Plant Physiol. Biochem. 38 (2000) 881−888 © 2000 Éditions scientifiques et médicales Elsevier SAS.

All rights reserved S0981942800011918/FLA

A potent antifungal protein from Helianthus annuus flowers is a trypsin inhibitor
Ana Marcela Giudici, Mariana Clelia Regente, Laura de la Canal*
Instituto de Investigaciones Biológicas, 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 flowers by its ability to inhibit the germination of fungal spores. This protein, SAP16, displays an associated activity of trypsin inhibitor and was further purified to apparent homogeneity by affinity chromatography on trypsin-agarose. SAP16 causes the complete inhibition of Sclerotinia sclerotiorum ascospores germination at a concentration of 5 µg·mL–1 (0.31 µM) and a clear reduction of mycelial growth at lower concentrations, indicating a strong antifungal potency against this natural pathogen of sunflower. 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 Éditions scientifiques et médicales Elsevier SAS antifungal protein / plant defense / Sclerotinia sclerotiorum / sunflower / trypsin inhibitor AFP, antifungal protein / CM, carboxymethyl / FPLC, fast protein liquid chromatography / PR-5, pathogenesis-related protein group 5 / PMSF, phenylmethylsulfonil fluoride / PA, phosphatidic acid / SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis / SAP16, 16-kDa sunflower 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 flowers. However, in those reports, none of these proteins have been tested for antifungal activity. Although the significance of the presence of these potential AFPs in flowers has not been established, a protective role against fungal attack has been suggested. In sunflower (Fam. Asteraceae), fertile flowers 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 sunflower [42]. The aim of this work was to determine whether antifungal proteins active against S. sclerotiorum were present in fertile flowers of sunflower 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.

Fusarium solani f. A. The antifungal fraction retained in the affinity column was further submitted to gel filtration on Biogel P-60 where a single protein peak was observed (not shown). another fungal pathogen. although growth was severely restricted and loss of viability was evidenced by methylene blue staining (not shown). Cation exchange chromatography of a heat-resistant protein fraction. Antifungal properties of SAP16 SAP16 produces the complete inhibition of S. different enzymatic assays were performed. although the unbound material also displayed antifungal activity (figure 1A). eumartii. / Plant. The basic heatresistant pool was further purified on Superose 12 FPLC (figure 1B) where two fractions with antifungal activity were detected (F18 and F22). where the strongest antifungal activity was detected in the basic fraction. This clarified extract was submitted to cation exchange chromatography on CM-Sepharose. the fraction eluted with 0. It has been shown that the activity of many cationic antimicrobial proteins is reduced or blocked in the . 2. The arrows indicate the elution with 0. some spores germinated (less than 10 %). Detection of antifungal proteins in flowers In order to detect proteins with constitutive expression able to inhibit in vitro the growth of S. 2.1. B. Hence. a tobacco PR-5 antibody was used to detect possible PR-5 antifungal proteins present in these fractions. Purification of an antifungal protein with trypsin inhibitory activity The ability of fraction F18 to inhibit trypsin was exploited as a purification strategy to verify if the antifungal activity was associated to the activity of trypsin inhibitor (TI). RESULTS 2. western blot analysis of fraction F18 and F22 showed no reactions with the antibody anti-PR-5. Figure 1. No activity of glucanase or chitinase associated with F18 and F22 could be detected. Bars indicate fractions with antifungal activity. retained in the column and eluted with 0. However.882 A. Moreover.M. this antifungal protein with an associated activity of proteinase inhibition was named SAP16 (Sunflower Antifungal Protein 16 kDa).2 M NaCl from A was further purified by gel filtration on Superose 12 FPLC. Hence.2 and 0. 38 (2000) 881–888 2. at a concentration of 5 µg·mL–1 only shortened hyphae could be observed (figure 3B). Giudici et al. sclerotiorum showed that after 24 h of incubation. In addition. a total protein extract from Helianthus annuus flowers was obtained and enriched in heat-resistant proteins. In order to characterize these proteins. This protein also exerts trypsin inhibitory activity (25 % inhibition on the activity of trypsin per µg protein.2 M NaCl.2 M NaCl from CM-Sepharose was submitted to affinity chromatography on a trypsin-agarose matrix. sclerotiorum.2. sclerotiorum spore germination at a concentration of 5 µg·mL–1 while at 3 µg·mL–1. The basic antifungal fraction eluted with 0. Biochem. under the assay conditions described in Methods). a reduction of hyphal growth is observed (figure 3A).3. Purification of antifungal proteins from sunflower flowers. sp. Physiol. was also inhibited by SAP16 but appears to be less sensitive than S.5 M NaCl. A more detailed microscopic examination of the effect of SAP16 on S. A sharp protein peak bound to the matrix displaying antifungal activity was recovered (figure 2A). but F18 showed trypsin inhibitory activity (not shown). on SDS-PAGE a unique band of 16 kDa estimated molecular mass was visualized (figure 2B). sclerotiorum.

38 (2000) 881–888 883 Figure 2. sclerotiorum is not a common feature of all TIs. SAP16 is indicated with the arrow. sclerotiorum. Mechanism of spore germination inhibition by SAP16. Figure 4. 3 or 5 µg·mL–1 of SAP16.4 kDa) were used as molecular mass standards. sclerotiorum after overnight incubation with 10 mM Na acetate (pH 5. 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. Spores of S. 37]. Magnification 50×.A. Giudici et al.2 M of NaCl from CM-Sepharose was subjected to affinity chromatography on a trypsin-agarose matrix. Physiol. Soybean TI (20. Biochem. Magnification 66×. presence of cations [32. under microscopic observation (figure 4C). To determine if the antifungal activity of SAP16 also shares this property. However. Spores were incubated with 10 mM Na acetate buffer (pH 5. Antifungal activity of SAP16. The basic antifungal fraction eluted with 0. 5 µg·mL–1 soybean TI (D). sclerotiorum ascospores germination. B. 5 µg·mL–1 SAP16 (B). A. its activity was evaluated in the presence of 1 mM CaCl2 added in the germinating medium. Controls with 1 mM CaCl2 or 95 µg·mL–1 PA germinated as control A.1 kDa) and lysozyme (14.M. 5 µg·mL–1 SAP16 + 1 mM CaCl2 (C). Peak 1 from A was submitted to size exclusion chromatography on Biogel P-60 and analyzed on 15 % SDS-PAGE.5 M NaCl (pH 2) is indicated. Figure 4 also illustrates that 5 µg·mL–1 of a commercial soybean trypsin inhibitor has no appreciable effect on S. Magnification 66×. no difference in the inhibitory activity of SAP16 could be detected in the presence of calcium cations. The elution (e) with 0. Purification of sunflower antifungal protein SAP16 by trypsin affinity chromatography. Effect of SAP16 on the fungus S. 0. . solani. B.2) (A). Spores were incubated with 10 mM Na acetate buffer (pH 5.2) (control) or 5 µg·mL–1 of SAP16. indicating that the inhibition observed on S. 5 µg·mL–1 SAP16 + 95 µg·mL–1 PA (F).2 mM TLCK (E).2) (control). A. Similar results were obtained with concentrations as high as 500 µg·mL–1. / Plant. Effect of SAP16 on the fungus F.

This observation prompted the investigation of whether SAP16. Giudici et al. antifungal TIs can exert their activity by the inhibition . as it has been reported for thaumatinlike proteins [32] and thionins [39]. 38 (2000) 881–888 tors. 3. although this reagent is inactivated in aqueous solution and would be acting only in the first hours of incubation. these observations suggest that no essential extracellular serine proteinase is needed for the germination of S. 15]. Physiol. two possible mechanisms can be suggested. it is unlikely that SAP16 exerts its antifungal activity by the inhibition of a proteinase required for fungal germination. By now. sclerotiorum ascospores was analyzed. As a consequence. the antifungal proteins with an associated activity of serine proteinase inhibition constitute a barely known family with only a few members isolated. being a basic AFP. Thus. similar to the most potent AFP active against S. PA is able to counteract SAP16 inhibition of ascospore germination. SAP16 displays a potent inhibitory activity causing the complete inhibition of S. 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]. and the inhibition of trypsin indicate that this antifungal protein is a serine proteinase inhibitor. Hence. Alternatively. 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. the previously described antifungal serine proteinase inhibitors are also small proteins (12–25 kDa) but none of them have been isolated from floral tissues. As for SAP16. This property might be particularly interesting for the production of transgenic plants overexpressing antifungal proteins to enhance resistance against fungal pathogens [11]. sclerotiorum spores germination at a concentration of 5 µg·mL–1 (0. A preliminary approach to determine the potential role of AFPs in controlling the growth of pathogens in planta is to evaluate their antifungal potency. sclerotiorum ascospores.M. 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 flavus and absent or present in low concentrations in six susceptible ones [8]. Figure 4E shows that when ascospores were incubated together with 0. suggesting that the protein interacts with PA instead of interacting with fungal membranes. sclerotiorum spores was investigated. The best characterized antifungal TI from corn requires concentrations greater than 100 µg·mL–1 to inhibit the germination of spores of nine fungal species [7]. could be acting at the level of the fungal plasma membrane by interaction with its anionic components. sclerotiorum reported [30]. 8] and barley [38] inhibit several fungi at concentrations between 40 and > 1 000 µg·mL–1. the presence of serine protease activity in the germination medium upon overnight incubation of S. sclerotiorum is not very sensitive to other described AFPs [37]. they may act directly at the level of fungal membrane affecting its permeability. On the other hand. reported data indicate that the widespread pathogen S. 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].31 µM). a constitutive anionic phospholipid of biological membranes. The trypsin affinity of SAP16. and those isolated from cabbage [23]. Taken together. The mechanism of action of the antifungal proteins with associated activity of TI has not been studied in detail yet but. we have analyzed the effect of SAP16 on the germination of ascospores in the presence of phosphatidic acid (PA). solani spores was 600 µg·mL–1 [23]. Among them are the serine proteinase inhibitors from corn [8. the ascospores germinated normally. used as purification strategy. As a second approach the effect of two irreversible inhibitors of serine proteinases (TLCK and PMSF) [44] on the germination of S. while the TIs isolated from corn [7. More recently. According to their basic features. the concentration of cabbage TIs producing a total inhibition of the germination of Botrytis cinerea and F. DISCUSSION The experimental approach employed allowed the isolation to apparent homogeneity of a 16-kDa protein from sunflower (SAP16) showing the inhibition of S. 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.2 mM TLCK (concentration producing 92 % inhibition of trypsin activity in vitro). First. Biochem. As shown in figure 4F. Hence. sclerotiorum spore germination. two experimental approaches were used. / Plant. in the standard conditions used in the bioassay.884 A. 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). TIs from barley [38] and wheat [10]. The same results was obtained with the addition of 1 mM PMSF to the germination medium. Interestingly. at least.

Fertile flowers were obtained from plants cultivated under standard greenhouse conditions. Our results also highlight a role for TIs as controllers of fungal attack and. Protein purification Fertile flowers were ground with three volumes of extraction buffer (100 mM Na acetate pH 5.3. [1] have isolated a 6-kDa protein from Nicotiana alata stigmas with inhibitory activity towards trypsin. When indicated. annuus flowers suggests a potential role in innate defense by a direct inhibition of fungal growth.21. METHODS 4. [31]. [20] and the reducing sugars generated were determined according to Somogyi [36]. Giudici et al. slides were evaluated under a light microscope for the presence of S. the assay was supplemented with different additions. The extracellular fraction obtained for germinating ascospores (16 h incubation) was assayed for its proteolytic activity by incubation in the presence of azocasein 0. the contribution of another mechanism cannot be excluded keeping in mind recent evidences reported for the 14-kDa TI from corn [9]. Argentina) was used in this work.3 mM β-mercaptoethanol. comm. [30] and . a direct effect of SAP16 on fungal membrane is suggested by the ability of exogenously added PA to displace the inhibition caused by SAP16. Chitinase activity was measured by the release of N-acetylglucosamine from colloidal chitin. This genotype has been characterized as tolerant to head rot 4. sclerotiorum ascospores.M. sp. to our best knowledge. The specific biological function of TIs in flowers 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]. Atkinson et al. On the other hand. Controls included enzyme and substrate blanks. The reaction was stopped by adding 500 µL TCA 10 % and the mixture was centrifuged at 1 200 × g for 15 min. eumartii.4) from bovine pancreas and testing for 20 min at 37 °C in 80 mM Tris-HCl (pH 8). produced by S. and the mixture was incubated 1 h at 37 °C with agitation.4.2. / Plant.4. The basic nature of SAP16 may then play a central role in determining the mode of its antifungal activity: electrostatic interactions with membrane phospholipids. Moreover. Argentina. Biological materials Sclerotinia sclerotiorum (Lib. 14. sclerotiorum hyphae. was grown at 25 °C on Petri plates containing potato dextrose agar and the spores were collected by suspension in water.2. 100 mM KCl). even if the significance of the presence of SAP16 in sunflower inflorescence remains to be determined. 1997) were used for the production of ascospores from apothecia [40]. SAP16 constitutes. the inhibition of fungal growth may be partially due to the inhibition of fungal α-amylase production and activity hence limiting fungal growth. The inhibitory activity was calculated as the difference between the proteolytic activity in the absence and presence of the inhibitor. Helianthus annuus L. After overnight extraction.A. the first TI isolated from flowers to display antifungal activity. a heat resistant protein fraction was obtained as previously described by Regente et al. isolate 3122 (INTA Collection. 4. Physiol. line AR10018 (Zeneca SAIC. sclerotiorum in field trials (Dr Bazzalo.) and displays low susceptibility to wilting in growth chamber tests [2]. 4. 4.3Glucanase activity was measured using laminarin (Sigma) as substrate as described Kauffmann et al. Biochem. in a final volume of 26 µL 10 mM Na acetate buffer (pH 5. In fact.5 % was then added as substrate. Controls were performed by replacing the protein sample with the same volume of buffer. Azocasein 0. alfalfa [26] and potato [28]. Zeneca SAIC. using the method described by Reissig et al. sclerotiorum ascospore germination was done on micro slides containing the protein sample. β-1. 38 (2000) 881–888 885 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. Argentina). The A350 nm of the supernatant was indicative of the residual proteinase activity. Fusarium solani f. Balcarce.2). Enzymatic activity Trypsin inhibitory activity was measured by preincubating 1 µg trypsin (EC 3.1. In this case. 103 ascospores and 6 % sucrose (w/v).5 % for 20 h at 37 °C. the occurrence of putative mRNAs from TIs has been reported in flowers of tomato [4]. Antifungal activities The test for inhibition of S. pers.) de Bary sclerotia obtained from a local virulent isolate (Balcarce. 1 mM CaCl2. in a final volume of 500 µL. After 16 h incubation at 25 °C in 100 % relative humidity. However. extracellular serine proteinases do not seem to be involved in the germination of S. its potent activity against a natural pathogen of H. However.

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