THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 267 , No. 22, Issue of August 5, pp.

15301-15309, 1992
8 1992 by The American Society for Biochemistry andMolecular Biology, Inc 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&#, HildeM. E. Schoofs$, Miguel F. C. DeBolle$, Fred Van Leuvennll,

Sarah B. Rees**, Jozef VanderleydenS, BrunoP. 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 iso- 1,3-glucanases (2), thionins (3, 4), permatins (5, 6), and ribo-
lated from radishseeds. some-inactivating proteins (1, 7). Recently, we have also
The first class consists of two homologous proteins characterized antimicrobialchitin-binding lectin-like pep-
(Rs-AFP1 and Rs-AFP2) that were purified to homo- tides from amaranth seeds (Ac-AMPs,’ Ref. 8) and a new
geneity. They are highly basicoligomericproteins class of insect neurotoxin-like antimicrobial peptides from
composed of small (6-kDa) polypeptides that are rich Mirabilisjalapa seeds (Mj-AMPs, Ref. 9). Interestingly, mem-
in cysteine. Both Rs-AFPs have abroadantifungal bers of at least the first four mentioned classes are induced in
spectrum and are among the most potent antifungal vegetative parts of plants upon challenge by fungi, bacteria,
proteins hitherto characterized. In comparison with or viruses (3, 10-14).
many other plant antifungal proteins, the activity of In thispaper, we describe the purification and characteriza-
the Rs-AFPs is less sensitive to the presence of cations.
Moreover, their antibioticactivity shows a high degree tion of two new classes of antifungal proteins from radish
of specificity to filamentousfungi. The amino-terminal seeds. Proteins from the first class have a potent antifungal
regions of the Re-AFPs show homology with the de- activity and show sequence homology to recently character-
rived amino acid sequences of two pea genes specifi- ized pea pod proteins that are induced upon fungal attack
cally induced upon fungalattack, to y-thionins and to (15). Homologous proteins are also present in seeds of mon-
sorghum a-amylase inhibitors. ocotyledonous plants. To our surprise, we found that thewell
The radish 2 s storage albumins were identified as characterized 2s seed storage albumins (18, 19) also exert
the second novel class of antifungal proteins. All iso- antifungal activity and classified them as thesecond new class
formsinhibitgrowthof different plantpathogenic of radish seed antifungal proteins.
fungi and some bacteria. However, their antimicrobial
activities are strongly antagonized by cations. EXPERIMENTAL PROCEDURES*

RESULTS
Purification of Radish Seed Antifungal Proteins-After ini-
Plant seeds are usually sown on a natural substrate thatis tial purification steps consisting of ammonium sulfate frac-
rich in microorganisms. The low water content of the seed tionation, heat treatment, and anion-exchange chromatogra-
and the hard seed coat provide effective physical barriers phy, the basic protein fraction from radish seeds was loaded
against bacterial and fungal invasion. During the imbibition on a cation-exchange column at pH6 and eluted by applying
phase preceding seed germination, however, these barriers are a linear gradient of sodium chloride. The unbound fraction
gradually disrupted and protection then mainly relies upon (not shown) was devoid of any substantial antifungal activity,
antimicrobial compounds including proteins. Many different whereas all desorbed material inhibited growth of the test
proteins with antifungal and/or antibacterial activity have fungus Fusarium culmorum grown in half-strengthpotato
already been detected in seeds. These are: chitinases (I),@- dextrose broth without additionalsalts (Fig. 1). However,
only the early eluting peaks 1 and 2 (see Fig. 1) still exerted
*This work was supported in part by the ECLAIR Programme antifungal activity when assayed in the same medium supple-
(AGRE-0005) of the Commission of the European Community. The mented with 1 mM CaCl, and 50 mM KCl.
costs of publication of this article were defrayed in part by the In a subsequent purification step, material of peaks 1 and
payment of page charges. This article must therefore be hereby 2 was applied on a reversed-phase chromatography (RPC)
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. column. Fig. 2A shows that the first cation-exchange peak
Received a predoctoral fellowship from the Belgian “Instituut ter elutes as a single symmetrical peak upon RPC, which co-
Aanmoediging van het Wetenschappelijk Onderzoek in de Nijverheid elutes with the antifungal activity. The active factor contained
en de Landbouw.”
(1 Supported by the Belgian “Nationaal Fonds voor Wetenschap- ’ The abbreviations used are: Ac-AMP,Amaranthus caudatusanti-
pelijk Onderzoek-Levenslijn” Action. microbial peptide; Mj-AMP, Mirabilis jalapa antimicrobial peptide;
$3 Received a postdoctoral fellowship from the Belgian “Instituut RPC, reversed-phase chromatography; Rs, Raphanus sativus; Rs-
ter Aanmoediging van het Wetenschappelijk Onderzoek in de Nijver- AFP, Raphanus sativus antifungal protein; SDS-PAGE, sodium do-
heid en de Landbouw.” decyl sulfate polyacrylamide gel electrophoresis.
$8 Research Associate of the Belgian “Nationaal Fonds voor We- * Portions of this paper (including “Experimental Procedures” and
tenschappelijk Onderzoek.” To whom correspondence should be ad- Figs. 1-3, 9, and 11) are presented in miniprint at the end of this
dressed F.A. Janssens Laboratory of Genetics, Catholic University paper. Miniprint is easily read with the aid of a standard magnifying
of Leuven, Willem De Croylaan 42, B-3001 Heverlee, Belgium. Tel.: glass. Full size photocopies are included in the microfilm edition of
32-16-28-66-11 (ext. 2403); Fax: 32-16-22-07-61. the Journal that is available from Waverly Press.

15301

This is an Open Access article under the CC BY license.

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 peaksof which the first
(designated Rs-AFP2) exerts themost pronounced antifungal
activity (Fig. 2B). Thesecond peak was only weakly active in
inhibiting fungal growth and was therefore not further char-
acterized. Striking observations are that Rs-AFP1 and Rs-
AFP2 both elute a t approximately 30% acetonitrile (in 0.1%
trifluoroacetic acid)and that theantifungal activities of both
components are relatively insensitive to thepresence 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 mate-
rial eluting in the interval of32-40% 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 mixtureof equal amounts of the five
2 s fractions yielded one single symmetrical peak which mi-
grated 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 (1mM CaCl, and 50 mM
KC1) were added to thefungal growth medium.
In conclusion, on the basis of the presentedchromato-
graphic data, two separate groups of radish seed antifungal
proteins can be distinguished; the first one consisting of the
relatively salt-insensitiveproteinsRs-AFP1 and Rs-AFP2,
and thesecond 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 apparentmolecular mass of 20kDa, whereas the unreduced
Rs-AFP2 (lane3) yielded a major band of 15 kDa and a minor
band of 20 kDa. The reduced and S-pyridylethylated deriva-
tives of both Rs-AFPs (lanes2 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 notshown). 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), andS-pyridylethylated derivatives were dis-
solved a t 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 aftercovering
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 oc-
curred when assaying the nativeproteins 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 activ-
ity 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, orprotein-
ase K) completely abolished antifungal activities.
Initial attempts to obtainNH2-terminalamino acid se-
quences of Rs-AFP1 and Rs-AFP2 by Edman degradation
failed, probably due to a blocked NH2-terminal residue. After
treatment of the S-pyridylethylated proteinswith the enzyme
pyroglutamate aminopeptidase, thefirst 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 theunreduced state (Fig. 7A), each fraction
represents a mixtureof two molecular mass forms of approx-
imately 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 thenative 2 s
Protein Activity
FIG. 5. Detectionofantifungal activity oftheRe-AFPs
after native cathodic gel electrophoresis.Rs-AFP1 (lanes1) 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
1 25
5 20
10 15
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 -R l 2 3 4 5
m
..88-19= 14.l7
4 --
a: 8 :
, "
6
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 theright); 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 aminoacid sequences of both subunits. To this
purpose, both subunitswere first separated by reversed-phase
chromatography after reduction and S-carboxyamidomethy-
lation (Fig. 8). SDS-PAGEanalysis of the different peak
fractions indicatedthat thefirst group of peaks represent the
4-kDa subunits,whereas the second group of peaks correspond
to the 10-kDasubunits. Sequence determination was per-
formed on samples from the first peak (small subunit) and
thethird peak (large subunit) of theRPC profile of S-
carboxyamidomethylated 2%. As could already be expected
on thebasis of their subunit structure, theirhigh 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% iden-
tical 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 extentradish seed
antifungal proteins are capable of inhibiting growth of differ-
ent fungi, dose-response curves were measured for 20 different
plant pathogenic fungi. From these curves, protein concentra-
tions 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 CaC12and 50 mMKC1. The results
of these tests aresummarized in TableI.
15303
30 35 40
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 pgof the S-carboxyamidomethylated 2S5
fraction was loaded on a P e p 4 reversed-phase chromatography col-
umn (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 ICsovalues ranging from 0.4 to 25 pg/ml. Generally, Rs-
AFPZ is 2-30-foldmore 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 1000pg/ml in the medium
with added salts, whereas ICsovalues 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 @-thioninfrom wheat and theM. 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
Proteins
Antifungal
TABLE
I
Antifungal activityof 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
Medium Fungus A" Medium Bb
Rs-AFP1 Rs-AFP2 Rs-2s Rs-AFP1 Rs-AFP2 Rs-~S
Pdml Irglml
Alternaria brassicola 15 2 10 >loo 20 >loo0
Ascochyta pisi 5 4 75 >loo 50 >loo0
Botrytis cinerea 8 2 >loo
>500 >loo >lo00
Cercospora beticola 2 2 ND 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 fijiensisvar. 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 >loo
60 >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 CaC12and 50 mMKC1.

ND, not determined.

TABLE I1
Variation of antifurwal activitvin the Dresence of Ca2+or Kt
ICw values
Fungus Antifungal Reference
medium" Reference medium supplemented with
protein
10KC1
mM 50 KC1
mM 1 mM CaC12 5 mM CaC12
rcglml
Rs-AFP1
culmorum
Fusarium 5 5 6 11 >loo
Rs-AFP2 2 2 2 2 5
Rs-2S5 35 40 >400 >400 >400
0-Purothionin 9 9 4 9 90
Mj-AMP2 3 4 12 11 >loo
Rs-AFP1
hamatum
Trichoderma 6 6 6 35 >loo
Rs-AFP2 2 2 3 2 >loo
Rs-2S5 30 30 >4o0 >400 >400
8-Purothionin 4 3 1.5 4 30
Mj-AMP2 2 2 25 25 >loo
a Reference medium: synthetic low ionic strength growth medium.

50 mM, whereas the activity of Mj-AMP2 is drastically re- illustrated by microphotographs in Fig. 10. The 2s albumins
duced. The antifungal activity of the 2s albumins is com- and thionins caused severely delayed growth of hyphae with
pletely abolished in the presence of 50 mM KCl. One milli- otherwise normal morphology. However, in the presence of
molar of CaC12has no effect on Rs-AFP2 and thionin,whereas thionins, large parts of the hyphae were stained by methylene
it reduces the activity of Rs-AFP1 and Mj-AMP2 and abol- blue, indicating loss of viability. This lethal effect was not
ishes that of the 2s albumins. CaC12concentrations of 5 mM seen, either with the 2s albumins or with the Rs-AFPs.
are necessary to cause activity reduction of Rs-AFP2 and Microcultures to which Rs-AFPs were added revealed a com-
thionin. Tests with other saltsincluding NaC1, NH,Cl, K2S0,, pletely different type of growth inhibition, featuring charac-
CaSO,, MgC12, and BaC12 indicated that salts of monovalent teristic claws of branched swollen hyphae. However, at high
cations had effects comparable with that of KC1and that salts concentrations of any of the antifungal proteins, no spore
of divalentcations had similar effects as those of CaCh germination occurs at all (Fig. 10).
(results not shown). The nonlethal effect of the Rs-AFPs and the 2 s albumins
During the course of this work, fungal growth inhibition was also confirmed in an experiment where these proteins
was routinely checked microscopically to confirm the micros- were added to duplicate cultures of F. culmorum at 10 pg/ml
pectrophotometric data. A striking difference in the morphol- (Rs-AFPl), 5 pg/ml (Rs-AFP~), or 100 pg/ml (2s albumin).
ogy of inhibited hyphae was apparent between fungi treated After 48 h of incubation, all cultures were inhibited by more
with Rs-AFPs and those treated with 2s albumins. This is than 90% relative to controls. After replacement of the me-
 Proteins
Antifungal Seed Radish
I
r, ;;
//
V By monitoring chromatographic separationsof radish seed
I proteins using an assay for growth inhibition of F. culmorum,
FIG. 10. Differences in morphologyofinhibitedhyphae.
Photomicrographs weretaken 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 bluewasadded to the
microcultures to raise contrast. The bur correspondsto approximately
25 pm.
dium (containing the proteins) by fresh half-strength potato
dextrose broth, growth of mycelium resumed, whereas the
duplicatecultures(stillcontaining 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 con-
centrations 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 lindemu-
thianum 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 thioninswill be presented in a separate paper.3
Effectson Bacteria, Yeast, Cultured Human Cells, and
Erythrocytes-To determine whether or not the radish seed
antifungal proteins exert biological activities other than fun-
gal 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 theGram-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 mMCaC1, and 50
mMKC1 were supplemented to the growth medium. Further-
more, Rs-AFP2 (and not Rs-AFP1) exerted antibacterial ac-
tivityagainst B. megaterium, although only a t fairly high
concentrations. IC,,, values of200 and 500 pg/ml were ob-
tained in the media without and with added salts (1mM 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.
15305
Yeast (Saccharomyces cerevisiae) cells were not affected in
their growth by either theRs-AFPs or the2s albumins at up
to 500 pg/ml. In contrast,Mj-AMP2, Ac-AMP2, and B-puro-
thionin had ICsovalues 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 thepresence 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
we purified two novel classes of broad spectrum antifungal
proteins which have clearly distinct biochemical and biolog-
ical properties.
Two members of the first novel class of antifungal proteins
were purified to homogeneity and designated as Rs-AFP1 and
Rs-AFP2. Bothproteins 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 onemore 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, andthe 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, bothrelative 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 theantago-
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 protec-
tion 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 substantiallylower than thatof 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 1mM CaC12and 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 de-
creased 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 thepresence 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 microorga-
nisms in planta, aswell 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
tissuesare 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+con-
centrations in plant vacuoles are about 0.06-1 mM, and apo-
plastic 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 interactwith the invading
hyphae. Undoubtedly, these conditions will influence the ac-
tivity 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-amylaseinhibitors 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 substitutionsare
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 contributeto thegeneral 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 activi-
tiesare, 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 pancreasor 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 thethionin family. Like the a- and@-thionins,these
proteinsinhibit protein synthesis in cell-free systems, al-
though 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 res-
idues (at positions 3 and 4) characteristic for the a- and @-
thionins and the related viscotoxins and crambin are not
found in they-thionins (31). The similarity at the amino acid
sequence level between the radish antifungal proteins, the
fungus-induced proteins from pea, the potato p322 geneprod-
uct, the y-type thionins from wheat and barley, and the a-
amylase inhibitors from sorghum suggests that this class of
proteins iswidespread 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 gen-
erally present in seeds of this family?
Based on structuralproperties, 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), antimicro-
bial 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 asa 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 calcu-
lated from cDNA-derived sequences (14.2 and 13.2 kDa for
the largest and thesmallest 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 identityto the cDNA-derivedsequences 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, manuscriptin preparation.
 Radish Seed Antifungal Proteins 15307
Darnme, J., Segura, M., Gheysen, G., Van Montagu, M., and Vandek-
of fungi in the IC,,) range from 3 to 200 pg/ml when assayed erckhove, J. (1988) Plant Physiol. 87,859-866
in the low ionic strength medium. However, the activity was 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
completely abolished in the presence of 1 mM CaCh and 50 Approach (Creighton, T. E., ed) pp. 1-22, IRL Press Ltd., Oxford
mM KC1. It is therefore doubtful that theseproteins by 21. Raynal, M., Depigny, D., Grellet, F., and Delseny, M. (1991) Gene (Amst.)
99,7746
themselves have a protective function in uiuo. However, the 22. Ericson, M. L., %din, J., Lenman, M., Glimelius, K., Josefsson, L. G., and
mentioned synergism between the 2s albumins and thionins Rask, L. (1986) J. Biol. Chem. 261,14576-14581
23. Robson, G.D., Wiebe, M. G., and Trlncl, A. P. J. (1991) Erp. Mycol. 16,
is also observed in high ionic strength media.3 263-272
24. Clarkson, D. T., and Hanson, J. B. (1980) Annu. Reu. Plant Physiol. 3 1 ,
239-298
Acknowledgments-We are grateful to Drs. A. Ludwig and T. Boller 25. Flowers, T. J., and Lauchli, A. (1983) in Encyclopedia of Plant Physiology,
for providing samples of pea chitinase and pea 8-1,3-glucanase. We New Series (Lauchli, A,, Bieleski, R. L., eds) Vol. 15B, pp. 651-681,
thank Dr. L. Nelles and Drs. J. Van Damme and P. Proost for Springer-Verla Heidelberg, Federal Republic of Germany
assistance with the cultures of the umbilical vein endothelial cells 26. Grignon, C., and kentenac, H. (1991) Annu. Reu. Plant Physiol. PlantMol.
Biol. 42,103-128
and thefibroblasts, respectively. We also thank J. Desair for skillfull 27. Macklom, A. E. S. (1984) Plant Cell Enuiron. 7,407-413
maintenance of the chromatographic equipment and I. Goderis for 28. Hepler, P. K., and Wayne, R. 0.(1982) Annu. Reu. Plant Physiol. 36,397-
A2CI
outstanding technical assistance. 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,
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S u p p l e m e n t a r yM a t e r i a lt o :

A n a l y ro
trw
rf o novel clarrex of plan
ant t l f v n g pa rl o t e i nf rsroa. d i s h
( R a p h a n u ss o t l r u s L.) reeds

F r a n k y R.G. Terras. Hllde M.E. Schoofr. M1guel F.C. De Bollc. F rV

edan
L c u r c nS. 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 ae n Y
dllle. F.
Broekaert
Radish Seed Antifungal Proteins

0.0
- 0.0

F I ~ . 1. S c p a r a t 7 o n of 1
.
0 rlarrer of antifungal p r o t e l n rf r o m R. sat~vus
reeds. The baric h e a t - s t a b l e protein fraction f r o m r a d l r hr e e d s 1
.1 loaded
on a 8 - S e p h a r o rHei gPhe r f o r m a n ccea t r o n - e x c h a n g e c o l u m n (IO x 1.6 C. :
PhamacJa) i n e q u ~ l i b n u .n t h 50 nM 2-(N-norphol1no)cthlne.ulphonlc acid
(MIS) a t pH 6. A f t e rt h ea b s o r b a n c eo ft h eu n b o u n df r a c t x o n( n o ts h o r n )f e l l
b e l o w 0.01 absorbance
u n i t st.h e
bound
fractlon was desorbed
at 2.5 ml/.in
n t h a I l n e a r g r a d l e n to f 1000 m l from 0 t o 5 0 0 1
11 NaCl ~n 50 nM ME8 (pH 6).
The e l u a t e w a s m o n 7 t o r e df o rp r o t e l n ra t 780 n. (lower p a n e l )a n dc o l l e c t e d
~n 1 0 m l f r a c t ? o n so fr h l c h2 0 0 111 was d l a l y z e da v e r n > g h t ~n a n,crod?alySir
apparatus. A f t e fr? l t e r - r t e r l l I z a t ? o n (0.22 urn). 70 "1 ot fh e rfer a c t i o n s
-a% terted ~n a .~c~~lpectr.ph~t..et~lc antifungal a c t l v l t ay s r a y (upper
panel) I" bothalf-strength
potato
dextrose
broth
-7th (----) or without
(-) 1 mM CaClZ
and 50 mM KC1. C h r o m a t o g r a p h y was p e r f o r m e d on a Y a t r r s
650 HPLC r t a t l o n .
 Radish Seed Antifungal Proteins 15309

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

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

!'I
B
I

I
P
,510