Characterization of Two Novel Defense Peptides from Pea (Pisum sativum) Seeds

Archives of Biochemistry and Biophysics Vol. 378, No. 2, June 15, pp. 278 –286, 2000 doi:10.1006/abbi.2000.1824, available online at http://www.idealibrary.com on

Characterization of Two Novel Defense Peptides from Pea (Pisum sativum) Seeds 1 Marcius S. Almeida, Ka´tia M. S. Cabral, Russolina B. Zingali, and Eleonora Kurtenbach 2 Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, RJ, Brasil

Received November 1, 1999, and in revised form March 3, 2000

A fraction that possesses antifungal activity against Aspergillus niger has been isolated from seeds of the pea (Pisum sativum) by ammonium sulfate fractionation followed by gel filtration on Sephadex G-75. On further purification by reverse-phase high performance liquid chromatography, two small cysteine-rich polypeptides were obtained (Psd1 and Psd2). They are localized primarily in vascular bundles and epidermis tissues of pea pods and exhibit high antifungal activity toward several fungi, displaying IC 50 values ranging from 0.04 to 22 ␮g/ml. This inhibitory activity decreases when A. niger growth medium is supplemented with cations such as Ca 2ⴙ, Mg 2ⴙ, Na ⴙ, and K ⴙ. Although the primary sequence of both Psd1 and Psd2 shows homology with other plant defensins, they cannot easily be assigned to any established group. © 2000 Academic Press

Key Words: antifungal activity; defensins; plant–fungal interaction; Pisum sativum.

Plant storage tissues possess a variety of defense mechanisms that can be triggered upon wounding or contact with microorganisms, which is associated with the increase of several proteins above their constitutive basal level. These proteins include trypsin, amylase inhibitors, toxic proteins, hydrolases, proteases, and small cysteine-rich antimicrobial peptides. This latter class appears to be widely distributed in the plant kingdom, where several homologous peptides have been isolated from plant seeds, or identified by cDNA sequencing and translation from both monocot and dicot species. These include sorghum (1), pea (2), to1 The SWISS-PROT Accession Numbers for the amino acid sequences of Psd1 and Psd2 are P81929 and P81930, respectively. 2 To whom correspondence should be addressed. Fax: (55-21) 2708647. E-mail: [email protected].

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bacco (3), potato (4), petunia (5), sugar cane (6), and several members of the family Brassicaceae (7, 8). Based on the inhibitory activity of some members of this Cys-rich peptide family toward plant pathogens, on their expression patterns and on their structural homology to mammalian and insect defensins, a new group called plant defensins has been proposed (8). This family is composed of small peptides of 45–54 amino acids, which include eight cysteines that give rise to four intramolecular disulfide bonds. The family can be subclassified into four groups, based on structural and functional considerations. Group I includes defensins that inhibit growth of Fusarium culmorum and cause morphological distortions of the fungal hyphae. Group II comprises those that merely inhibit fungal growth without inducing morphological changes. Plant defensins without antifungal activity are included in Group III (9 –11). A fourth group (group IV) described more recently (12) includes defensins isolated from Spinacia oleracea that exhibit both antifungal and antibacterial activities. The antifungal activity exhibited by some members of this large family decreases in the presence of cations such as Ca 2⫹ and K ⫹ as shown for Rs-AFP1, Rs-AFP2, and Rs-AFP3, three defensins isolated from radish (Raphanus sativus L.) seeds (7). Here we describe the purification and characterization of two new Cys-rich proteins from seeds of the garden pea (Pisum sativum), which we have named Psd1 and Psd2. The N-terminal sequence of the first one reveals 95% identity with the C-terminal region of the predicted transcript of the Disease Resistance Response Gene 230-b (2, 13). The peptide Psd2 shows 65% identity with the amino acid sequence deduced from a cDNA clone for a storage mRNA (14) present in cotyledons of Vigna unguiculata seeds (pSAS10). Both peptides exhibit high antimicrobial activity against several fungi, including pea pathogens, and are found in 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

CHARACTERIZATION OF PEA DEFENSINS

epidermal tissues and vascular bundles, which suggests a role in the pea defense mechanisms. MATERIALS AND METHODS Materials. Leupeptin, pepstatin, PMSF, 3 TFA, paraformaldehyde, guanidinium hydrochloride, trypsin (TPCK treated), aminomethyl propanediol, goat anti-rabbit IgG immunoglobulin-alkaline phosphatase conjugate, tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG, and L-cysteine were from Sigma Chemical Co. Sephadex G-75 superfine and protein A–Sepharose were from Amersham Pharmacia Biotech. Acetonitrile, ammonium sulfate, and DTNB were from E. Merck. Potato dextrose medium and YNB were from DIFCO Laboratories. Purification. Purification methodology used in this work is based in part on the purification of soyatoxin (15). Mature, dry seeds of P. sativum biovar MIKADO (kindly provided by Dr. L. Giordano, CNPH/EMBRAPA) were ground in a laboratory mill (Wiley) fitted with a 0.75-mm mesh screen, defatted with petroleum ether (10%, w/v) under continuous stirring for 72 h, and finally air-dried at room temperature. The defatted meal was resuspended and stirred for 4 h at 4°C in extraction buffer [1:5 (w/v), meal:buffer] containing 25 mM Tris–Cl, pH 7.5, 5 mM sodium azide, and protease inhibitors (leupeptin 2 ␮g/ml, pepstatin 2 ␮g/ml, and PMSF 100 ␮g/ml, final concentration). The suspension was centrifuged at 14,000g for 30 min. The supernatant, referred to as crude extract, was collected and precipitated at 4°C by adding ammonium sulfate at a rate of 1 g/min to 35, 65, and 85% of saturation. The individual precipitated fractions (F1, F2, and F3, respectively) were collected by centrifugation at 14,000g for 30 min and dissolved in a small amount of buffer (25 mM Tris–Cl, pH 7.5). Sephadex-G75 gel filtration. The F2 sample was subjected to gel filtration on a Sephadex G-75 Superfine column (1.6 ⫻ 100 cm) at a flow rate of 0.7 ml/min, using 25 mM Tris–Cl, pH 7.5, as the eluant. The absorbance of collected fractions (3.0 ml each) was measured at 280 nm. High-performance liquid chromatography (HPLC). Fractions containing high antifungal activity (F-PII) were pooled and centrifuged at 11,000g for 5 min and further purified by reversed-phase HPLC on an analytical C 8 column (VYDAK) using as eluants buffer A (0.1% TFA) and buffer B (0.1% TFA plus 90% acetonitrile). The flow rate was 0.7 ml/min and the absorbance of fractions was measured at 214 nm. Samples of 1 ml were collected, dried under vacuum, and dissolved in water for bioassays. Protein determination. Protein content of the fractions was determined by the method of Lowry et al. (14), using bovine serum albumin as standard. Polyacrylamide gel electrophoresis. SDS–PAGE was carried out on 0.75-mm-thick slab gels containing a 12–20% (w/v) gradient of acrylamide with a stacking gel of 3.3% acrylamide in the Laemmli

3 Abbreviations used: BSA, bovine serum albumin; DRRG, disease resistance response gene; DTNB, 5,5⬘ dithiobis(2-nitrobenzoic acid); EDTA, ethylenediaminetetraacetic acid; GCG, Genetics Computer Group; HPLC, high performance liquid chromatography; Idd, identity; IC 50, protein concentration required for 50% of fungal growth inhibition; PD, potato dextrose; PIR, Protein Information Resource; PMSF, phenylmethylsulfonyl fluoride; PTH, phenylthiohydantoin; PVDF, polyvinylidene difluoride; RP-HPLC, reverse-phase high performance liquid chromatography; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; Tris-Cl, Tris-[hydroxymethyl] aminomethane hydrochloride; TBS, Tris-buffered saline; TPCK, 1-tosylamide-2-phenylethyl chloromethyl ketone; TRITC, tetramethylrhodamine isothiocyanate; YNB, yeast nitrogen base.

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system (17). Gels were stained with silver (18). Molecular mass standards were: myosin heavy chain, 205.0 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.3 kDa; and aprotinin, 6.5 kDa. Trypsin hydrolysis. Native Psd2 was dissolved in 8 M urea and 25 mM ammonium bicarbonate, pH 7.5, and reduced with 7.5 mM DTT for 15 min at 50°C. The reduced Psd2 was S-carboxyamidomethylated as reported (22), by adding 14.3 mM iodoacetamide and incubating at room temperature for 15 min. The S-carboxyamidomethylated Psd2 was purified by reverse-phase HPLC on an analytical C 18 column (VYDAK) and the corresponding peak (OD 214 nm) was dried under vacuum. This peptide (3.2 ␮g) was then subjected to digestion with 3.7 ␮g/ml trypsin in 100 mM ammonium bicarbonate, pH 7.5, for 12 h at 37°C. The resulting peptides were fractionated by RP-HPLC on an analytical C 18 column (VYDAK) and sequenced. Sequencing. After SDS–PAGE, the proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane sheet for 45 min at 150 mA, using a Pharmacia semidry blotting system. Protein bands were cut out and sequenced by automated Edman degradation in a Porton Integrated microsequencing system (Model PI2090). Proteins obtained from reverse-phase chromatography (⬃13 ␮g each) were also blotted onto a PVDF membrane and sequenced. Cysteine residues were identified after alkylation of free ⫺SH groups in situ (in the reaction cartridge of the sequencer) using acrylamide in ethyl acetate (19) following Edman degradation and PTH derivatization to form the PTH-Cys-S-Pam derivative. Determination of free cysteine thiol groups. Release of 2-nitro-5thiobenzoate upon reaction of free thiols with DTNB was carried out according to a modification of Ellman’s method (21). Reduced or unreduced peptides (150 ␮g each) were dissolved in 6 N guanidinium hydrochloride containing 25 mM Tris–Cl (pH 7.5) and 1 mM EDTA. Reaction was allowed to take place at 25°C in the presence of 95 ␮M DTNB, and the absorbance was read at 412 nm. L-Cysteine (0 –36 ␮M final concentration) was used as a standard. Antibody production. Two rabbits were immunized with purified proteins (Psd1 and Psd2) from the HPLC run (80 ␮g each) emulsified in complete Freund’s adjuvant and injected subcutaneously at multiple sites in the animal’s back. Three weeks later, this procedure was repeated using incomplete Freund’s adjuvant. The rabbits were first bled 1 week after the second injection. An intramuscular booster injection was given after 1 month (20). In an enzyme-linked immunoabsorbent assay (ELISA), midpoint titration of the antiserum obtained occurred at a dilution of 1:500 and 1:100 when the purified proteins Psd1 and Psd2 were used as antigen, respectively. The immunoglobulin G fraction was isolated using a protein A–Sepharose column and analyzed by 10% SDS–PAGE, which revealed two protein bands of 75 and 25 kDa, corresponding to the light and heavy chains, respectively. Western blots. The protein bands from SDS-PAGE were electrotransferred (150 mA, 1 h) onto a PVDF membrane and further blocked with 2% low-fat dry milk for 16 h. The membranes were incubated with rabbit anti-Psd1 IgG or rabbit anti-Psd2 IgG as primary antibodies and, after several washes, were exposed to goat anti-rabbit IgG conjugated to alkaline phosphatase. The color reaction was developed with nitro blue tetrazolium and 5-bromo-4chloro-3-indoyl phosphate in water. Optical microscopy. Mature, dry pea pod tissues were fixed with 4% paraformaldehyde, embedded in 8% agarose, and sectioned (200 ␮m) across the long axis. Sections were incubated in 0.1 M sodium phosphate buffer, pH 7.4, for 1 h at room temperature, washed with TBS, and blocked for 3 h in 0.1 M PBS containing 3% bovine serum albumin. Then the sections were incubated overnight with purified IgG anti-Psd1 (1:100) or IgG anti-Psd2 (1:100) as primary antibodies, and for 2 h with TRITC-conjugated goat anti-rabbit IgG (1:300) as the secondary antibody. Control sections were incubated with TRITC-conjugated goat anti-rabbit IgG (1:300). The sections were

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mounted in 0.1 M sodium phosphate buffer, pH 7.4, on a glass, and examined by laser scanning confocal microscopy (LSM 410; Zeiss Inc.) using a filter set selective for rhodamine (543 nm) or by stereomicroscopy (Stemi Sv11; Zeiss Inc.). Computer analysis. All sequence comparisons were carried out using the GCG package from the University of Wisconsin (Madison, WI). Microorganisms. The following fungal and yeast strains were used: Aspergillus niger (EK0197), Aspergillus vesicolor (strain 40028 LMR/INCQS, Oswaldo Cruz collection), Fusarium solani (strain 2389 UFPe), Fusarium moniliforme (strain 2414 UFPe), Fusarium oxysporum (strain 2665 UFPe), Neurospora crassa (strain 74 kindly provided by Dr. H. Terenzi, Universidade de Sa˜o Paulo/Ribeira˜o Preto), Saccharomyces cerevisae PF2700, and Trichophyton mentagrophytes (strain 40131 LMR/INCQS, Oswaldo Cruz collection). Antifungal assays. Antifungal assays were carried out by microspectrophotometry (20). Routinely, tests were performed with 20 ␮l of the filter-sterilized putative antimicrobial peptide in water and 100 ␮l of the test fungal spore suspension (3 ⫻ 10 4 spores/ml) in potato dextrose (PD) (Table II) or YNB medium (Fig. 5). Controls were tested identically except that the peptide was omitted. Cultures were incubated at 25°C until absorbance readings (OD 540 nm) indicated that 50% of maximum fungal growth has been attained, based on the growth curve for each microorganism tested. Percentage growth inhibition is defined as 100 times the ratio of the corrected absorbance at 540 nm of the control microculture, minus the corrected absorbance of the test microculture, divided by the corrected absorbance of the control microculture. The corrected absorbance values are equal to the absorbance of the culture measured after the appropriate time minus the absorbance at 540 nm measured after 30 min. Values of growth inhibition lower than 10% were not considered significant. All experiments were done in triplicate. The antifungal activity of 30 ␮g/mL Psd2 against A. niger was also assayed in 0.5⫻ YNB containing 0.45–17.1 mM CaCl 2, 2.1–18.8 mM MgCl 2, 3.65–337 mM KCl, or 0.85–334 mM NaCl.

RESULTS

Purification and Molecular Characterization of the P. sativum Peptides The starting material for the isolation of the P. sativum antimicrobial peptides was the extract fraction in 25 mM Tris–Cl, pH 7.5, obtained from the defatted mature seeds followed by 35– 65% ammonium sulfate fractionation. Purification was performed by gel filtration column chromatography on Sephadex G-75 Superfine and the eluted fractions were monitored for the ability to inhibit the growth of A. niger as well as by OD at 280 nm. Gel filtration resolved the mixture into two main peaks (PI and PII) followed by OD at 280 nm, with a prominent peak of antifungal activity (F-PII) located between these two peaks (Fig. 1). On SDS– PAGE analysis, the F-PII fraction consisted of a single broad band of 5 kDa (Fig. 2, lane 5), which was resolved into two bands (F-PIIA, top band; F-PIIB, bottom band) when a lower amount of the sample was applied (Fig. 2, lane 6). Further purification of F-PII was accomplished using reverse phase high-performance liquid chromatography (RP-HPLC). Four peaks were detected (Fig. 3). Fractions containing antifungal activity (peaks P2 and

FIG. 1. Gel filtration chromatography of F2 sample. F2 (600 mg) was dissolved in 25 mM Tris–Cl, pH 7.5 and applied on a Sephadex G-75 Superfine column (1.6 ⫻ 100 cm) at a flow rate of 0.7 ml/min. The absorbance of collected fractions (3 ml) was monitored at 280 nm (—, left axis). Except for P1, for which fractions 5–16 were pooled (large dot at left), 25 ␮g of each fraction was used in the microspectrophotometric antifungal activity assay using Aspergillus niger (---, right axis) (triplicates, mean ⫾ SD).

P3) were lyophilized, diluted in electrophoresis loading buffer, and examined by SDS–PAGE. Silver staining (Fig. 2, lanes 7 and 8) showed that P2 corresponds to F-PIIB and P3 to F-PIIA (bottom and top bands in lane 6 of Fig. 2). Samples containing F-PIIA and F-PIIB were electrotransferred to a PVDF membrane after separation by SDS–PAGE. For comparison, aliquots of the P2 and P3 fractions from the RP-HPLC run were spotted on a PVDF membrane and all four samples were sequenced by automatic N-terminal Edman degradation. Analysis of both F-PIIB and P2 both revealed an identical unambiguous primary sequence of 46 amino acids (line 1 in Fig. 4). This protein has a calculated molecular weight of 5108 in agreement with the molecular mass of 5.0 kDa estimated for F-PIIB by SDS–PAGE (Fig. 2). A search of the Gen-EMBL Bank and SWISS-Prot data bases using the TFASTA program from GCG re-

CHARACTERIZATION OF PEA DEFENSINS

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FIG. 2. Polyacrylamide gel electrophoresis and Western blots of pea proteins. (A) Silver-stained SDS–polyacrylamide gels. Protein samples were analyzed by electrophoresis on 12–20% linear gradient polyacrylamide gel stained with silver (⬃4 ␮g in lanes 1– 6). Lane 1, molecular mass markers; lane 2, crude extract; lane 3, F1 sample, lane 4, F2 sample, lanes 5 and 6 F-PII sample from Sephadex G-75 column; lane 7, P3 from HPLC run (2.1 ␮g); lane 8, P2 from HPLC (2.6 ␮g). Molecular mass markers: myosin, 205.0 kDa; BSA, 66.2 kDa; ovalbumin, 45.0 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.3 kDa; aprotinin, 6.5 kDa. (B) Western-blot analysis of peptides purified by RP-HPLC. Lane 1, Psd1 and lane 2, Psd2 (2.5 ␮g each).

vealed 95% identity of the purified peptide with the sequence corresponding to the C-terminal region (Gln 29–Cys 74) of the deduced transcript of the Disease Resistance Response Gene DRR 230-b from P. sativum (L.). This gene rapidly accumulates after inoculation of pea (P. sativum) pods with spores of the bean pathogen F. solani (2, 13). We have designated this peptide P. sativum defensin one (Psd1) (Fig. 4). The amino-terminal sequence of P3 was determined out to 47 cycles and the carboxy-terminal amino acid

FIG. 3. Reverse-phase high-performance liquid chromatography (RP-HPLC). F-PII (280 ␮g) obtained from the gel filtration column shown in Fig. 1 was analyzed by RP-HPLC on an analytical VYDAK-C 8 column equilibrated with 0.1% TFA and 22.5% acetonitrile. The retained proteins were eluted at a flow rate of 0.7 ml/min using a linear acetonitrile gradient (22.5–35%, v/v, in 0.1% TFA) followed by 90% acetonitrile/0.1% TFA (right axis). The protein content was followed by measurement of OD at 214 nm (left axis).

sequence was confirmed by sequencing one heptapeptide generated after the hydrolysis of P3 by trypsin. It exhibits 65% identity with the amino acid sequence deduced from the mRNA of pSAS10, which was isolated from the cotyledons of Vigna unguiculata seeds and characterized by Ishibashi et al. (1990) (14). We propose to call it P sativum defensin 2 (Psd2) (line 2 in Fig. 4). No free cysteines were detected in either Psd, based on quantification of thiol residues following a modification of Ellman’s method (21). This indicates that all cysteines participate in the formation of disulfide bridges. The alignment of the amino acid sequences of plant defensins from other sources (found in SWISS-Prot and PIR databases using the BLASTP command) revealed significant identities to Psd1 and Psd2 (Fig. 4). As can be seen from this alignment, all sequences possess eight conserved Cys residues, an aromatic residue at position 12, and a Gly residue at position 39. Also, residues conserved in most, but not all, plant defensins include an acidic residue at position 6, Ser at position 9, Gly at position 14, hydrophobic residues at positions 15 and 34, and Glu at position 31. Some of the amino acid residues fully conserved among these plant defensins have been used by other authors to classify them into three groups (I, II, and III) (9 –11). Despite some features in common with other plant defensins the Psd1 peptide reveals some unique characteristics, including the presence of an Ala residue instead of a Ser residue at position 9 and the lack of a Glu residue at position 31. Furthermore, neither Psd1

FIG. 4. Comparison of the complete amino acid sequences of various plant defensins including the pea defensins Psd1 and Psd2 isolated and characterized in this work. Asterisk (*) indicates sequences deduced from cDNA. Residues conserved among all plant defensins are shown in black boxes. Residues in gray boxes are those conserved in most plant defensins. Highly conserved residues that are relevant for the classification of defensins are in unshaded boxes. Dashes indicate gaps introduced to maximize homology. Conserved residues are shown at the bottom of sequence alignment. Symbols used to classify conserved amino acids: (@) aromatic residues; (⫹) positively charged residues; (⫽) hydrophobic residues; (⫺) negatively charged residues. Accession numbers refer to SWISS-PROT, GENBANK and PIR databases. Activity: (A/⫺A) with/without inhibitory activity on ␣-amylase; (B/⫺B) antibacterial activity strong/weak or absent; (C/⫺C) toxic/nontoxic to animal cells; (F/⫺F) antifungal activity strong/weak or absent; (N) Na ⫹ channel inhibitor; (?) unknown.

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CHARACTERIZATION OF PEA DEFENSINS TABLE I

Antifungal Activity of the Cysteine-Rich Peptides from P. sativum Seeds IC 50 (␮g/ml)

PD medium Fungus

Psd1

Psd2

PD medium supplemented with 1 mM CaCl 2 Psd1

Psd2

Aspergillus niger 12.1 10.2 7.4 Aspergillus versicolor ⬍5.0 0.34 6.0 Fusarium moniliforme 21.7 10.0 33.2 Fusarium oxysporum ⬎100 ⬎100 ⬎100 Fusarium solani 12.1 8.5 79.2 Neurospora crassa 0.04 ⬍0.5 0.05 Saccharomyces cerevisae ⬎100 ⬎100 NT Trichophyton mentagrophytes ⬎100 ⬍100 NT

7.8 5.9 25.0 ⬎100 5.0 ⬍0.5 NT NT

Note. Protein concentration (␮g/ml) required for 50% growth inhibition (IC 50) was determined from the corresponding dose–response curves (percentage growth inhibition versus protein concentration). Concentrations of Psd higher than 100 ␮g/ml were not tested. NT, not tested; PD, potato dextrose.

nor Psd2 possesses all of the highly conserved sequence motifs that were utilized as the basis for classification of groups I–III of plant defensins. This is not surprising since the Psd sequences show low identity (around 35%) when compared with the majority of isolated plant defensins (column labeled Idd % in Fig. 4). Purified Psd1 and Psd2 were used to obtain antiserum in rabbits and the corresponding IgGs were isolated by protein-A affinity chromatography. In spite of the identity found in the Psd1 and Psd2 amino acid sequences (Fig. 4, Idd 45%), rabbit IgG raised against each peptide recognized only itself, as shown in an immunoblot assay (Fig. 2B, lanes 1 and 2). No crossreactivity was observed when anti-Psd2 IgG and antiPsd1 IgG were used as primary antibody with Psd1 and Psd2, respectively (data not shown). Hence, these IgGs are reliable tools for use in immunolocalization experiments. Antimicrobial Properties of the Purified Peptides The antifungal activity of Psd1 and Psd2 was examined by determining the protein concentration required for 50% growth inhibition (IC 50) of eight species of fungi. Generally, both Psd peptides exhibited similar antifungal potency in the absence of Ca 2⫹, with IC 50 values ranging from 0.04 to 22 ␮g/ml (Table I). Fusarium oxysporum, S. cerevisae, and T. mentagrophytes were not inhibited by Psd1 at concentrations below 100 ␮g/ml. In some cases, higher IC 50 values are obtained

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for Psds in the medium with added Ca 2⫹. For example, addition of 1 mM CaCl 2 to the PD medium markedly decreased the activity of Psd1 on F. solani but had little effect on that of Psd2. In contrast, the activity of Psd2 on A. vesicolor was strongly affected by the presence of Ca 2⫹. The small decrease in the IC 50 of both Psds against A. niger in the presence of Ca 2⫹, however, shows that the effect of Ca 2⫹ is strongly dependent on the fungus tested. Both Psds were highly active against Neurospora crassa, with an IC 50 ⬃0.04 ␮g/ml. Amphotericin B (400 ␮g/ml), a known antifungal drug, was nearly 100% effective against S. cerevisae and inhibited growth of F. solani and A. niger by 75%. As shown in Fig. 5, the Psd2 antifungal activity against A. niger was inhibited in a dose-dependent manner by calcium, magnesium, potassium, and sodium, showing EC 50 values of 2.0, 2.3, 15, and 21 mM, respectively. Immunolocalization of Psds The tissue localization of Psd1 and Psd2 in pea pods was investigated by confocal laser scanning microscopy, using the purified IgGs described above. Strong labeling was achieved when tissues obtained from fresh pods were incubated with IgG anti-Psd1 (Fig. 6A) and IgG anti-Psd2 (Fig. 6B) following incubation with TRITC-conjugated anti-rabbit IgG as secondary antibody. This positive rhodamine labeling, indicative of

FIG. 5. Cation sensitivity of the antifungal activity of Psd2. Growth inhibition of A. niger caused by 30 ␮g/ml of Psd2 in 0.5⫻ YNB medium with the concentrations of CaCl 2, MgCl 2, NaCl, and KCl added to the final concentration shown on the abscissa. Percentage growth inhibition was measured by the microspectrophotometry assay. All values were recorded after control microcultures (growing without Psd2) reached an OD 540 nm of 0.6. Zero percent growth inhibition was assigned to this control value. One hundred percent growth inhibition was assigned to microcultures growing in 0.5 ⫻ YNB, with 30 ␮g/ml of Psd2. Growth inhibition in controls containing the same ionic concentration but no defensins has been subtracted from the values shown. Data show x ⫾ SE of triplicates.

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FIG. 6. Immunofluorescence of pea pod tissue challenged with serum containing antibodies against Psd1 and Psd2. Images were collected by confocal laser scanning microscopy from sections treated with rabbit IgG anti-Psd1 (A), rabbit IgG anti-Psd2 (B), and secondary antibody alone (TRITC-conjugated anti-rabbit IgG) (C and D). In A–C, images showing the fluorescence of TRITC. In D, reflection image obtained using the same wavelength as the incident light (green channel). All images were also visualized in bright field mode (E); the box outlines the region used in the other panels (A–D). Arrowheads in A, B, D, E, indicate localization of epidermis (e) and vascular bundles (v); seed (s). All figures are oriented identically; asterisk (*) in all figures, indicates the funiculus. The recorded images were processed in a LSM v. 3.80 (Carl Zeiss) system and edited by Adobe Photoshop 5.0 software.

immunoreactivity, was observed especially in epidermis (e) and vascular bundles (v), thus indicating the presence of Psd1 and Psd2 in these tissues. When pea pod tissue was incubated with secondary antibody alone (Fig. 6C) no labeling could be detected, confirming the specificity of the response obtained when IgGs

anti-Psd1 and anti-Psd2 were used. Figure 6D shows a bright field image where more detail can be seen, since the immunofluorescence response (red channel) was suppressed. The pea pod cross sections were also visualized by light stereomicroscopy (Fig. 6E) where the box shows the region observed in Figs. 6A– 6D.

CHARACTERIZATION OF PEA DEFENSINS

DISCUSSION

We have purified two peptides, namely Psd1 and Psd2, by monitoring chromatographic separation of pea seed proteins using antifungal activity. This purification procedure described here yields an amount of Psd1 and Psd2 corresponding to approximately 0.5% of the total protein content of the seed, comparable to the content of different plant defensins described in the literature (8). The two peptides are found together at a reasonable purity level in fraction FPII (Fig. 2). They are 45% identical to each other (Fig. 4) and are very effective as fungal growth inhibitors (Table I). In four of eight microorganisms tested (A. niger, A. vesicolor, F. solani, and N. crassa), the peptides showed antifungal potency with IC 50 varying from 0.04 to 12.5 ␮g/ml (Table I). A similar range of antifungal activity (IC 50 varying generally from 0.5 to 25 ␮g/ml) has been ascribed to other defensins against several fungi cultivated in potato dextrose broth (7, 11). The high homology of Psd1 with DRR protein 39, the putative mature product of the pea disease resistance response gene DRR 230-b (Fig. 4), suggests that it may be derived from the same or a very similar gene. The C-terminus of DRR protein 39 corresponds to the plant defensin domain itself and the N-terminus carries a putative signal peptide domain. Chiang and Hadwiger (2) have shown that the mRNA corresponding to DRR 230-b increases markedly upon exposure of pea tissues to the phytopathogenic fungus F. solani, which suggests its participation in the plant disease resistance response. The second peptide, Psd2 (P3 from the HPLC run), shares homology (Fig. 4) with pSAS10, the V. unguiculata cDNA from stored cotyledon poly(A) ⫹ RNA briefly described by Ishibashi et al. in 1990 (14). The pSAS10 mRNA was induced just before germination of cowpea seeds and its level began to decline when seeds germinated. Ishibashi and coworkers have proposed that pSAS10 mRNA directs the synthesis of a protein that is required for germination, but its functional role remains unclear. The antifungal activities of the two peptides described here exhibited a calcium modulator effect (Table I). Their cation sensitivity varied greatly with the test fungus used. For example, addition of 1 mM Ca 2⫹ to the growth medium reduced the activity of Psd1 against F. solani, but stimulated its activity against A. niger. The cation-dependent decrease of Psd2 antifungal activity was also achieved for Na ⫹, K ⫹, and Mg 2⫹ (Fig. 5). In A. niger, the inhibition of antifungal activity of Psd2 by divalent cations (EC 50 of 2.0 mM and 2.3 mM for Ca 2⫹ and Mg 2⫹, respectively) required lower concentration than the inhibition by monovalent cations (EC 50 of 15 mM and 21 mM for K ⫹ and Na ⫹, respectively) (Fig. 5). However, the antifungal activity of Psd2 could

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be entirely suppressed by all cations at physiologically compatible concentrations (for example, in apoplasts, where [K ⫹] ⫽ 2–100 mM) (7). From these observations, it seems likely that the antagonistic effect of cations is the result of a weakening of the electrostatic interactions between the positively charged Psds (data obtained from amino acid sequence analysis and from ion-exchange chromatography—not shown) and their putative membrane receptors. The reduction of antimicrobial activity in the presence of divalent cations, especially calcium, has been reported for several plant defensins (7, 11, and 23) and also for insect (28) and mammalian defensins (29). An understanding of this phenomenon may reveal how plant defensins contribute to survival of the organism during different phases of its life cycle, since the tissues where the defensins occur are subject to a range of ionic environments (7). In this work we have shown that both Psds are localized predominantly in vascular bundles and epidermal tissues of pea pods (Fig. 6). Together with their antifungal properties, the immunolocalization findings are consistent with the participation of these peptides in the defense machinery of the pea, since these tissues are the first barriers to pathogen invasion (epidermal tissues), and dissemination (vascular tissues). According to the sequence homologies presented in Fig. 4, the Psds can be included in a large family of small, basic, Cys-rich polypeptides that includes plant defensins, neurotoxins from scorpion venoms, and insect defensin A. The fact that the primary sequences of these proteins are conserved throughout the animal and plant kingdoms points to a similar three-dimensional organization and a similar defense function. The high antifungal activity (Table I) described in this paper plus the homology with several plant defensins (Fig. 4) argues for the inclusion of Psd1 and Psd2 in this family. As shown in Fig. 4, several plant defensins have been classified previously by taking into account the organisms whose growth they inhibit and the presence of highly conserved motifs in their amino acid sequences (9 –12). In spite of the homology that is evident from our sequence alignment (Fig. 4), Psds show several structural features that are distinct from the plant defensins of Groups I, II, or III. These include the presence of an Ala residue instead of a Ser residue at position 9 and the lack of a Glu residue at position 31 (in Psd1). Terras et al. (30) analyzed the antifungal activities of wild-type and site-directed mutants of RsAFP2 and identified five amino acid residues (Thr11, Ala34, Tyr43, Phe45, and Pro46) that are very important for this biological function. Only Thr11 is present in the peptides described here.

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In conclusion, the two new peptides described in this work show reasonable similarity in anti-fungal activity and primary sequence with those reported for other defensins, but they exhibit enough disparity in amino acid sequence to suggest that they belong in a different defensin subgroup. ACKNOWLEDGMENTS We thank Mr. Denis L. S. Dutra for amino acid sequence analysis. Rui M. Domingues, Rosangela Rosa, and Ana Lucia O. Carvalho provided valuable technical assistance. We thank Dr. Martha M. Sorenson and Dr. Ana Paula Valente for critical reading of the manuscript and Mrs. Marilia Martins Nishikawa and Maria Helena Simo˜es Villas Boas for advice on growth and maintenance of fungi. We also thank Evander de Jesus Oliveira Batista for his help with microscopy. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq; PADCT/ CNPq), Financiadora de Estudos e Projetos (PRONEXII/FINEP), Fundac¸a˜o Jose´ Bonifa´cio (FUJB) and Fundac¸a˜o de Amparo a Pesquisa do Rio de Janeiro (FAPERJ).

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