Analysis of Two Novel Classes of Plant Antifungal … · 15302 Radish Seed Antifungal Proteins by this peak is henceforward referred to as Rs-AFP1 (Ra- phunus satiulls antifungal

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267 , No. 22, Issue of August 5, pp. 15301-15309, 1992 Printed in U. S. A.

Analysis of Two Novel Classes of Plant Antifungal Proteins from Radish (Raphanus sativus L.) Seeds*

(Received for publication, December 13, 1991)

Franky R. G. Terra&#, Hilde M. E. Schoofs$, Miguel F. C. De Bolle$, Fred Van Leuvennll, Sarah B. Rees**, Jozef VanderleydenS, Bruno P. A. Cammue$ $$, and Willem F. BroekaertSQQ From the SF. A. Janssens Laboratory of Genetics, Catholic University of Leuven, Willem De C r o y h n 42, B-3001 Heverlee, Belgium, the llCenter of Human Genetics, Catholic University of Leuven, Herestraut 49, B-3000 Leuven, Belgium, and **ICI Agrochemicals, Jealott’s Hill Research Station, Bracknell, Berks RG12 6EY, United Kingdom

Two novel classes of antifungal proteins were isolated from radish seeds.

The first class consists of two homologous proteins (Rs-AFP1 and Rs-AFP2) that were purified to homogeneity. They are highly basic oligomeric proteins composed of small (6-kDa) polypeptides that are rich in cysteine. Both Rs-AFPs have a broad antifungal spectrum and are among the most potent antifungal proteins hitherto characterized. In comparison with many other plant antifungal proteins, the activity of the Rs-AFPs is less sensitive to the presence of cations. Moreover, their antibiotic activity shows a high degree of specificity to filamentous fungi. The amino-terminal regions of the Re-AFPs show homology with the derived amino acid sequences of two pea genes specifi- cally induced upon fungal attack, to y-thionins and to sorghum a-amylase inhibitors.

The radish 2 s storage albumins were identified as the second novel class of antifungal proteins. All isoforms inhibit growth of different plant pathogenic fungi and some bacteria. However, their antimicrobial activities are strongly antagonized by cations.

Plant seeds are usually sown on a natural substrate that is rich in microorganisms. The low water content of the seed and the hard seed coat provide effective physical barriers against bacterial and fungal invasion. During the imbibition phase preceding seed germination, however, these barriers are gradually disrupted and protection then mainly relies upon antimicrobial compounds including proteins. Many different proteins with antifungal and/or antibacterial activity have already been detected in seeds. These are: chitinases (I), @-

*This work was supported in part by the ECLAIR Programme (AGRE-0005) of the Commission of the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received a predoctoral fellowship from the Belgian “Instituut ter Aanmoediging van het Wetenschappelijk Onderzoek in de Nijverheid en de Landbouw.”

(1 Supported by the Belgian “Nationaal Fonds voor Wetenschap- pelijk Onderzoek-Levenslijn” Action.

$3 Received a postdoctoral fellowship from the Belgian “Instituut ter Aanmoediging van het Wetenschappelijk Onderzoek in de Nijver- heid en de Landbouw.”

$8 Research Associate of the Belgian “Nationaal Fonds voor We- tenschappelijk Onderzoek.” To whom correspondence should be ad- dressed F.A. Janssens Laboratory of Genetics, Catholic University of Leuven, Willem De Croylaan 42, B-3001 Heverlee, Belgium. Tel.: 32-16-28-66-11 (ext. 2403); Fax: 32-16-22-07-61.

1,3-glucanases (2), thionins (3, 4), permatins (5, 6), and ribo- some-inactivating proteins (1, 7). Recently, we have also characterized antimicrobial chitin-binding lectin-like peptides from amaranth seeds (Ac-AMPs,’ Ref. 8) and a new class of insect neurotoxin-like antimicrobial peptides from Mirabilis jalapa seeds (Mj-AMPs, Ref. 9). Interestingly, members of at least the first four mentioned classes are induced in vegetative parts of plants upon challenge by fungi, bacteria, or viruses (3, 10-14).

In this paper, we describe the purification and characterization of two new classes of antifungal proteins from radish seeds. Proteins from the first class have a potent antifungal activity and show sequence homology to recently characterized pea pod proteins that are induced upon fungal attack (15). Homologous proteins are also present in seeds of mon- ocotyledonous plants. To our surprise, we found that the well characterized 2s seed storage albumins (18, 19) also exert antifungal activity and classified them as the second new class of radish seed antifungal proteins.

EXPERIMENTAL PROCEDURES*

RESULTS

Purification of Radish Seed Antifungal Proteins-After initial purification steps consisting of ammonium sulfate frac- tionation, heat treatment, and anion-exchange chromatography, the basic protein fraction from radish seeds was loaded on a cation-exchange column at pH 6 and eluted by applying a linear gradient of sodium chloride. The unbound fraction (not shown) was devoid of any substantial antifungal activity, whereas all desorbed material inhibited growth of the test fungus Fusarium culmorum grown in half-strength potato dextrose broth without additional salts (Fig. 1). However, only the early eluting peaks 1 and 2 (see Fig. 1) still exerted antifungal activity when assayed in the same medium supplemented with 1 mM CaCl, and 50 mM KCl.

In a subsequent purification step, material of peaks 1 and 2 was applied on a reversed-phase chromatography (RPC) column. Fig. 2A shows that the first cation-exchange peak elutes as a single symmetrical peak upon RPC, which co- elutes with the antifungal activity. The active factor contained

’ The abbreviations used are: Ac-AMP, Amaranthus caudatus antimicrobial peptide; Mj-AMP, Mirabilis jalapa antimicrobial peptide; RPC, reversed-phase chromatography; Rs, Raphanus sativus; Rs- AFP, Raphanus sativus antifungal protein; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

* Portions of this paper (including “Experimental Procedures” and Figs. 1-3, 9, and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

15301

15302 Radish Seed Antifungal Proteins

by this peak is henceforward referred to as Rs-AFP1 (Ra- phunus satiulls antifungal protein 1). RPC of the second peak yielded two well resolved symmetrical peaks of which the first (designated Rs-AFP2) exerts the most pronounced antifungal activity (Fig. 2B). The second peak was only weakly active in inhibiting fungal growth and was therefore not further characterized. Striking observations are that Rs-AFP1 and Rs- AFP2 both elute a t approximately 30% acetonitrile (in 0.1% trifluoroacetic acid) and that the antifungal activities of both components are relatively insensitive to the presence of salts in the fungal growth medium.

All attempts to obtain homogeneous preparations from each one of the five major cation-exchange peaks (indicated as 2S1-2S5 in Fig. 1, see below for the justification of these designations) by any chromatographic method failed. How- ever, all five fractions gave a similar RPC profile, with material eluting in the interval of 32-40% acetonitrile (in 0.1% trifluoroacetic acid; Fig. 3), suggesting that we were dealing with isoforms. Further evidence for this assumption was pro- vided by gel filtration; a mixture of equal amounts of the five 2 s fractions yielded one single symmetrical peak which migrated at an apparent molecular mass of 14.8 kDa (results not shown). Furthermore, and in sharp contrast to Rs-AFP1 and Rs-AFP2, the antifungal activity exerted by the 2s proteins was completely abolished when salts (1 mM CaCl, and 50 mM KC1) were added to the fungal growth medium.

In conclusion, on the basis of the presented chromatographic data, two separate groups of radish seed antifungal proteins can be distinguished; the first one consisting of the relatively salt-insensitive proteins Rs-AFP1 and Rs-AFP2, and the second one comprising the salt-sensitive 2s proteins. Approximate yields of the purification procedure were 40 mg of Rs-AFP1, 30 mg of Rs-AFP2, and 3 g of the pooled 2s fractionslkg of seeds. Molecular Characterization of Rs-AFPl and Rs-AFP2-

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of Rs-AFP1 and Rs-AFP2 is shown in Fig. 4. The unreduced Rs-AFP1 (lane 1 ) yielded a single band with an apparent molecular mass of 20 kDa, whereas the unreduced Rs-AFP2 (lane 3) yielded a major band of 15 kDa and a minor band of 20 kDa. The reduced and S-pyridylethylated derivatives of both Rs-AFPs (lanes 2 and 4 ) migrated as single bands with a molecular mass of approximately 5 kDa. However, when both Rs-AFPs were reduced without further derivati- zation, the 5-kDa polypeptide is always accompanied by a 15- kDa band (results not shown). Rs-AFP1 and Rs-AFP2 appear to be oligomeric proteins built up of 5-kDa protomers. Intact disulfide bridges seem to be necessary for stabilization of the oligomeric structure. The 15-kDa band may represent a tri- mer, whereas the 20-kDa band may be a tetrameric form.

kDa R 1 2 3 4 R

17 - 14.4 ;

8, 6,

2.5

FIG. 4. SDS-PAGE analysis of the purified Rs-AFPs. Unre- duced proteins were dissolved at 200 pg/ml in sample buffer without dithioerythritol (DTE), and S-pyridylethylated derivatives were dissolved at 200 pg/ml in sample buffer with DTE. Separation of samples (200 ng) was performed on Phastgel High Density (Pharmacia LKB Biotechnology Inc.) and silver-stained in the gel after fixing with 12.5% glutaraldehyde. Lane R, myoglobin fragments with molecular masses indicated in kilodaltons (kDa) at the left; lane 1, unreduced Rs-AFP1; lane 2, S-pyridylethylated Rs-AFP1; lane 3, unreduced Rs- AFP2; lane 4, S-pyridylethylated Rs-AFP2.

Molecular weight estimation of small unreduced proteins containing disulfide bridges should be interpreted with care since such proteins do not bind optimal amounts of SDS (8, 18). Native gel electrophoresis of Rs-AFP1 and Rs-AFP2 yielded two single protein bands (Fig. 5) with Rs-AFP2 mi- grating faster toward the cathode than Rs-AFP1. Antifungal activity could be attributed to the pure proteins after covering the gel with an agar layer containing fungal spores (Fig. 5). Both Rs-AFPs have isoelectric points that are higher than

.10.5, as was determined by isoelectric focusing (results not ‘shown).

In contrast to the reduced forms, no positive reaction occurred when assaying the native proteins for free thiols, indicating that all cysteine residues are involved in the for- mation of disulfide bridges.

The reduced proteins were inactive in the antifungal activity assay. Heating of the two Rs-AFPs a t 100 “C for 10 min did not affect their antifungal properties, whereas treatment with proteases (trypsin, chymotrypsin, Pronase E, or proteinase K) completely abolished antifungal activities.

Initial attempts to obtain NH2-terminal amino acid sequences of Rs-AFP1 and Rs-AFP2 by Edman degradation failed, probably due to a blocked NH2-terminal residue. After treatment of the S-pyridylethylated proteins with the enzyme pyroglutamate aminopeptidase, the first 43 and 35 NH2- terminal amino acids of Rs-AFP1 and Rs-AFP2, respectively, could be determined (Fig. 6). Rs-AFP1 and Rs-AFP2 appear to be highly homologous proteins, since only two differences occur within the first 35 residues.

Identification of 2 s Storage Albumins as Antifungal Pro- tei--Proteins of all five 2 s peaks (see Fig. 1) were subjected to SDS-PAGE. In the unreduced state (Fig. 7A), each fraction represents a mixture of two molecular mass forms of approximately 17 and 18 kDa. However, a clear shift can be seen in the relative amounts of both forms; fractions 2S1-2S4 contain relatively more of the smallest form, whereas fraction 2S5 is mainly composed of the largest form. After reduction, the five 2 s fractions each yielded two bands of approximately 10 and 4 kDa, respectively (Fig. 7B). To overcome problems with poor fixation of the reduced polypeptides, staining was done after diffusion blotting onto nitrocellulose. From this SDS- PAGE analysis, it appears that the 2s fractions represent heterodimeric proteins composed of a small (4-kDa) and a large (10-kDa) subunit linked together by disulfide bonds. By thiol dosage, no free cysteines were detected in the native 2 s

Protein Activity

FIG. 5. Detection of antifungal activity of the Re-AFPs after native cathodic gel electrophoresis. Rs-AFP1 (lanes 1) and Rs-AFP2 (lanes 2 ) were loaded at 5 pg/lane and separated on a 10% polyacrylamide gel a t pH 7. A diffusion blot was prepared and developed by silver staining to localize protein bands (“Protein” panel). The gel was covered with an agar layer containing spores of T. hamatum to detect growth inhibition zones (“Actiuity”pane1).

Radish Seed Antifungal Proteins 15303 1 5 10 15 20 25 30 35 40

Rs-AFPl ( Q ) K L C E R P S G T U S G V C G N N N A C K N Q C I N L E K A R H G S C N V V F P A H K RSdFP2 ( Q ) K L C Q R P S G T W S G V C G N N N A C K N Q C I R L E K A R H G S C

4 4 FIG. 6. Amino-terminal amino acid sequences of Rs-AFP1 and Rs-AFP2. NH2-terminal sequences of both Rs-AFPs were deter-

mined after treatment with Dvroelutamate aminoDeDtidase. The first residue (between brackets) is suggested to be a cyclisized glutamine. Differences between Rs-AFPiand Rs-AFP2 are inbicated by arrows.

A B

1 2 3 4 5 R m.-- kDa m-

a: 8 :

R l 2 3 4 5

..88-19= l7 - , 6

14.4 - " 2.5

FIG. 7. SDS-PAGE analysis of the antifungal 2 s albumin fractions. A, fifty ng of the different 2 s fractions dissolved in sample buffer without DTE were separated on Phastgel High Density (Phar- macia) and silver-stained in the gel after fixing with ethanol/acetic acid/water (30:1060). Lane R, myoglobin fragments as molecular mass markers (indicated in kilodaltons (kDa) at the right); lanes 1-5, 2S1-2S5. B, fifty ng of the different 2 s fractions dissolved in sample buffer with DTE were separated on Phastgel High Density, diffusion- blotted, and detected by silver staining the blot. Lane R, myoglobin fragments; lanes 1-5, 2S1-2S5.

fractions, indicating that all cysteines participate in the for- mation of disulfide bridges.

Heating of the 2s fractions a t 100 "C for 10 min did not affect their antifungal activity. Upon treatment with trypsin, chymotrypsin, proteinase K, or Pronase E, no residual fungal growth inhibition activities could be observed.

As judged by reversed-phase chromatography (Fig. 3) and SDS-PAGE (Fig. 7), the 2S5 fraction seemed to be the least heterogeneous and was therefore chosen to determine the NH2-terminal amino acid sequences of both subunits. To this purpose, both subunits were first separated by reversed-phase chromatography after reduction and S-carboxyamidomethy- lation (Fig. 8). SDS-PAGE analysis of the different peak fractions indicated that the first group of peaks represent the 4-kDa subunits, whereas the second group of peaks correspond to the 10-kDa subunits. Sequence determination was performed on samples from the first peak (small subunit) and the third peak (large subunit) of the RPC profile of S- carboxyamidomethylated 2%. As could already be expected on the basis of their subunit structure, their high abundance and the existence of multiple isoforms, sequence homology was found with the well characterized 2 s storage albumins. In Fig. 9, the determined partial sequences are compared with those derived from the radish 2s albumin cDNA clone pBA3 (21) and to the determined rapeseed napin sequence (22). The obtained partial sequence of the small subunit of Rs-2S5 is 100% identical to the pBA3-derived sequence and 87% identical to the napin sequence. The large subunit shows 90% identity with the corresponding pBA3 cDNA-derived and the napin sequences.

Antifungal Properties of Rs-AFPl, Rs-AFP2, and Radish 2s Albumins-In order to determine to what extent radish seed antifungal proteins are capable of inhibiting growth of different fungi, dose-response curves were measured for 20 different plant pathogenic fungi. From these curves, protein concentrations required for 50% inhibition of fungal growth (ICso) were derived. This was done in parallel using both a low ionic strength synthetic growth medium and the same medium supplemented with 1 mM CaC12 and 50 mM KC1. The results of these tests are summarized in Table I.

I I . . . . . I I

0 10 20 30 40 50 66 70 00 90 ELUTION TIME (mlnuter)

FIG. 8. Separation of small and large subunits of the 255 fraction. Two hundred pg of the S-carboxyamidomethylated 2S5 fraction was loaded on a P e p 4 reversed-phase chromatography column (C&!le 5-pm porous silica, 25 X 0.4 cm; Pharmacia) in equilib- rium with 0.1% trifluoroacetic acid. The column was eluted at 1 ml/ min with the following gradient (solvent B is acetonitrile containing 0.1% trifluoroacetic acid): 0-2 min, 0% solvent B; 2-92 min, 0-45% solvent B. The eluent was monitored for proteins by absorbance measurement a t 214 nm. Peak fractions were collected manually, vacuum-dried, and redissolved in 50 pl of Milli-Q water. Inset, SDS- PAGE analysis of the separated subunits. Three p1 of each peak fraction was mixed with 1 pl of 4-fold concentrated sample buffer without dithioerythritol and separated on a Phastgel High Density (Pharmacia). Detection of the subunits was done by silver staining of the diffusion blot. Lanes R, myoglobin fragments as molecular mass markers. Lanes 1-5 correspond to peaks 1-5.

Rs-AFP2 seems to be the most potent antifungal protein with ICso values ranging from 0.4 to 25 pg/ml. Generally, Rs- AFPZ is 2-30-fold more active than Rs-AFP1 (ICso values from 0.3 to 100 pg/ml). Exceptions are Rhizoctonia solani and Sclerotinia sclerotwrum on which Rs-AFP2 does not have any appreciable effect a t 100 pg/ml. The higher potency of Rs- AFPZ relative to Rs-AFP1 is even more pronounced in the medium with added salts. In this medium, 12 out of 20 fungi and only 5 out of 19 fungi are still inhibited by Rs-AFP2 and Rs-AFP1, respectively, at concentrations below 100 pg/ml. Consistently higher ICso values are obtained in the medium with added salts (from 20 to 100 pg/ml for Rs-AFP1 and from 3 to 50 pg/ml for Rs-AFP2). The high salt sensitivity of the 2 s albumins is confirmed by this test; none of the fungi is affected by the 2s albumins at up to 1000 pg/ml in the medium with added salts, whereas ICso values vary from 3.3 to 200 pg/ ml in the medium without added salts.

In a second test, only performed on two fungi (F. culmorum and Trichoderma hamatum), different concentrations of var- ious divalent and monovalent metal ions were added to the synthetic low ionic strength growth medium. Again, the ICs0 values were determined and are given in Table 11. The IC50 values obtained for a @-thionin from wheat and the M. jalapa antimicrobial peptide 2 (Mj-AMP2, Ref. 9) are also included for comparative purposes. The antifungal activity of the Rs- AFPs and thionin is not affected by addition of KC1 at up to

15304 Radish Seed Antifungal Proteins TABLE I

Antifungal activity of the radish seed antifungal proteins Protein concentrations required for 50% growth inhibition (ICso) after 48 h of incubation were determined from the dose-response curves

(percent growth inhibition versus protein concentration). Growth of the slowly growing fungi Septoria nodorum and Venturia inaequalis was measured after 5 and 15 days of incubation, respectively.

ICso values

Fungus Medium A" Medium Bb Rs-AFP1 Rs-AFP2 Rs-2s Rs-AFP1 Rs-AFP2 R s - ~ S

Pdml Irglml Alternaria brassicola 15 2 10 >loo 20 >loo0 Ascochyta pisi 5 4 75 >loo 50 >loo0 Botrytis cinerea 8 2 >500 >loo >loo >lo00 Cercospora beticola 2 2 N D 100 3 ND Colletotrichum lindemuthianum 100 3 15 >loo >loo >loo0 Fusarium culmorum 5 2 35 70 5 >loo0 Fusarium oxysporum f.sp. lycopersici 30 2 >500 >loo >lo0 >loo0 Fusarium oxysporum f.sp. pisi 15 2 200 >loo >loo >loo0 Mycosphaerella fijiensis var. fijiensis 4 1.5 150 30 10 >500 Nectria haematococca 6 2 33 >loo 30 >loo0 Phoma betae 2 1 500 20 6 >loo0 Phytophthora infestans 3 25 60 >loo >loo >500 Pyrenophora tritici-repentis 3 1.5 ND 30 7 ND Pyricularia oryzae 0.3 0.4 10 >loo 7 >loo0 Rhizoctonia solani 100 >loo ND >loo >loo ND Sclerotinia sclerotiorum 20 >loo ND >loo >loo ND Septoria nodorum 20 15 ND 100 20 ND Trichoderma hamatum 6 2 30 20 4 >loo0 Verticillium dahliae 5 1.5 3.3 >loo 50 >loo0 Venturia inaequalis ND 25 ND ND >50 ND

a Medium A Synthetic low ionic strength growth medium. * Medium B: medium A supplemented with 1 mM CaC12 and 50 mM KC1. ND, not determined.

TABLE I1 Variation of antifurwal activitv in the Dresence of Ca2+ or Kt

ICw values

Reference medium supplemented with

10 mM KC1 50 mM KC1 1 mM CaC12 5 mM CaC12

Fungus Antifungal Reference protein medium"

Fusarium culmorum Rs-AFP1 5 Rs-AFP2 2 Rs-2S5 35 0-Purothionin 9 Mj-AMP2 3

Trichoderma hamatum Rs-AFP1 6 Rs-AFP2 2 Rs-2S5 30 8-Purothionin 4 Mj-AMP2 2

5 2 40 9 4 6 2

30 3 2

rcglml 6 2

>400 4

12

6 3

>4o0 1.5

25

11 2

>400 9

11

35 2

>400 4

25

>loo 5

>400 90

>loo >loo >loo >400

30 >loo

a Reference medium: synthetic low ionic strength growth medium.

50 mM, whereas the activity of Mj-AMP2 is drastically reduced. The antifungal activity of the 2s albumins is completely abolished in the presence of 50 mM KCl. One milli- molar of CaC12 has no effect on Rs-AFP2 and thionin, whereas it reduces the activity of Rs-AFP1 and Mj-AMP2 and abol- ishes that of the 2s albumins. CaC12 concentrations of 5 mM are necessary to cause activity reduction of Rs-AFP2 and thionin. Tests with other salts including NaC1, NH,Cl, K2S0,, CaSO,, MgC12, and BaC12 indicated that salts of monovalent cations had effects comparable with that of KC1 and that salts of divalent cations had similar effects as those of CaCh (results not shown).

During the course of this work, fungal growth inhibition was routinely checked microscopically to confirm the micros- pectrophotometric data. A striking difference in the morphology of inhibited hyphae was apparent between fungi treated with Rs-AFPs and those treated with 2s albumins. This is

illustrated by microphotographs in Fig. 10. The 2s albumins and thionins caused severely delayed growth of hyphae with otherwise normal morphology. However, in the presence of thionins, large parts of the hyphae were stained by methylene blue, indicating loss of viability. This lethal effect was not seen, either with the 2s albumins or with the Rs-AFPs. Microcultures to which Rs-AFPs were added revealed a completely different type of growth inhibition, featuring characteristic claws of branched swollen hyphae. However, at high concentrations of any of the antifungal proteins, no spore germination occurs at all (Fig. 10).

The nonlethal effect of the Rs-AFPs and the 2 s albumins was also confirmed in an experiment where these proteins were added to duplicate cultures of F. culmorum at 10 pg/ml (Rs-AFPl), 5 pg/ml (Rs-AFP~), or 100 pg/ml (2s albumin). After 48 h of incubation, all cultures were inhibited by more than 90% relative to controls. After replacement of the me-

Radish Seed Antifungal Proteins 15305

I

r,

I

;; //

V

FIG. 10. Differences in morphology of inhibited hyphae. Photomicrographs were taken after 24 h of incubation of a Pyricularia oryzae spore suspension (in half-strength potato dextrose broth) in the presence of water (control) ( A ) , 25 pg/ml 8-purothionin ( B ) , 200 pg/ml 2S5 (C), 3 pg/ml Rs-AFP1 (D), 50 pg/ml 0-purothionin ( E ) , 500 pg/ml 2S5 (F), and 50 pg/ml Rs-AFP1 ( G ) . Ten minutes before taking photomicrographs, 0.01% methylene blue was added to the microcultures to raise contrast. The bur corresponds to approximately 25 pm.

dium (containing the proteins) by fresh half-strength potato dextrose broth, growth of mycelium resumed, whereas the duplicate cultures (still containing the proteins) remained inhibited. Hence, the antifungal effect of the Rs-AFPs and the 2s albumins must be qualified as fungistatic rather than fungicidal.

Two-by-two combinations of different subinhibitory concentrations of pea chitinase, pea @-1,3-glucanase, Urtica dioica agglutinin, a- and P-purothionin, Mj-AMP2, the Rs-AFPs, the radish 2 s storage albumins, and the nonproteinaceous chitin synthase-inhibiting compound nikkomycin Z were tested against Botrytis cinerea and Colletotrichum lindemuthianum to discover possible synergistic effects between any of the cited antifungal agents. Synergism was only observed for the combination of 2s albumins with thionins. A more detailed analysis of the synergism between the 2s albumins and thionins will be presented in a separate paper.3

Effects on Bacteria, Yeast, Cultured Human Cells, and Erythrocytes-To determine whether or not the radish seed antifungal proteins exert biological activities other than fungal growth inhibition, the effect of these proteins on other cell types was examined.

Of eight bacterial species tested, only growth of the Gram- positive Bacillus megaterium and the Gram-negative Erwinia carotovoru was suppressed by the radish 2 s albumins (ICso values of 10 and 250 pg/ml, respectively). However, growth of these two bacteria was unaffected when 1 mM CaC1, and 50 mM KC1 were supplemented to the growth medium. Further- more, Rs-AFP2 (and not Rs-AFP1) exerted antibacterial activity against B. megaterium, although only a t fairly high concentrations. IC,,, values of 200 and 500 pg/ml were obtained in the media without and with added salts (1 mM CaC1, and 50 mM KCl), respectively.

' F. R. G. Terras, R. W. Osborn, J. Vanderleyden, B. P. A. Cammue, and W. F. Broekaert, manuscript in preparation.

Yeast (Saccharomyces cerevisiae) cells were not affected in their growth by either the Rs-AFPs or the 2s albumins at up to 500 pg/ml. In contrast, Mj-AMP2, Ac-AMP2, and B-purothionin had ICso values of 20, 18, and 70 pg/ml, respectively. Neither human umbilical vein endothelial cells nor human skin-muscle fibroblasts showed a decreased viability when incubated in the presence of any of the isolated radish proteins at up to 500 pg/ml, whereas 6-purothionin has an ICso value of about 25 pg/ml on both cell types. Finally, no hemolysis occurred when any of the purified radish proteins, Mj-AMP2 or Ac-AMP2, was added to human erythrocytes at up to 500 pg/ml. Fifty percent of the erythrocytes were lysed by the p- purothionin at 5 pg/ml.

DISCUSSION

By monitoring chromatographic separations of radish seed proteins using an assay for growth inhibition of F. culmorum, we purified two novel classes of broad spectrum antifungal proteins which have clearly distinct biochemical and biological properties.

Two members of the first novel class of antifungal proteins were purified to homogeneity and designated as Rs-AFP1 and Rs-AFP2. Both proteins comprise highly basic (isoelectric points higher than 10.5) 5-kDa polypeptides that are assem- bled in an oligomeric quaternary configuration. Disulfide bridges stabilize this configuration. Forty-three and 35 amino acids were obtained by amino-terminal sequencing of Rs- AFPl and Rs-AFP2, respectively, after treatment of these proteins with pyroglutamate aminopeptidase. The molecular mass of the Rs-AFP1 peptide calculated on the basis of the partial amino acid sequence (4,993 Da) is very close to the value estimated by SDS-PAGE (about 5,000 Da), which in- dicates that the proposed sequence encompasses the major part of the polypeptide. However, it is anticipated that Rs- AFPl contains at least one more cysteine residue (in addition to the 5 cysteines determined by Edman degradation), since the absence of free thiol groups requires an even number of cysteines. Thus, Rs-AFP1 should have a t least three disulfide bridges. The primary structures of Rs-AFP1 and Rs-AFP2 only differ at two positions within the first 36 residues; the glutamate at position 5 in Rs-AFP1 is a glutamine in Rs- AFP2, and the asparagine a t position 27 in Rs-AFP1 is substituted by an arginine in Rs-AFP2. Both changes result in a higher net positive charge of Rs-AFP2 in comparison with Rs-AFP1. This is consistent with their differential be- havior upon cation-exchange chromatography and cathodic gel electrophoresis.

The more basic nature of Rs-AFP2 may also explain its higher specific activity on fungi and its lower sensitivity to cations, both relative to Rs-AFP1. Generally, divalent cations antagonize the antifungal activity of the Rs-AFPs more strongly than do monovalent cations. The cation sensitivity of the antifungal activity of the Rs-AFPs seems to vary greatly with the test fungus used. For instance, addition of 5 mM CaCl, to the growth medium caused only a 1.7-fold reduction of the activity of Rs-AFP2 against F. culmorum but a more than 50-fold reduction of activity against T. hamatum (Table 11). From these observations, it seems likely that the antago- nistic effect of cations is not the result of a hypothetical conformational change of the protein by direct interaction with the cations. Rather, an interaction occurs between the fungus and the cations, whereby the fungus acquires protection against the action of the protein. The highly branched morphology of the hyphae treated with Rs-AFPs suggests that these proteins may interfere with morphogenetic Ca'+ signal- ing. Recent evidence shows that branching of fungal hyphae

15306 Radish Seed Antifungal Proteins

is regulated by specific Ca2+ channels (23). This hypothesis may also explain the lack of inhibitory activity of the Rs- AFPs on yeast cells.

The cation sensitivity of Rs-AFP2 is comparable with that of the B-purothionin and substantially lower than that of Mj- AMP2 (Table 11). In contrast to Rs-AFPs, the Ac-AMPs and Mj-AMPs are not inhibitory to most fungi listed in Table I at concentrations below 100 pg/ml when assayed in a growth medium containing 1 mM CaC12 and 50 mM KCl.4 Exceptions are C. beticola, which is inhibited by the Ac-AMPs, and C. lindemuthiunum, which is inhibited by the Mj-AMPs.

Zeamatin, a permatin from maize seeds (5), has also been reported to be particularly salt-sensitive; its activity is decreased 40-fold upon addition of 100 mM NaCl to the growth medium (5). Even the antifungal activity of pea chitinase on T. hamatum seems to be severely repressed in the presence of cation^.^

The antifungal potency of a protein in the presence of cations is of particular importance for the evaluation of its possible contribution to defense reactions against microorganisms in planta, as well as for its possible use as a transferable resistance trait for molecular breeding of crop plants. Concen- trations of K+, the most abundant cellular and apoplastic cation, reach about 100 mM in the cytosol (24) and vary from 10 to 100 mM in vacuoles (25) and from 2 to 100 mM in the apoplast (26). The most abundant divalent cations in plant tissues are Ca2+ and Mg"'. In the cytosol, the free Ca2+ concentration is very low (between 0.1 and 1 p ~ ; Ref. 27), whereas free Mg2' reaches about 1 mM (28). Free Ca2+ concentrations in plant vacuoles are about 0.06-1 mM, and apoplastic free Ca2+ ranges between 0.02 and 1.3 mM (29). It appears thus that relatively high ionic strength conditions occur in all cellular compartments. However, in many cases fungal infection leads to the disruption of intact cells and contact of the cellular contents with the environment (e.g. the external imbibing soil water in the case of seeds). This makes it very difficult to predict the exact ionic conditions under which the antifungal proteins interact with the invading hyphae. Undoubtedly, these conditions will influence the activity of cation-sensitive antifungal proteins.

The Rs-AFPs show striking sequence homology with the cDNA-derived amino acid sequences of the pea genes pI39 and pI230 that are induced upon interaction with Fusarium solani (15), with the tuber- and stem-specific p322 gene of potato (30), with the insect a-amylase inhibitors from sorghum (17), and with a class of proteins called y-thionins (16, 31). The alignment of all sequences is shown in Fig. 11. Noteworthy are the invariant cysteine residues at positions 4, 15,21,25,37 (and 46,48,52) and a glycine residue at position 35. Other well conserved residues are found at positions 8 (serine), 13 (glycine), and 29 (glutamate). The occurrence of aromatic residues is fairly well conserved at positions 11 and 41. Highest identity (30%) with the partial Rs-AFP1 amino acid sequence is observed for the pea gene products and the yl-purothionin. If conserved amino acid substitutions are taken into account, relatively high homologies (from 35 to 54%) with the Rs-AFP1 sequence are found for all proteins.

Although the pea gene products were not characterized, it was assumed that they contribute to the general resistance of plants (15). A speculative role as proteinase inhibitor was suggested for the potato p322 gene product (30) and for the pea pI39 and pI230 gene products (15) due to their (weak) homology with the Bowman-Birk type trypsin-chymotrypsin inhibitor. Neither trypsin nor chymotrypsin inhibiting activities are, however, exerted by the Rs-AFPs, nor does the

W. F. Broekaert, unpublished results.

soybean Bowman-Birk inhibitor inhibit fungal growth." Consistent with the results obtained for the sorghum a-

amylase inhibitors (17), the Rs-AFPs do not inhibit the a- amylases from porcine pancreas or Bacillus specie^.^ The effect of the Rs-AFPs on insect a-amylases remains to be investigated.

The y-thionins (16, 31) are classified as putative members of the thionin family. Like the a- and @-thionins, these proteins inhibit protein synthesis in cell-free systems, although to a different quantitative extent. However, careful analysis of the amino acid sequence data makes this classifi- cation doubtful. Only 5 out of the 8 cysteine residues of the y-thionins can be aligned with those of the a- and 8-thionins (31), and several major gaps have to be introduced to obtain this alignment. Furthermore, the two successive cysteine residues (at positions 3 and 4) characteristic for the a- and @- thionins and the related viscotoxins and crambin are not found in the y-thionins (31). The similarity at the amino acid sequence level between the radish antifungal proteins, the fungus-induced proteins from pea, the potato p322 gene product, the y-type thionins from wheat and barley, and the a- amylase inhibitors from sorghum suggests that this class of proteins is widespread among plants and may fulfill a similar function. Based on the fungistatic properties of the Rs-AFPs and the responsiveness of the pI230 and pI39 genes to fungal attack, we assume that these proteins play a role in plant defense. Future investigations on the antibiotic effects of the proteins homologous to the Rs-AFPs are needed to point out if they can be classified as antifungal proteins too. Examina- tions of other Brassicaceae species indicated that Rs-AFP- like proteins exhibiting similar antifungal activities are generally present in seeds of this family?

Based on structural properties, the Rs-AFPs seem to belong to a superfamily of highly basic cysteine-rich small-sized proteins with antibiotic properties including thionins (32), defensins found in mammals (33) and insects (34), antimicrobial peptides from seeds of Amaranthus caudatus (8) and M. jalapa (9), and a secreted Aspergillus giganteus antifungal protein (35). However, compared with these proteins, the Rs- AFPs are unique in the sense that they do not seem to be active on organisms other than filamentous fungi.

It is generally accepted that the widely occurring 2 s seed storage albumins (19) serve as a source of nitrogen, and possibly sulfur, for the developing germling (36). The only biological activity hitherto known of these proteins is that they act as allergens toward hypersensitive individuals (37- 39). So, we describe here for the first time that the seed 2s storage albumins have antifungal activity.

The identification of the isolated antifungal proteins as 2s albumins is based on the following lines of evidence. 1) The isolated proteins exist as at least five different isoforms, which is consistent with the estimated size of the 2s albumin gene family in radish (at least five to six genes; Ref. 21); 2) the mass of the native proteins estimated by gel filtration (14.8 kDa) is in good agreement with the theoretical values calculated from cDNA-derived sequences (14.2 and 13.2 kDa for the largest and the smallest isoform, respectively; Ref. 21); 3) the occurrence of a small 4-kDa and a large 10-kDa subunit is characteristic for many 2 s albumins (18); and 4) the partial amino acid sequences of the large and the small subunit showed near identity to the cDNA-derived sequences of radish 2s albumins.

The radish 2 s albumins inhibit growth of a large spectrum

F. R. G. Terras, unpublished results. 6F. R. G. Terras, I. J. Goderis, F. Van Leuven, J. Vanderleyden,

B. P. A. Cammue, and W. F. Broekaert, manuscript in preparation.


of fungi in the IC,,) range from 3 to 200 pg/ml when assayed in the low ionic strength medium. However, the activity was completely abolished in the presence of 1 mM CaCh and 50 mM KC1. It is therefore doubtful that these proteins by themselves have a protective function in uiuo. However, the mentioned synergism between the 2s albumins and thionins is also observed in high ionic strength media.3

Acknowledgments-We are grateful to Drs. A. Ludwig and T. Boller for providing samples of pea chitinase and pea 8-1,3-glucanase. We thank Dr. L. Nelles and Drs. J. Van Damme and P. Proost for assistance with the cultures of the umbilical vein endothelial cells and the fibroblasts, respectively. We also thank J. Desair for skillfull maintenance of the chromatographic equipment and I. Goderis for outstanding technical assistance.

REFERENCES 1. Roberts, W. K., and Selitrennikoff, C. P. (1986) Biochim. Biophys. Acta

2. Manners, D. J., and Marshall, J. J. (1973) Phytochemistry (Orf . ) 12,547- 880,161-170

3. Bohlmann, H., Clausen, S., Behnke, S., Giese, H., Hiller, C., Reimann- 553

Philipp, U., Schrader, G., Barkholt, V., and Apel, K. (1988) EMBO J. 7, 15559-1 5G.5

4. Fernandez de Caleya, R., Gonzalez-Pascual, B., Garcia-Olmedo, F., and

5. R?+fis,-W_ K., and Selitrennikoff, C. P. (1990) J. Gen. Microbiol. 136 ,

""

Carbonero, P. (1972) Appl. Microbiol. 23,998-1000

6. Vigers, A. J., Roberts, W. K., and Selitrennikoff, C. P. (1991) Mol. Plant-

7. Leah. R.. Tommeruo. H.. Svendsen. I.. and Mundv, J. (1991) J. Biol. Chem.

1'1 11-1/'/8

Microbe interact. 4,315-323

8.

9.

10.

11.

12.

13.

14.

15.

2 6 6 , i564-1573 . ' '

. .

Broekaert, W. F., Marien, W., Terras, F. R. G., De Bolle, M. F. C., Proost, P., Van Damme, J., Dillen, L., Claeys, M., Rees, S. B., Vanderleyden, J., and Cammue, B. P. A. (1992) Biochemistry 31,4308-4314

Cammue, B. P. A., De Bolle, M. F. C., Terras, F. R. G., Proost, P., Van Damme, J., Rees, S. B., Vanderleyden, J., and Broekaert, W. F. (1992)

Hedrick, S. A,, Bel, J . N., Boller, T., and Lamb, C. J. (1988) Plant Physiol. J. Biol. Chem. 267 , 2228-2233

Joosten, M. H. A. J., and De Wit, P. J. G. M. (1989) Plant Physiol. 89, 86, 182-186

Ebrahim-Nesbat, F., Behnke, S., Kleinhofs, A., and Apel, K. (1989) Planta 945-951

Reimann-Philipp, U., Schrader, G., Martinoia, E., Barkholt, V., and Apel,

Woloshuk, C. P. J., Meulenhoff, J. S., Sela-Buurlage, M., Van den Elzen,

Chiang, C. C., and Hadwlger, L. A. (1991) Mol. Plant-Macrobe Interact. 4 ,

179 , 203-210

K. (1989) J. Biol. Chem. 264,8978-8984

P. J . M., and Cornelissen, B. J. C. (1991) Plant Cell 3,619-628

.13A-.1.11 " _ "_ 16. Colilla, F. J., Rocher, A., and Mendez, E. (1990) FEBS Lett. 270,191-194 17. Bloch, C., Jr., and Richardson, M. (1991) FEBS Lett. 279,101-104 18. Krebbers, E., Herdies, L., De Clercq, A,, Seurinck, J., Leemans, J., Van

Darnme, J., Segura, M., Gheysen, G., Van Montagu, M., and Vandek- erckhove, J. (1988) Plant Physiol. 87,859-866

19. Youle, R. J., and Huang, A. H. C. (1981) Am. J. Bot. 68,44-48 20. See, Y. P., and Jackowski, G. (1990) in Protein Structure: A Practical

21. Raynal, M., Depigny, D., Grellet, F., and Delseny, M. (1991) Gene (Amst.) Approach (Creighton, T. E., ed) pp. 1-22, IRL Press Ltd., Oxford

22. Ericson, M. L., %din, J., Lenman, M., Glimelius, K., Josefsson, L. G., and 9 9 , 7 7 4 6

23. Robson, G. D., Wiebe, M. G., and Trlncl, A. P. J. (1991) Erp. Mycol. 16,

24. Clarkson, D. T., and Hanson, J. B. (1980) Annu. Reu. Plant Physiol. 31 ,

25. Flowers, T. J., and Lauchli, A. (1983) in Encyclopedia of Plant Physiology, New Series (Lauchli, A,, Bieleski, R. L., eds) Vol. 15B, pp. 651-681, Springer-Verla Heidelberg, Federal Republic of Germany

26. Grignon, C., and kentenac, H. (1991) Annu. Reu. Plant Physiol. Plant Mol.

Rask, L. (1986) J. Biol. Chem. 261,14576-14581

263-272

239-298

27. Macklom, A. E. S. (1984) Plant Cell Enuiron. 7,407-413 28. Hepler, P. K., and Wayne, R. 0. (1982) Annu. Reu. Plant Physiol. 36,397-

Biol. 42,103-128

A2CI 29. HaiFer, F. R., and Venis, H. A. (1991) Plant CeU Enuiron. 14,525-530 30. Stiekema, W. J., Heidekamp, F., Dirkse, W. G., Van Beckurn, J., De Haan,

269 P., Ten Bosch, C., and Leuwerse, J. D. (1988) Plant Mol. Bud. 11, 255-

31. Mendez, E., Moreno, A., Colilla, F., Pelaez, F., Limas, G. G.,, Mendez, R., Soriano, F., Salinas, M, and de Haro, C. (1990) Eur. J. Bmhem. 194 , 533-539

32. Apel, K., and Bohlmann, M. (1991) Annu. Reu. Plant Physiol. Plant Mol.

33. Lehrer, R. I., Ganz, T., and Selsted, M. E. (1991) Cell 6 4 , 229-230 Biol. 42,227-240

34. Lambert, J., Keppi, E., Dimarcq, J.-L., Wicker, C., Reichhart, J. M., Dunbar, B., Lepage, P., Van Dorsselaer, A., Hoffmann, J., Fothergill, J.,

35. Nakaya, K., Omata, K., Okahashi, I., Nakamura, Y., Kolkenbrock, H., and and Hoffmann, D. (1989) Proc. Natl. Acad. Sei. U. S. A. 86,262-266

Ulbrich, N. (1990) Eur. J. Biochem. 193, 31:38

37. You%, R. J., and Huang, A. H. C. (1978) Plant Physrol. 6 1 , 1040-1042 36. Hig .ns, T J. V (1984) Annu. Reu. Plant Physrol. 3 6 , 191-221

38. Youle, R. J., and Huan , A H C. (1979) J. Agric. Food Chem. 27,500-503 39. Menendez-Arias, L., hfoneo, i., Dominguez, J., and Rodriguez, R. (1988)

40. Peumans, W. J., De Ley, M., and Broekaert, W. F. (1983) FEBS Lett. 177,

41. Redman, D. G., and Fisher, N. (1969) J. Sci. Food Agric. 20,427-432 42. Broekaert, W. F., Terras, F. R. G., Cammue, B. P. A., and Vanderleyden,

43. Alessi, M., Declerck, P. J., De Mol, M., Nelles, L., and Collen, D. (1988)

Eur. J. Bwchem. 177,159-166

99-103

J. (1990) FEMS Microbiol. Lett. 69, 55-60

44. Van Damme, J., Cayphas, S., Opdenakker, G., Billiau, A., and Van Snick, Eur. J. Biochem. 175,531-540

45. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. J. (1987) Eur. J. Zmrnunol. 17 , 1-7

H.. Provenzano. M. D.. Fuiitomo. E. K.. Goeke. N. M.. Olson. B. J.. and Klenk, D. C. (1985) Ahl. Biochem. 150,76-85

, .

47. Kovarik. A.. Hlubinova, K.. Vrbenska, A., ancfPrachar, J. (1987) Folia 46. Heukeshoven J., and Dernick, R. (1985) Ekctro horesis 6,103-112

Biologics (Pr ue) 33,253-257

Broekaert W. F. (1991) Electrophoresis 12,442-444 48. De Bolle, M. F.%, Goderis, I. J., Terras, F. R. G., Cammue, B. P. A., and

49. Fullmer, C. A. (1984) Anal. Biochem. 142,336-339 50. Creighton, T. E. (1989) in Protein Structure: A Practical Approach (Creigh-

ton, T. E., ed) pp. 155-167, IRL Press Ltd, Oxford

S u p p l e m e n t a r y M a t e r i a l t o :

A n a l y r r r o f t w o novel clarrex o f p l a n t a n t l f v n g a l p r o t e i n s f r o . r a d i s h

( R a p h a n u s s o t l r u s L . ) r e e d s

F r a n k y R.G. Terras. H l l d e M.E. S c h o o f r . M1guel F.C . De Bollc. F r e d V a n

L c u r c n . S a r a h B. Rae%. Jozef V a n d e r l e y d e n . Bruno P.A. C a m w e a n d Y l l l e . F.

B r o e k a e r t

Radish Seed Antifungal Proteins

0.0

- 0.0

reeds. The baric h e a t - s t a b l e protein fraction f r o m r a d l r h r e e d s 1.1 l o a d e d

F I ~ . 1. S c p a r a t 7 o n o f 1.0 rlarrer o f antifungal p r o t e l n r f r o m s a t ~ v u s R .

on a 8-Sepharore H igh Per fo rmance ca t ron-exchange column (IO x 1.6 C. : P h a m a c J a ) i n e q u ~ l i b n u . n t h 50 nM 2-(N-norphol1no)cthlne.ulphonlc a c i d

( M I S ) a t pH 6. A f t e r t h e a b s o r b a n c e o f t h e u n b o u n d f r a c t x o n ( n o t s h o r n ) f e l l

b e l o w 0.01 a b s o r b a n c e u n i t s . t h e b o u n d f r a c t l o n was d e s o r b e d a t 2.5 ml/.in n t h a Ilnear g r a d l e n t o f 1000 m l from 0 t o 500 111 NaCl ~n 50 nM ME8 (pH 6). The e l u a t e was m o n 7 t o r e d f o r p r o t e l n r a t 780 n. ( l o w e r p a n e l ) a n d c o l l e c t e d

~n 10 m l f r a c t ? o n s o f r h l c h 2 0 0 111 was d l a l y z e d a v e r n > g h t ~n a n , c r o d ? a l y S i r

a p p a r a t u s . A f t e r f ? l t e r - r t e r l l I z a t ? o n (0.22 urn). 70 "1 o f t h e r e f r a c t i o n s

-a% t e r t e d ~n a . ~ c ~ ~ l p e c t r . p h ~ t . . e t ~ l c antifungal a c t l v l t y a s r a y (upper

p a n e l ) I " b o t h h a l f - s t r e n g t h p o t a t o d e x t r o s e b r o t h - 7 t h (----) o r w i t h o u t

(-) 1 mM CaClZ and 50 mM KC1. Chromatography was per formed on a Y a t r r s

650 HPLC r t a t l o n .


B I !'I , 5 1 0 P I

Large subunlt Rr-ZS5 P Q 6 V Q Q R V P L L Q Q C C N N L L Q PO13 V P G V Q Q R V P L L P P C C N f L k Q napln P Q O V Q P R S P L L P P C C N f l k Q

L H R Q L H R Q L H K O

Documents

Analysis of Two Novel Classes of Plant Antifungal … · 15302 Radish Seed Antifungal Proteins by this peak is henceforward referred to as Rs-AFP1 (Ra- phunus satiulls antifungal