Plant Physiol. Biochem. 38 (2000) 881888 2000 ditions scientiques et mdicales Elsevier SAS.

All rights reserved S0981942800011918/FLA

A potent antifungal protein from Helianthus annuus owers is a trypsin inhibitor

Ana Marcela Giudici, Mariana Clelia Regente, Laura de la Canal*
Instituto de Investigaciones Biolgicas, Universidad Nacional de Mar del Plata, Casilla Correo 1245, 7600 Mar del Plata, Argentina

Received 7 April 2000; accepted 27 July 2000 Abstract A 16-kDa protein was isolated from Helianthus annuus owers by its ability to inhibit the germination of fungal spores. This protein, SAP16, displays an associated activity of trypsin inhibitor and was further puried to apparent homogeneity by affinity chromatography on trypsin-agarose. SAP16 causes the complete inhibition of Sclerotinia sclerotiorum ascospores germination at a concentration of 5 gmL1 (0.31 M) and a clear reduction of mycelial growth at lower concentrations, indicating a strong antifungal potency against this natural pathogen of sunower. Our data suggest that the antifungal ability of SAP16 would not be the result of the inhibition of a fungal protease. This study contributes to the characterization of the emerging family of antifungal proteins with an associated activity of trypsin inhibition and emphasizes their role in plant resistance against fungal attack. 2000 ditions scientiques et mdicales Elsevier SAS antifungal protein / plant defense / Sclerotinia sclerotiorum / sunower / trypsin inhibitor AFP, antifungal protein / CM, carboxymethyl / FPLC, fast protein liquid chromatography / PR-5, pathogenesis-related protein group 5 / PMSF, phenylmethylsulfonil uoride / PA, phosphatidic acid / SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis / SAP16, 16-kDa sunower antifungal protein / TCA, trichloroacetic acid / TI, trypsin inhibitor / TLCK, N-tosyl-lysine chloromthyl ketone

1. INTRODUCTION
During the last decade, several plant proteins capable of inhibiting fungal growth in vitro, have been isolated and characterized. Among these proteins generically called antifungal proteins (AFPs), glucanases and chitinases [25], thaumatin-like proteins [14, 41, 43], several families of basic-cysteine-rich peptides [5], chitin-binding proteins [29], ribosome-inactivating proteins [22] and proteinase inhibitors [38] are found. AFPs may be part of the preformed defense barriers or induced upon perception of microorganisms and accumulated evidences suggest that these proteins may have a direct antimicrobial activity in vivo, especially the enhanced resistance to microbial pathogens conferred to transgenic plants overexpressing some of these antimicrobial proteins [12]. Constitutive expression of some defense-related proteins (such as proteinase inhibitors [1, 28], defensins
* Correspondence and reprints: fax +54 223 4753150. E-mail address: ldelacan@mdp.edu.ar (Laura de la Canal).

[13, 18], tobacco PR-1, chitinases and glucanases [24, 27], and osmotin [6]) or their transcripts has been described in owers. However, in those reports, none of these proteins have been tested for antifungal activity. Although the signicance of the presence of these potential AFPs in owers has not been established, a protective role against fungal attack has been suggested. In sunower (Fam. Asteraceae), fertile owers have been described as an entrance for the fungal pathogen Sclerotinia sclerotiorum [34], who is responsible for severe yield losses in crops. Only a few studies has been reported that investigated the bioactivity of AFPs on this soil-borne pathogen with a host range encompassing about 400 species including many important crops besides sunower [42]. The aim of this work was to determine whether antifungal proteins active against S. sclerotiorum were present in fertile owers of sunower and potentially involved as part of the preformed defense barriers. Thus, a proteinase inhibitor with strong antifungal activity against S. sclerotiorum has been detected and characterized.

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2. RESULTS 2.1. Detection of antifungal proteins in owers

In order to detect proteins with constitutive expression able to inhibit in vitro the growth of S. sclerotiorum, a total protein extract from Helianthus annuus owers was obtained and enriched in heat-resistant proteins. This claried extract was submitted to cation exchange chromatography on CM-Sepharose, where the strongest antifungal activity was detected in the basic fraction, retained in the column and eluted with 0.2 M NaCl, although the unbound material also displayed antifungal activity (gure 1A). The basic heatresistant pool was further puried on Superose 12 FPLC (gure 1B) where two fractions with antifungal activity were detected (F18 and F22). In order to characterize these proteins, different enzymatic assays were performed. No activity of glucanase or chitinase associated with F18 and F22 could be detected, but F18 showed trypsin inhibitory activity (not shown). In addition, a tobacco PR-5 antibody was used to detect possible PR-5 antifungal proteins present in these fractions. However, western blot analysis of fraction F18 and F22 showed no reactions with the antibody anti-PR-5.

2.2. Purication of an antifungal protein with trypsin inhibitory activity

The ability of fraction F18 to inhibit trypsin was exploited as a purication strategy to verify if the antifungal activity was associated to the activity of trypsin inhibitor (TI). Hence, the fraction eluted with 0.2 M NaCl from CM-Sepharose was submitted to affinity chromatography on a trypsin-agarose matrix. A sharp protein peak bound to the matrix displaying antifungal activity was recovered (gure 2A). This protein also exerts trypsin inhibitory activity (25 % inhibition on the activity of trypsin per g protein, under the assay conditions described in Methods). The antifungal fraction retained in the affinity column was further submitted to gel ltration on Biogel P-60 where a single protein peak was observed (not shown). Moreover, on SDS-PAGE a unique band of 16 kDa estimated molecular mass was visualized (gure 2B). Hence, this antifungal protein with an associated activity of proteinase inhibition was named SAP16 (Sunower Antifungal Protein 16 kDa).
Figure 1. Purication of antifungal proteins from sunower owers. A, Cation exchange chromatography of a heat-resistant protein fraction. The arrows indicate the elution with 0.2 and 0.5 M NaCl. B, The basic antifungal fraction eluted with 0.2 M NaCl from A was further puried by gel ltration on Superose 12 FPLC. Bars indicate fractions with antifungal activity.

2.3. Antifungal properties of SAP16

SAP16 produces the complete inhibition of S. sclerotiorum spore germination at a concentration of

5 gmL1 while at 3 gmL1, a reduction of hyphal growth is observed (gure 3A). Fusarium solani f. sp. eumartii, another fungal pathogen, was also inhibited by SAP16 but appears to be less sensitive than S. sclerotiorum; at a concentration of 5 gmL1 only shortened hyphae could be observed (gure 3B). A more detailed microscopic examination of the effect of SAP16 on S. sclerotiorum showed that after 24 h of incubation, some spores germinated (less than 10 %), although growth was severely restricted and loss of viability was evidenced by methylene blue staining (not shown). It has been shown that the activity of many cationic antimicrobial proteins is reduced or blocked in the

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Figure 2. Purication of sunower antifungal protein SAP16 by trypsin affinity chromatography. A, The basic antifungal fraction eluted with 0.2 M of NaCl from CM-Sepharose was subjected to affinity chromatography on a trypsin-agarose matrix. The elution (e) with 0.5 M NaCl (pH 2) is indicated. B, Peak 1 from A was submitted to size exclusion chromatography on Biogel P-60 and analyzed on 15 % SDS-PAGE. Soybean TI (20.1 kDa) and lysozyme (14.4 kDa) were used as molecular mass standards. SAP16 is indicated with the arrow.

Figure 4. Mechanism of spore germination inhibition by SAP16. Spores of S. sclerotiorum after overnight incubation with 10 mM Na acetate (pH 5.2) (A), 5 gmL1 SAP16 (B), 5 gmL1 SAP16 + 1 mM CaCl2 (C), 5 gmL1 soybean TI (D), 0.2 mM TLCK (E), 5 gmL1 SAP16 + 95 gmL1 PA (F). Controls with 1 mM CaCl2 or 95 gmL1 PA germinated as control A. Magnication 66.

presence of cations [32, 37]. To determine if the antifungal activity of SAP16 also shares this property, its activity was evaluated in the presence of 1 mM CaCl2 added in the germinating medium. However, under microscopic observation (gure 4C), no difference in the inhibitory activity of SAP16 could be detected in the presence of calcium cations. Figure 4

also illustrates that 5 gmL1 of a commercial soybean trypsin inhibitor has no appreciable effect on S. sclerotiorum ascospores germination. Similar results were obtained with concentrations as high as 500 gmL1, indicating that the inhibition observed on S. sclerotiorum is not a common feature of all TIs. It is currently not known whether the mechanism of growth inhibition of this kind of antifungal proteins is related to their enzymatic activity as proteinase inhibi-

Figure 3. Antifungal activity of SAP16. A, Effect of SAP16 on the fungus S. sclerotiorum. Spores were incubated with 10 mM Na acetate buffer (pH 5.2) (control), 3 or 5 gmL1 of SAP16. Magnication 66. B, Effect of SAP16 on the fungus F. solani. Spores were incubated with 10 mM Na acetate buffer (pH 5.2) (control) or 5 gmL1 of SAP16. Magnication 50.

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tors. Hence, in order to evaluate if the antifungal property of SAP16 is a consequence of its activity as a serine proteinase inhibitor acting on a fungal proteinase, two experimental approaches were used. First, the presence of serine protease activity in the germination medium upon overnight incubation of S. sclerotiorum spores was investigated. This assay failed to detect any azocaseinolytic activity in the extracellular medium even upon a 5-fold increase in the concentration of spores (not shown). As a second approach the effect of two irreversible inhibitors of serine proteinases (TLCK and PMSF) [44] on the germination of S. sclerotiorum ascospores was analyzed. Figure 4E shows that when ascospores were incubated together with 0.2 mM TLCK (concentration producing 92 % inhibition of trypsin activity in vitro), in the standard conditions used in the bioassay, the ascospores germinated normally. The same results was obtained with the addition of 1 mM PMSF to the germination medium, although this reagent is inactivated in aqueous solution and would be acting only in the rst hours of incubation. Taken together, these observations suggest that no essential extracellular serine proteinase is needed for the germination of S. sclerotiorum ascospores. As a consequence, it is unlikely that SAP16 exerts its antifungal activity by the inhibition of a proteinase required for fungal germination. This observation prompted the investigation of whether SAP16, being a basic AFP, could be acting at the level of the fungal plasma membrane by interaction with its anionic components. Hence, we have analyzed the effect of SAP16 on the germination of ascospores in the presence of phosphatidic acid (PA), a constitutive anionic phospholipid of biological membranes. As shown in gure 4F, PA is able to counteract SAP16 inhibition of ascospore germination, suggesting that the protein interacts with PA instead of interacting with fungal membranes.

3. DISCUSSION
The experimental approach employed allowed the isolation to apparent homogeneity of a 16-kDa protein from sunower (SAP16) showing the inhibition of S. sclerotiorum spore germination. The trypsin affinity of SAP16, used as purication strategy, and the inhibition of trypsin indicate that this antifungal protein is a serine proteinase inhibitor. By now, the antifungal proteins with an associated activity of serine proteinase inhibition constitute a barely known family with only a few members isolated. Among them are the

serine proteinase inhibitors from corn [8, 15], TIs from barley [38] and wheat [10], and those isolated from cabbage [23]. As for SAP16, the previously described antifungal serine proteinase inhibitors are also small proteins (1225 kDa) but none of them have been isolated from oral tissues. TIs are frequently found in plant seeds and tubers where they are believed to act as defensive agents against herbivores by the inhibition of their proteinases [16]. More recently, it has been shown that certain TIs are inhibitors of fungal growth and it has been suggested that they may be part of a set of defense-related proteins that provide a barrier to fungal entrance. A relevant evidence supporting this hypothesis is the observation that the 14-kDa antifungal TI from corn kernels is abundant in seven genotypes resistant to Aspergillus avus and absent or present in low concentrations in six susceptible ones [8]. A preliminary approach to determine the potential role of AFPs in controlling the growth of pathogens in planta is to evaluate their antifungal potency. Thus, the concentration of cabbage TIs producing a total inhibition of the germination of Botrytis cinerea and F. solani spores was 600 gmL1 [23], while the TIs isolated from corn [7, 8] and barley [38] inhibit several fungi at concentrations between 40 and > 1 000 gmL1. The best characterized antifungal TI from corn requires concentrations greater than 100 gmL1 to inhibit the germination of spores of nine fungal species [7]. On the other hand, SAP16 displays a potent inhibitory activity causing the complete inhibition of S. sclerotiorum spores germination at a concentration of 5 gmL1 (0.31 M), similar to the most potent AFP active against S. sclerotiorum reported [30]. Interestingly, reported data indicate that the widespread pathogen S. sclerotiorum is not very sensitive to other described AFPs [37]. Another interesting feature of SAP16 is its weak sensitivity to cations compared to other antifungal TIs whose activities are practically blocked in the presence of millimolar concentrations of CaCl2 [38]. This property might be particularly interesting for the production of transgenic plants overexpressing antifungal proteins to enhance resistance against fungal pathogens [11]. The mechanism of action of the antifungal proteins with associated activity of TI has not been studied in detail yet but, at least, two possible mechanisms can be suggested. According to their basic features, they may act directly at the level of fungal membrane affecting its permeability, as it has been reported for thaumatinlike proteins [32] and thionins [39]. Alternatively, antifungal TIs can exert their activity by the inhibition

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of serine proteinases involved in fungal growth or differentiation. Our results suggest that the mechanism of inhibition of spore germination by SAP16 is not mediated by its activity as fungal proteinase inhibitor. In fact, extracellular serine proteinases do not seem to be involved in the germination of S. sclerotiorum ascospores. On the other hand, a direct effect of SAP16 on fungal membrane is suggested by the ability of exogenously added PA to displace the inhibition caused by SAP16. The basic nature of SAP16 may then play a central role in determining the mode of its antifungal activity: electrostatic interactions with membrane phospholipids. However, the contribution of another mechanism cannot be excluded keeping in mind recent evidences reported for the 14-kDa TI from corn [9]. In this case, the inhibition of fungal growth may be partially due to the inhibition of fungal -amylase production and activity hence limiting fungal growth. SAP16 constitutes, to our best knowledge, the rst TI isolated from owers to display antifungal activity. However, the occurrence of putative mRNAs from TIs has been reported in owers of tomato [4], alfalfa [26] and potato [28]. Moreover, Atkinson et al. [1] have isolated a 6-kDa protein from Nicotiana alata stigmas with inhibitory activity towards trypsin. The specic biological function of TIs in owers is still unknown but research has focused on their contribution as a barrier against herbivorous insects because similar TIs are effective against proteinases of insect origin [33]. Our results also highlight a role for TIs as controllers of fungal attack and, even if the signicance of the presence of SAP16 in sunower inorescence remains to be determined, its potent activity against a natural pathogen of H. annuus owers suggests a potential role in innate defense by a direct inhibition of fungal growth.

produced by S. sclerotiorum in eld trials (Dr Bazzalo, Zeneca SAIC, pers. comm.) and displays low susceptibility to wilting in growth chamber tests [2]. Fertile owers were obtained from plants cultivated under standard greenhouse conditions.

4.2. Antifungal activities

The test for inhibition of S. sclerotiorum ascospore germination was done on micro slides containing the protein sample, 103 ascospores and 6 % sucrose (w/v), in a nal volume of 26 L 10 mM Na acetate buffer (pH 5.2). Controls were performed by replacing the protein sample with the same volume of buffer. When indicated, the assay was supplemented with different additions. After 16 h incubation at 25 C in 100 % relative humidity, slides were evaluated under a light microscope for the presence of S. sclerotiorum hyphae.

4.3. Enzymatic activity

Trypsin inhibitory activity was measured by preincubating 1 g trypsin (EC 3.4.21.4) from bovine pancreas and testing for 20 min at 37 C in 80 mM Tris-HCl (pH 8), 1 mM CaCl2. Azocasein 0.5 % was then added as substrate, in a nal volume of 500 L, and the mixture was incubated 1 h at 37 C with agitation. The reaction was stopped by adding 500 L TCA 10 % and the mixture was centrifuged at 1 200 g for 15 min. The A350 nm of the supernatant was indicative of the residual proteinase activity. The inhibitory activity was calculated as the difference between the proteolytic activity in the absence and presence of the inhibitor. Controls included enzyme and substrate blanks. The extracellular fraction obtained for germinating ascospores (16 h incubation) was assayed for its proteolytic activity by incubation in the presence of azocasein 0.5 % for 20 h at 37 C. -1,3Glucanase activity was measured using laminarin (Sigma) as substrate as described Kauffmann et al. [20] and the reducing sugars generated were determined according to Somogyi [36]. Chitinase activity was measured by the release of N-acetylglucosamine from colloidal chitin, using the method described by Reissig et al. [31].

4. METHODS 4.1. Biological materials

Sclerotinia sclerotiorum (Lib.) de Bary sclerotia obtained from a local virulent isolate (Balcarce, Argentina, 1997) were used for the production of ascospores from apothecia [40]. Fusarium solani f. sp. eumartii, isolate 3122 (INTA Collection, Balcarce, Argentina), was grown at 25 C on Petri plates containing potato dextrose agar and the spores were collected by suspension in water. Helianthus annuus L. line AR10018 (Zeneca SAIC, Argentina) was used in this work. This genotype has been characterized as tolerant to head rot

4.4. Protein purication

Fertile owers were ground with three volumes of extraction buffer (100 mM Na acetate pH 5.2, 14.3 mM -mercaptoethanol, 100 mM KCl). After overnight extraction, a heat resistant protein fraction was obtained as previously described by Regente et al. [30] and

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subjected to cation-exchange chromatography on CM-Sepharose equilibrated in 20 mM Na acetate (pH 5.2). The column was step eluted using 0.2 and 0.5 M NaCl solutions. The unbound proteins and the fractions eluted at 0.2 and 0.5 M NaCl were concentrated and extensively dialyzed against 20 mM Na acetate (pH 5.2) for microscopic determination of the antifungal activity. The fractions eluted with 0.2 M NaCl were further puried on FPLC using a Superose 12 column equilibrated in 20 mM Na acetate (pH 5.2) or, alternatively, submitted to affinity chromatography on a trypsin-agarose (ICN) matrix. This column was equilibrated in 10 mM Tris-HCl (pH 8), 0.1 M KCl and the elution was carried out with 0.5 M NaCl (pH 2). A nal purication step was performed by gel ltration in a Biogel P-60 column, equilibrated with 20 mM Na acetate (pH 5.2).

del Plata, CONICET and Fundacin Antorchas, Argentina.

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4.5. Protein analysis, electrophoresis and immunobloting

Protein concentration was determined by the bicinchoninic acid method [35] adapted to microplates using bovine serum albumin as reference protein. SDS-PAGE was performed on 15 % acrylamide gels according to Laemmli [21]. The sample buffer contained 60 mM Tris-HCl (pH 6.8), 2 % (w/v) SDS, 0.1 % (w/v) bromophenol blue and 25 % (v/v) glycerol. Silver staining of separated proteins was performed according to Blum et al. [3]. For western blot analysis, fractionated proteins were electrotransferred to nitrocellulose membranes (0.45 m pore size) and incubated with an antiserum against tobacco PR S (PR-5) [19] at a 1:2 000 dilution. Antibody binding was revealed using the appropriate alkaline phosphatase-conjugate secondary antibody (Sigma). This antibody used at a 1:2 000 dilution recognizes acidic, neutral and basic fractions of PR S in sunower [17].

Acknowledgments. We thank Dr M.E. Bazzalo (Zeneca SAIC, Centro Biotecnolgico Balcarce, Argentina) for the gift of H. annuus owers, and Dr Casalongu and colleagues (IIB) for kindly providing the F. solani isolate. The antiserum against tobacco PR S was a gift of Dr P. Geoffroy, IBMP, CNRS, Strasbourg, France. AMG is a fellow of the Comisin de Investigaciones Cientcas de la Provincia de Buenos Aires, and MCR and LdlC are a fellow and career investigator respectively of the National Research Council from Argentina (CONICET). This research was supported by grants from the University of Mar

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