heinrich 1988). Some proteases are important in the formation and germination of spores, in pathogenesis of several microorganisms and in post-translational regulation (Yuan and Cole 1989, Vartivarian 1992, White and Agabian 1995, Monod et al 1998, Suarez-Rendueles et al 1991). Proteolysis is an essential life process. The study of Saccharomyces cerevisiae has been important in the elucidation of proteinase multiplicity and proteinase function in eukaryotic cells. Survival of cells in their natural environment depends highly on their ability to adapt to frequent changes. The ability of

Departamento de Ingenierı´a Gene´tica, Unidad Irapuato, Centro de Investigacio´n y Estudios Avanzados, IPN. Irapuato, Guanajuato, Me´xico

Lourdes Villa-Tanaca1 Departamento de Microbiologı´a. Escuela Nacional de Ciencias Biolo´gicas, IPN. Casco de Santo Toma´s. Me´xico DF

Abstract: The proteolytic system of the phytopathogenic and dimorphic fungus Ustilago maydis is not known. In this work, we report the presence of at least four proteases from two haploid strains of U. maydis. Activities of two proteinases pumA and pumB, aminopeptidase pumAPE, and dipeptidylaminopeptidase pumDAP were measured under several nutritional and morphological conditions, including the yeast-mycelium transition. The activity of pumA was found in the intracellular and extracellular fractions, pumAi and pumAe, respectively. The latter activity was detected only during the yeast-mycelium dimorphic transition induced by growth at acid pH in a medium containing ammonium as the sole nitrogen source. Activity of pumAe was partially inhibited by Pepstatin A, which also inhibited mycelium formation. Activity of pumAi was inhibited by this specific inhibitor of aspartyl-proteases. Activity of pumB was detected in intracellular and extracellular fractions, mostly bound to an endogenous inhibitor, which was removed by treatment at acid pH. This fungus contains at least two soluble pumAPE, which might be metallo-proteases, because they were inhibited by EDTA and 1–10, phenanthroline. When the fungus was grown in media containing proline or corn infusion as the nitrogen source, an intracellular pumDAP activity was detected. No carboxypeptidase activity was found with N-benzoyl-L-tyrosine-4-nitroanilide as substrate in any of the conditions tested in any of the U. maydis strains analyzed. Accepted for publication August 31, 2002. 1 Corresponding author, E-mail: [email protected]

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cells to respond to environmental extremes is vital to their survival. Proteolysis plays an essential role in this response to stress. Conditions of cellular stress can be caused by starvation and physical or chemical conditions, such as heat, extreme pH values or UV radiation. Nutritional stress probably leads to reorganization of cell metabolism and therefore to the reorganization of enzyme composition. The most obvious example of this form of stress occurs when a diploid yeast cell is transferred from a glucose-rich medium to a medium with a poor carbon source supplied by acetate. Under these conditions, and with removal of nitrogen, the yeast cell begins to differentiate and four spores appear. Here, intensive proteolysis of unnecessary vegetative proteins provides the cell with all amino acids required for new protein synthesis and probably for the generation of energy. As a further response to nutritional stress, yeast cells have been reported to form pseudo-hyphae when starved of nitrogen. It has been suggested that this filamentous growth enables otherwise sessile cells to forage for nutrients (for review see Hilt and Wolf 1992). Although studies on several phytopathogenic fungi suggest the role of proteases in pathogenesis, the proteolytic system in U. maydis is unknown. The phytopathogenic fungi in which proteases have been described include: Botrytis cinerea (Urbanek and Kaczmare 1985), Fusarium culmorum (Urbanek and Yirdaw 1984), Endothia parasitica (Choi et al 1993, Jara et al 1996), Glomerella cingulata (Clark et al 1997) and Sclerotinia sclerotiorum (Poussereau et al 2001). Detailed analyses of proteases in U. maydis might let us determine the proteases that this fungus produces and to examine the production of these enzymes in several nutritional conditions and during the dimorphic transition of yeast to mycelia, which is considered to be an important factor in the phytopathogenicity of this fungus. Moreover, it might be possible to relate protease synthesis with the life cycle of this fungus and with its response to diffusible compounds produced by the host plant, Zea mays. MATERIALS AND METHODS

Fungal strains. These strains and mating types of U. maydis were used in this study: FB1 a1b1 and FB2 a2b2. These strains kindly were provided by Dr. Flora Banuett, University of California at San Francisco, USA. Culture media. The strains were maintained at 270 C in 50% (v/v) glycerol. The cells were grown on complete Y PD medium (1% yeast extract, 2% bactopeptone, 2% glucose) or minimal medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, plus 2% glucose) (Suarez-Rendueles et al 1991) with different sources of nitrogen:

2% peptone, 2% proline, 0.5% ammonium sulfate or corn infusion (equivalent to 4 mg of protein per mL of medium). The corn infusion was obtained by boiling for 20 min 500 g of corncobs in 1 L of water and then filtering. This solution was supplemented with 0.17% yeast nitrogen base without amino acids and ammonium sulfate, plus 2% glucose. Growth conditions. Cells inoculated into liquid medium promoted growth as a homogeneous population of budding, yeast-like cells (culture pH 7.0) or as a population in the mycelial form (pH 3.0) as previously described (RuizHerrera et al 1995). To measure cell growth, samples of the cultures were centrifuged at 20003 g. Sedimented cells were washed twice with distilled water, and the protein content was measured. Changes in pH were recorded with a pH meter coupled to a recorder. Enzyme assays. Crude cell-free extracts and soluble extracts were prepared as described by Arbesu´ et al (1993). Enzymatic activities were determined as described by Hirsch et al (1989). These substrates were used for the different proteases: acid denatured hemoglobin for proteinase A activity (pumA); Hide Powder Azure (HPA) for proteinase B activity (pumB); L-lysyl-4-nitroanilide (Lys-4-NA) for aminopeptidase activity (pumAPE); L-alanyl-prolyl-4-nitroanilide (Ala-pro-4-NA) for dipeptidylaminopeptidase activity (pumDAP); and N-benzoyl-L-tyrosine-4-nitroanilide (N-BzTyr-4-NA) for carboxypeptidase activity (pumCP). Activation of fresh crude extracts was carried out as described by Saheki and Holzer (1975). Lysine amino peptidase activity in polyacrylamide gels was detected with L-lysyl-b-napthylamide (Lys-b-NphA) and Fast Garnet as described by Garcı´a-Alvarez et al (1991). As a source of crude inhibitor of pumB, we used cell-free extracts of the fungus boiled and centrifuged as described by Escudero et al (1993). Enolase activity was determined with D-glyceric acid 2-phosphate as substrate as described by Dmitriy and Nowak (1998) and was used as marker enzyme for the intracellular localization. Light and scanning electron microscopy. Cells were observed in unstained preparations by interference microscopy. For scanning electron microscopy (SEM), cells were observed in a JEON model JSM-5800LV, cells were fixed with 1% (v/ v) glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.0. They were washed in a solution containing 0.10 M Na2HPO4 and 20% sucrose, pH 7.0, and post-fixed with 2% (w/v) osmium tetroxide. Cells were washed and dehydrated in a graded alcohol series. For SEM, the dehydrated cells were mounted on specimen holders and coated with gold. The images were captured and digitized by a computer coupled to the microscope. Miscellaneous. Protein content was estimated by the method of Lowry et al (1951). Hemoglobin, Hide Powder Azure (HPA), L-aminoacyl-4-nitroanilides, and L-aminoacyl-b-napthylamides, (L-A-4NA; L-A-b-NphA) were used as proteinase and peptidase substrates. They were obtained from either Sigma Chemical Co. (St. Louis, MO, USA) or Bachem (King of Prussia, PA, USA). D-glyceric acid 2-phosphate (Sigma) was used as the enolase substrate. Growth media were from

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Difco (Detroit, MI, USA). Protease inhibitors were from Bo¨ehringer Mannheim (Indianapolis, IN, USA). The Protease inhibitor kits contained Na2EDTA at 0.0–10.0 mg/mL (0.0–23 mM); E64 at 0.0–10.0 mg/mL (0.0–28 mM); Pefabloc at 0.0–1.0 mg/mL (0.0–4.0 mM); 1–10, phenanthroline at 0.0–1.5 mg/mL (0.0–7.5 mM) or Pepstatin A at 0.0– 1.0 mg/mL (0.0–1.42 mM). Other chemicals used were of the highest purity available. Unless otherwise indicated, data reported are representative of results from at least three different experiments with determinations in duplicate. RESULTS

Throughout this work, the proteases of U. maydis are identified by use of the prefix ‘‘pum’’ (for protease Ustilago maydis), ‘‘e’’ for extracellular activity and ‘‘i’’ for intracellular activity. Our initial efforts to detect intracellular proteases in U. maydis led us to investigate activities similar to those described in S. cerevisiae. PrA, PrB and CpY, ( Johansen et al 1976, Kominami et al 1981, Magni et al 1982) were present in U. maydis. The results presented in FIG. 1A show the existence of proteolytic activity against acid-denatured hemoglobin, which we identified as proteinase pumA. Also, proteolytic activity toward HPA (proteinase pumB) was detected in the fungus. The former activity did not change after incubation of extracts at 4 C at pH 5.0 during 12 h. In contrast, activity with HPA increased several fold under those conditions, reaching a maximum at about 12 h. In general, determination of proteinase activities in fresh crude extracts from different fungi is difficult because of endogenous inhibitors that bind firmly to proteinases upon cell disintegration (Schu et al 1991). This problem has been overcome in S. cerevisiae proteinases PrA, PrB and carboxypeptidase CpY by incubation of crude extracts at pH 5.0. No carboxypeptidase activity (pumCP) was found in U. maydis under our assay conditions with N-BzTyr-4-NA as substrate, even after incubation at pH 5.0 or treatment with deoxycholate, a procedure that activates carboxypeptidase in S. cerevisiae (Aibara et al 1971). Proteinase pumB activity fell when different concentrations of boiled extracts of the crude inhibito r were added to the reaction mixture (FIG. 1B), suggesting the presence of an endogenous inhibitor with a similar mode of action to the one described in S. cerevisiae and Schizosaccharomyces pombe (Magni et al 1986, Escudero et al 1993). We measured protease with denatured hemoglobin as substrate (pumAi), using cell-free extracts obtained from U. maydis cells grown in batch culture for different periods of time in either minimal or complete medium. Activity under these conditions

FIG. 1. A) Activation of proteases in a soluble extract of U. maydis by incubation at pH 5.0. Yeast was cultured at 28 C in complete Y PD medium for 24 h. Cells were harvested, washed and soluble extracts were prepared as indicated in Materials and Methods. Five ml of soluble extract (60 mg protein) were incubated at 4 C with 5 mL 0.1 M citrate buffer, pH 5.0. Proteinase pumAi (V), Proteinase pumBi (v), Carboxypeptidase pumCPi (l). B) Effect of the endogenous inhibitor on activity of proteinase pumB. The crude extract of pumB inhibitor was obtained from a soluble crude extract of U. maydis grown in Y PD medium for 24 h. Inhibitory activity of endogenous inhibitor (V), residual activity of proteinase pumBi (v). Inhibitory activity was determined as described previously (Escudero et al 1993).

was similar for the cells of either mating type (FB1 and FB2), indicating that amounts of protease do not depend on mating type (not shown). Activity also appeared to be independent of the nitrogen source, although higher levels were obtained when cells were grown in a medium containing proline (FIG. 2A). Growth period was critical for proteinase pumAi activity. As shown in FIG. 2A, higher specific-activity levels of the enzyme were detected during early growth and disappeared, or at least reached very low levels in older cultures.

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FIG. 2. Kinetic study of the production of proteases of U. maydis FB1 in different nitrogen sources. A) pumAi, B) pumBi (side left) and pumBe (side right), C) pumAPEi, D) pumDAPi. Specific activity (v), Total activity (l),Growth (M) and pH (V). The intracellular activities (pumAi, pumBi, pumAPEi and pumDAPi) were determined in the supernatant of 23 0003 g fraction (free cells extract) and the extracellular activity (pumBe) was determined in the supernatant of 20003 g. (supernatant of media).

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FIG. 2.

Ultracentrifugation of the cell-free extract showed that most of the activity was localized in the supernatant, although a small amount sedimented at low and high speeds of centrifugation (TABLE I). The same distribution of activity was found for all culture media and incubation times in this study (not shown). Activity of pumAe (extracellular) was detected only in cells grown in YNB medium containing ammonium sulfate. Specific activity began to increase after 12 h of growth and reached maximal values after 24 h, remaining almost constant during the stationary phase (FIG. 3). Under these conditions, the culture medium became acidic as a function of incubation

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Continued

time in contrast to the other media, in which pH remained near neutrality (FIGS. 2 and 3). As previously described (Ruiz-Herrera et al 1995), yeast-tomycelium dimorphic transition took place (FIG. 3). Pepstatin A, which specifically inhibits aspartyl proteases, inhibited proteinase pumAi activity and about 40% of proteinase pumAe activity in the two U. maydis strains (TABLE II). With the cysteine-protease inhibitor E-64, the percentage of residual activity of the intracellular proteinase pumAi varied between 50 and 60% in the two U. maydis strains, while EDTA inhibited about 50% of the protease activity (TABLE II). Proteinase pumAe activity increased considerably with addition of EDTA to the reaction mixture (TA-

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TABLE I.

Distribution of enzyme activities in cellular fractions of U. maydis FB1

Cellular fraction

pumA ammonium activity %

pumB Y PD activity %

pumAPE peptone activity %

pumDAP proline activity %

Enolase Y PD activity %

Cellular homogenate S 23 000 3 g P 23 000 3 g S 100 000 3 g P 100 000 3 g

100.0 84.9 15.0 70.6 6.4

100.0 82.0 17.9 76.2 0.7

100.0 98.0 0.0 82.2 3.6

100.0 75.1 25.0 35.2 22.3

100.0 99.0 0.0 90.0 0.0

The cellular fractions were prepared from cells growing in the optimal media for each enzyme. Biomass from the culture media was recovered by centrifugation at 5 C (2000 3 g), washed twice with Tris 0.1 M, pH 7.0 buffer, and disrupted with glass beads to produce a cell homogenate. This fraction was centrifuged at 23 000 3 g, and the corresponding supernatant of 23 000 3 g was centrifuged at 100 000 3 g to separate the soluble fraction (cytoplasmic) from the membrane fraction. BLE II). The other inhibitors did not affect proteinase pumAe and pumAi activity. A kinetic study of the production of proteinase pumB activity in YNB containing ammonium sulfate was carried out. Ustilago maydis produced both intracellular activity (S 23 0003 g) and extracellular activ-

ity (FIG. 2B). The latter reached its greatest specific activity during the early and late stationary-growth phases. Synthesis of pumBe increased up to four fold in YNB medium containing corn infusion relative to the ammonium-containing medium (FIG. 2B). Proteinase pumBi accumulated mainly in the 100 0003

FIG. 3. Proteinase pumAe, cell morphology and pH of U. maydis FB2 grown in YNB-ammonium media. At the times indicated, samples were recovered and supernatant was used to determine proteinase activity and pH. A) Morphology of yeast-like cells of U. maydis FB2 in unstained preparations, observed by interference microscopy (403) at 0 h of incubation at 28 C. B) Morphology of mycelial grown of U. maydis FB2 in unstained preparations, observed by interference microscopy (403 at 24 h of incubation at 28 C. C). Proteinase pumAe activity of U. maydis FB2. Specific activity mU/mg protein (v), Total activity (l), Growth (M), and pH (V).

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Effect of proteolytic inhibitors on U. maydis FB1 proteases

Protease pumAe (S 2000 3 g)

Inhibitors/ Activators Pepstatin A (mg/ml)

EDTA (mg/ml) pumAi (S 23 000 3 g)

E-64 (mg/ml)

EDTA (mg/ml)

Pepstatin A (mg/ml) pumAPE (S 10 000 3 g)

EDTA (mg/ml)

1–10, phenantroline (mg/ml) pumDAP (P 23 000 3 g)

E-64 (mg/ml)

Pefabloc (mg/ml)

EDTA (mg/ml)

1–10, phenantroline (mg/ml) pumDAP (P 100 000 3 g)

Pefabloc (mg/ml)

Concentration of inhibitor/ activator

Percent activity

0.0 0.7 1.0 0.0 5.0 10.0 0.0 5.0 10.0 0.0 5.0 10.0 0.0 0.7 1.0 0.0 5.0 10.0 0.0 0.5 1.5 0.0 5.0 10.0 0.0 0.5 10.0 0.0 5.0 10.0 0.0 0.5 1.5 0.0 0.5 10.0

100.0 60.7 54.7 100.0 141.3 213.8 100.0 72.9 62.3 100.0 58.9 40.9 100.0 0.0 0.0 100.0 13.8 0.0 100.0 0.0 0.0 100.0 74.9 49.5 100.0 45.8 30.1 100.0 0.0 0.0 100.0 0.0 0.0 100.0 22.1 0.0

Proteolytic activities were determined in different nitrogen sources. pumA (YNB-ammonium); pumAPE (YNB-peptone); pumDAP (YNB-corn infusion).

g supernatant fraction, although considerable activity also was found in the fraction that sedimented at 23 0003 g (TABLE I). Activity of pumAPE (aminopeptidase) was found only in the supernatant fraction (S 23 0003 g) in cells grown in all culture media. Similar levels of activity were found in both U. maydis strains (data not shown). Activity reached maximal values during the exponential growth phase of the fungus for all the media used (FIG. 2C). Maximal activity was detected in the 100 0003 g supernatant fraction, as with pumA and pumB (TABLE I). When cell-free extracts were subjected to PAGE and pumAPE activity was revealed

in gels with the Lys-b-NphA substrate, two bands of activity were detected (FIG. 4). Activity of pumAPE was blocked by two metalloprotease inhibitors, EDTA and 1–10, phenanthroline (TABLE II). Protease pumDAP activity, detected with the chromogenic substrate Ala-pro-4-NA for dipeptidylaminopeptidase, was observed in U. maydis strains (FB1 and FB2). Highest levels of specific activity were observed during the late stationary-growth phase in YNB-proline medium (FIG. 2D). In contrast, when YNB-corn infusion medium was used, highest levels of activity were reached during the exponential growth phase (FIG. 2D).

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FIG. 4. Activity staining of aminopeptidase activities from U. maydis separated by non-denaturing polyacrylamide gel electrophoresis. The gels were run at 87 V, and electrophoresis was carried out for 2 h at 4 C. Aminopeptidase activity staining was carried out by incubating the gel for 30 min at 37 C with Tris-HCl 5 mM, pH 7.2, containing 0.05% of Lys-b-NphA and 0.1% of Fast Garnet. A) 50 mL of soluble extract (S 100 0003 g) of U. maydis FB1 (315 mU/ mL). B) 50 mL of soluble extract (S 100 0003 g) of U. maydis FB2 (535 mU/mL).

In contrast to the other proteases, pumDAP activity from cells grown in either YNB-proline or YNB-corn infusion media was localized in the particulate fraction that sedimented at 23 0003 g (TABLE I). Activity of pumDAP in the particulate fraction that sedimented at 100 0003 g was inhibited by Pefabloc, which is a specific serine protease inhibitor (TABLE II), but not by E-64, which is a cysteine- protease inhibitor, EDTA or 1–10, phenanthroline (data not shown). On the other hand, pumDAP in the 23 0003 g particulate fraction was only partially inhibited by Pefabloc and E-64 and completely inhibited by EDTA or 1–10, phenanthroline (TABLE II). In contrast to the presence of acid, alkaline and amino-peptidases, no carboxypeptidase activity was detected when extracts of either strain of U. maydis were incubated with the synthetic chromogenic substrate N-Bz-Tyr-4-NA. As shown in FIG. 3, proteinase pumAe was produced only in YNB-ammonium medium after 12 h of

culture, when the fungus acidifies the medium and the transition from yeast to mycelial form takes place. Levels of proteinase pumAe and pumAi activity were measured during development of the mycelial form in U. maydis. Proteinase pumAe in the two U. maydis strains was formed only under the conditions that gave rise to the mycelial phase (FIG. 5-2). This event was reversible in both strains of U. maydis. When cells were transferred to a neutral pH medium, mycelial cells reverted to yeast-like growth and levels of pumAe were reduced (FIG. 5-4). As indicated above, proteinase pumAe was partially inhibited by Pepstatin A (TABLE II). To determine whether this inhibitor had any effect on the dimorphic transition of U. maydis, Pepstatin was added to the culture medium at concentrations of 1 and 2 mg/ mL. As a control, medium without inhibitor was included, as well as a culture in which the yeast phase was maintained by incubation at pH 7.0. Microscopic observations and determinations of activities of proteinase pumAe and pumAi were performed in all these cultures. Activity of enolase was determined as an intracellular marker in supernatants of culture media; the absence of this enzyme demonstrated the integrity of the cells (data not shown). Formation of mycelium was partially inhibited when Pepstatin A was added to cultures of either strain. Furthermore, the specific activity values of the intracellular and extracellular acid proteinases were reduced compared to those in the absence of the inhibitor (FIG. 6). In contrast, cell viability was not affected by addition of Pepstatin A. (data not shown), and levels of pumBi, pumBe, pumAPE and pumDAP did not change (data not shown). As previously indicated, no pumCP proteolytic activity was detected in these experiments with N-Bz-Tyr-4-NA as substrate. DISCUSSION

Ustilago maydis has been a useful model for the study of relationships between phytopathogenic fungi and their host plants. It also has been used to study the relationship between dimorphic transition and phytopathogenicity (Ruiz-Herrera et al 1995, Ruiz-Herrera and Martinez-Espinoza 1998, Bo¨lker 2001). However, enzymatic activities involved in pathogenesis and the dimorphic transition are largely unknown. Therefore, it is not surprising that we have little knowledge about the proteases produced by this fungus. In fact, the study of proteolytic enzymes in phytopathogenic microorganisms has been neglected, and only a few reports on this topic have appeared in the recent literature. Considering the importance of the role of proteases in multiple aspects of fungal biology ( Jones

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FIG. 5. Proteinase pumAi and pumAe activity, pH and morphology of mycelial and yeast-like cells of U. maydis FB1. 1) and 3) Yeast-like growth at pH 7.0. 2) and 5) Induced yeast-to-mycelial transition by transfer of cultures from synthetic medium, pH 7.0, to synthetic medium, pH 3.0. 4) Induced mycelial-to-yeast transition by transferring cultures from synthetic medium, pH 3.0, to synthetic medium, pH 7.0. Induced yeast-to-mycelial transition culture was obtained as described RuizHerrera et al (1995). Unstained cell preparations observed by interference microscopy (403), after incubation at 28 C. Proteinase pumA activity was measured against acid-denatured hemoglobin, as described in Materials and Methods.

1991, Knop et al 1993, van den Hazel et al 1996, Rao et al 1998), we have analyzed the proteases produced by U. maydis. In our study, different growth conditions were used and the localization of proteases in different cellular fractions was assessed. No differences in the types of proteases were found for the two U. maydis strains belonging to different mating types. Both produced the same proteinases— an acid protease (pumA), a neutral protease (pumB), an aminopeptidase (pumAPE) and a dipeptidylaminopeptidase (pumDAP). No carboxypeptidase was detected under any growth condition with N-Bz-Tyr4-NA as substrate. The acid protease pumAi was synthesized under all growth conditions and reached maximum levels of specific activity at the exponential-growth phase. This activity is probably similar to that of yscA from S. cerevisiae, which is a soluble vacuolar endopeptidase (Sua´rez-Rendueles and Wolf 1988). In other yeasts, such as Candida albicans, Schizosaccharomyces pombe and Kluyveromyces lactis, intracellular proteinase A activity also has been found (Portillo and Gancedo 1986, Sua´rez-Renduelles et al 1991, Flores et al 1999). Like the enzymes from S. cerevisiae and C. albicans, (Portillo and Gancedo 1986, Hirsch et al 1989), ac-

tivity of proteinase pumAi was inhibited by Pepstatin A, a specific inhibitor of aspartyl proteases. In contrast, proteinase pumAi activity of the haploid strains of U. maydis produced under the same conditions showed important differences in their sensitivity to the metallo- and cysteine-protease inhibitors EDTA and E-64, respectively. The activity of proteinase yscA of S. cerevisiae is particularly cryptic in cell extracts due to the presence of the endogenous inhibitor. Incubation of cell extracts at an acidic pH increased proteinase yscA activity (Saheki and Holzer 1975, Hirsch et al 1989). Incubation of cell-free extracts at acidic pH did inactivate pumAi. On the contrary, activity declined, suggesting that, in U. maydis, an endogenous inhibitor of the intracellular acid protease is lacking or that activation of this enzyme requires different conditions. The two U. maydis strains secreted an acid proteinase (pumAe) during exponential growth. This protease was produced only in a medium containing ammonium sulfate as nitrogen source, when the pH reached 3.5. Previous studies indicated that, in the presence of an ammonium salt of strong anion, the fungus acidifies the medium due to the activity of a

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FIG. 6. Effect of Pepstatin A on proteinase pumAi and pumAe, and cell morphology of U. maydis FB1. (1 and 3) Mycelial growth of haploid strain FB1 in synthetic medium, pH 3.0, without Pepstatin A. (2 and 4) Mycelial growth partially inhibited by Pepstatin A, 2.0 mg/mL. (1 and 2) Scanning electron microscopy micrographs, bar 10 mm (17003). (3 and 4) Unstained cell preparations observed by interferential phase contrast microscopy (403). Proteinase activity pumA was measured against acid-denatured hemoglobin.

proton pump (Ruiz-Herrera et al 1995). The enzyme also was synthesized when the fungus was grown in an acid medium, following the protocol employed for induction of mycelial growth in vitro (Ruiz-Herrera et al 1995). These results suggest that the protease is induced by an acidic pH. Unlike the intracellular enzyme, the extracellular acid protease of U. maydis was only partially inhibited by Pepstatin A, indicating that it represents a mixture of enzymes with similar catalytic activity and that only one of these is an aspartyl proteinase. Partial inhibi-

tion of the dimorphic transition by Pepstatin A, suggests that the aspartyl-proteinase might be involved in processes regulating dimorphism. In S. cerevisiae, vacuolar aspartyl proteinase yscA (PrA) is encoded as a preproenzyme by the PEP4 gene. When this gene is over expressed in wild-type yeasts, the mature form of the enzyme is found in the culture medium in addition to a pseudo-PrA form (Wolff et al 1996). A similar phenomenon might operate in U. maydis, and the extracellular aspartyl proteinase pumAe might be the same as the vacuolar enzyme pumAi. It

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also is possible that culture conditions in our studies resulted in overexpression and secretion of the enzyme. Proteolysis plays an essential role in responses to stress, such as nutrient deficiency, high temperature, extreme pH, UV irradiation or the presence of heavy metal ions or amino acid analogues. Nutritional stress probably induces a reorganization of cellular metabolism and existing enzymes (Hilt and Wolf 1992). Under the protocol used to obtain the mycelial phase of U. maydis, it is possible that the cells are stressed and that the acidic pH induces the synthesis of pumAe. Extracellular aspartyl proteases have been reported in other phytopathogenic fungi. For example, in Botrytis cinerea an aspartyl protease has been found to be involved in the hydrolysis of proteins in apple cell walls (Urbanek and Kaczmarek 1985). Likewise, Fusarium culmorum produces an acid protease that hydrolyzes numerous vegetable proteins (Urbanek and Yirdaw 1984). Also, Glomerella cingulata has an aspartyl protease whose transcript is induced by exogenous proteins and repressed by ammonium salts (Clark et al 1997). Cryphonectria parasitica secretes acid proteases, which have been purified and characterized, and their genes have been cloned (eapA, eapB and eapC) (Choi et al 1993, Jara et al 1996). It is possible that U. maydis synthesizes more than one extracellular acid protease, because as mentioned above it was only partially inhibited by Pepstatin A. Proteinase pumAe of the two U. maydis strains tested was activated by EDTA. This observation suggests the existence of another protease that participates in the regulation of this enzyme that might be inhibited by EDTA. As indicated above, concomitant with the dimorphic transition of yeast to mycelium, the activity of this enzyme increases, and maximum specific activity levels are reached when only mycelia are found in the culture. Because an acidic pH develops under these conditions, we suggest that the enzyme might be regulated by pH, as has been reported for other fungal proteases (for review, see Denison 2000). Nevertheless, additional evidence suggests a possible relationship between pumAe and the filamentation of the fungus. The most appealing observation is that, when Pepstatin A was added to cultures maintained at pH 3.0, the formation of mycelium was inhibited. An opposite, but related, example of pH-controlled morphogenesis is C. albicans. Unlike U. maydis, C. albicans grows in the yeast form when cultured in acidic pH; whereas neutral pH favors growth of mycelium (Soll 1985). Interestingly, however, expression of vacuolar aspartyl proteinase of this yeast is induced in the yeast form by growth at acidic pH,

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but the evidence suggests that this enzyme has no direct role in the dimorphic transition of the organism (Niimi et al 1997). On the other hand, the secreted aspartyl proteinases SAP 4, 5 and 6 that are produced by C. albicans, seem to be implicated in the differentiation that occurs during the formation of the germination tube (Hube et al 1994). This could be the case in U. maydis, in which aspartyl proteinase pumAi is not associated with the dimorphism events of this fungus, whereas pumAe might be involved in the phenomenon. Ustilago maydis contains a second extracellular proteinase that we called pumBe, which is active against the HPA substrate. Highest levels of the enzyme were found in media containing ammonium salts or corn infusion. Unlike the pH of media containing ammonium, the pH of corn infusion media reaches a value of 6.0. In a similar way to that described for proteinase yscB of S. cerevisiae (Magni et al 1986, Hirsch et al 1989), pumBi activity in U. maydis is associated with an endogenous inhibitor, because detection of activity of this enzyme required incubation of the extracts at pH 5.0. Also in S. pombe, Saccharomyces carlsbergensis and C. albicans, there are specific endogenous inhibitors of this type of protein (Matern et al 1979, Farley et al 1986, Escudero et al 1993). In both U. maydis strains used, pumBi activity was found in YNB-ammonium medium during the early and late stationary phases, whereas in the YNB-corn infusion medium, the intracellular activity reached its maximum value during the exponential-growth phase. Similarly, in S. cerevisiae expression of the gene coding for the vacuolar proteinase yscB is regulated by nitrogen or carbon sources and depends on the growth phase of the yeast (Naik et al 1997). The activity of proteinase pumB does not seem to be associated with any events during the dimorphic transition of yeast to mycelia in the two U. maydis strains. Both U. maydis strains contained at least two soluble pumAPEi activities that reached maximal activities during the exponential growth phase and were inhibited by EDTA and 1–10, phenanthroline. Protease pumAPE, like proteinase pumB, is not clearly associated with any of the dimorphic transition phases in the U. maydis strains. Another protease detected in the two U. maydis strains was pumDAPi. In YNB-proline medium, highest levels of this enzyme were reached during the late stationary phase, whereas optimal levels were detected during the exponential-growth phase in the YNBcorn infusion medium. This stimulatory effect of corn infusion is interesting, because it might be due to a thermo-stable low molecular-weight compound

338

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from the host. That this effect is involved in the hostpathogen interaction is an attractive possibility. Activity of pumDAP was localized both in the cytosol and the membrane fractions. Activity of pumDAP in the membrane fraction was inhibited by Pefabloc, suggesting that it is a serine protease. It is interesting to note that the dipeptidylaminopeptidase of S. cerevisiae, called yscIV, is a membrane serine exopeptidase that participates in the processing of the pheromone (Sua´ rez-Rendueles and Wolf 1988). A similar role for that membrane-bound pumDAP in U. maydis would be an intriguing. The pumDAP in the pellet obtained at low speed was inhibited by EDTA and 1–10, phenanthroline and partially inhibited by the serine and cysteine protease inhibitors, Pefabloc and E-64, respectively. This pattern of inhibition suggests the existence of a mixture of enzymes with the same catalytic activity, one of them a serine-metallo-protease and the other a cysteine-metallo-protease. S. cerevisiae produces dipeptidylaminopeptidase yscII, a metalloprotease whose location and function are unknown (Sua´rez-Rendueles and Wolf 1988). Activity of pumDAPi was detected in both the yeast and mycelial forms of U. maydis without significant differences in their specific activities. This result suggests that the enzyme(s) is (are) not involved in the dimorphic transition of the fungus. At least seven carboxypeptidases have been described in S. cerevisiae (Sua´rez-Rendules and Wolf 1988). In contrast, no carboxypeptidase activity was found in our analysis of U. maydis. This result does not appear to be due to the presence of an endogenous inhibitor, such as that described for S. cerevisiae (Lenney 1975). Addition of sodium deoxycholate to the reaction mixture, as well as incubation at an acidic pH, failed to reveal any carboxypeptidase activity. Hydrophobic properties of the surface of U. maydis depend on the proteolysis of Rep1. This protein must be processed by proteases to generate small peptides that are important in the formation of hydrophobic aerial hyphae, which enhance the contact between the fungus and the plant when infection is initiated (Wo¨sten et al 1996). To date, the only protease described that is responsible for processing Rep1 is a protease that is equivalent or similar to Kex2 of S. cerevisiae (Park et al 1994), which has been described in the processing of the pheromone and killer factor in this yeast. This membrane peptidase cleaves the carboxyl terminus of synthetic substrates (Sua´rezRendueles and Wolf 1988). Accordingly, either Rep1 is processed by a different mechanism in U. maydis or the carboxypeptidase from this fungus is not active against the synthetic substrate used in this study.

ACKNOWLEDGMENTS

The authors acknowledge Dr. Alfredo Martı´nez-Espinoza and Miss Claudia Leo´n-Ramı´rez for their valuable advice. M. C. Yuridia Mercado-Flores was a fellow from CONACyT and PIFI, IPN. This work was partially supported by grants from COFAA and EDD, Instituto Polite´cnico Nacional to Dr. Lourdes Villa-Tanaca and Dr. Ce´sar Herna´ndez-Rodrı´guez and from CONACYT to Dr. Jose´ Ruiz-Herrera

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