(Received for publication, July 16, 1996, and in revised form, October 9, 1996)
From the F. A. Janssens Laboratory of Genetics,
Katholieke Universiteit Leuven, Willem De Croylaan 42, B-3001
Heverlee, Belgium, the § Department of Organic Chemistry,
Biomolecular NMR Unit, Universiteit Gent, Krijgslaan 248, S4bis,
B-9000 Gent, Belgium, ¶ Zeneca Agrochemicals, Jealott's Hill
Research Station, Bracknell, Berkshire, RG42 6ET, United Kingdom, and
the
Department of Biochemistry and Molecular Biology,
University College London, Gowerstreet,
London WC1 E6BT, United Kingdom
Mutational analysis of Rs-AFP2, a radish
antifungal peptide belonging to a family of peptides referred to as
plant defensins, was performed using polymerase chain reaction-based
site-directed mutagenesis and yeast as a system for heterologous
expression. The strategy followed to select candidate amino acid
residues for substitution was based on sequence comparison of Rs-AFP2
with other plant defensins exhibiting differential antifungal
properties. Several mutations giving rise to peptide variants with
reduced antifungal activity against Fusarium culmorum were
identified. In parallel, an attempt was made to construct variants with
enhanced antifungal activity by substituting single amino acids by
arginine. Two arginine substitution variants were found to be more
active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to
be important for antifungal activity, namely the region around the type
VI -turn connecting
-strands 2 and 3, on the one hand, and the
region formed by residues on the loop connecting
-strand 1 and the
-helix and contiguous residues on the
-helix and
-strand 3, on
the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to
20-fold. An arginine substitution variant with enhanced antifungal
activity caused increased Ca2+ uptake by up to 50-fold,
whereas a variant that was virtually devoid of antifungal activity did
not stimulate Ca2+ uptake.
During the last decades, it has been recognized that many living
organisms produce small antimicrobial peptides to protect their tissues
from infectious microbial agents. Well known examples of peptides with
antimicrobial properties are the cecropins of invertebrates (reviewed
in Ref. 1) and magainins of amphibians (reviewed in Ref. 2). Another
class comprises cysteine-rich peptides, among which are the mammalian
and insect defensins (3, 4, 5), both small, basic proteins with a
cysteine-stabilized three-dimensional folding pattern involving
antiparallel -sheets. Insect defensins are produced upon perception
of pathogens by the insect fat body and are secreted in the hemolymph
(5). Mammalian defensins are present in phagocytic blood cells and are
also produced by epithelial cells of the intestines and airways (4).
Antimicrobial peptides have also been found in plants. Thionins, for
instance, are highly basic 5-kDa peptides toxic to both Gram-positive
and Gram-negative bacteria, fungi, yeast, and various mammalian cell
types (6). A number of plant species produce thionins constitutively in
their seed as well as in their leaves in a pathogen-inducible way (7).
Other potent antimicrobial peptides found in plants are structurally
related to defensins of mammals and insects and are therefore termed
plant defensins (8). Plant defensins are small cysteine-rich peptides
consisting of 45-54 amino acids with four intramolecular disulfide
bridges. They are encountered in different plant species and various
tissues such as seed, flowers, and pathogen-stressed leaves. Comparison of the known primary sequences of a series of plant defensins shows
that the arrangement of the cysteines is highly conserved and reveals
the existence of a cysteine-stabilized
-helix motif (9), which is
also present in insect defensin A (10). Their three-dimensional
structure consists of three antiparallel
-strands and an
-helix
(11) and is similar to that of insect defensins (5) and some scorpion
toxins (e.g. charybdotoxin; Ref. 12). Most plant defensins
hitherto isolated exhibit antifungal activity. Some of them, for
example SI
21 (Sorghum bicolor
inhibitor 2 of
-amylase), are inhibitors of
-amylases but do not
inhibit fungal growth (13, 14). The plant defensins with antifungal
activity can be divided in two groups. The first group causes
morphological distortions of the fungal hyphae resulting in swollen and
hyperbranched fungal structures (9, 14). The second group merely
inhibits fungal growth without inducing morphological changes. Mode of
action studies performed on a representative of each class (Rs-AFP2
from radish and Dm-AMP1 from dahlia) has shown that plant defensins
cause rapid ion fluxes upon addition to fungal hyphae, resulting in
Ca2+ uptake, K+ efflux, and medium
alkalinization (15).
In this study we have performed a structure-function analysis of Rs-AFP2, a plant defensin isolated from radish seed and member of the plant defensin group causing hyperbranching of fungal hyphae. It is the most potent among a number of plant defensin isoforms occurring in radish, including Rs-AFP1 isolated from seed and Rs-AFP3 and Rs-AFP4 isolated from infected leaves (8). In order to investigate which amino acids are essential for the antifungal activity of Rs-AFP2, we have undertaken a mutational analysis of this peptide. Information on amino acid substitutions resulting in either a decreased or enhanced antifungal activity, taken together with preliminary data on the three-dimensional configuration of Rs-AFP2, allows prediction of the sites which possibly interact with the yet unknown target site on the fungal hyphae.
DNA manipulations were performed in
Escherichia coli strain DH5. The yeast strain used for
expression of the Rs-AFP2 variants was Saccharomyces
cerevisiae c13-ABYS86 (genotype: MAT
,
pra1, prb1, prc1, cps1,
ura3, leu2, his3) (16). The fungal
strains used for antifungal activity assays were Alternaria
brassicicola (MUCL 20297), Ascochyta pisi (MUCL 20164),
Botrytis cinerea (JHCC 8973), Fusarium culmorum
(IMI 180420), Nectria hematococca (Collection van Etten
160-2-2), Phoma betae (MUCL 9916), and Verticillium dahliae (MUCL 19210). Authentic Rs-AFP2 was purified from radish seed as described previously (17).
Mutagenesis of the Rs-AFP2 coding
sequence was performed by two sequential polymerase chain reactions
(PCR) as described in Ref. 18. Primers used in the PCR reaction are
listed in Table I. In a first PCR, part of the Rs-AFP2 coding sequence
was amplified using a sense mismatch primer containing the desired
mutation and primer OWB35, a derivative of the M13 reverse primer
elongated with a 5 tag (28 cycles; 1 min at 94 °C, 1 min at
55 °C, 1 min at 72 °C). For design of the mismatch primer, the
yeast preferential codon usage was taken into account (19). Ten ng of
PvuI-linearized plasmid pBluescript/RsAFP* (20) was used as
a template for the first PCR. The amplified product containing the
mismatch served as a megaprimer to further elongate the Rs-AFP2
sequence (5 cycles; 1 min at 94 °C, 1 min at 55 °C, 1 min at
72 °C). In a second PCR, this elongated fragment was amplified by
primer OWB61, binding to the 5
end of the Rs-AFP2 gene, and OWB36, an
oligonucleotide identical to the 5
tag of OWB35 (28 cycles; 1 min at
94 °C, 1 min at 55 °C, 1 min at 72 °C). OWB61 contains a
restriction site allowing in-frame cloning into the HindIII
site in the MF
1 pro-sequence region of pVD4 (20). Amplification
products of the second PCR were digested with
HindIII-BamHI and introduced in the corresponding sites of pVD4. After verification of the occurrence of the desired mutations by nucleotide sequence determination, the expression blocks
containing the MF
1 promoter and prepro-sequence followed by the
mutated Rs-AFP2 gene were isolated by SalI-BamHI
restriction digestion and subcloned into the
SalI-BglII-digested yeast shuttle vector pTG3828
(21). After subcloning, the sequence of the mutated Rs-AFP2 domain was
verified by nucleotide sequencing. Restriction enzymes were purchased
from Boehringer Mannheim (Mannheim, Germany), T4 DNA ligase from Life
Technologies, Inc. (Life Technologies, Merelbeke, Belgium), and
Taq DNA polymerase from Appligene (Pleasanton, CA). DNA
sequencing was performed on a Pharmacia A.L.F. DNA sequencer using the
AutoRead Sequencing Kit (Pharmacia, Uppsala, Sweden) according to the
manufacturer's instructions.
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Transformation of S. cerevisiae, growth of
the yeast cultures, and purification of the Rs-AFP2 variants from the
culture supernatants were essentially done as described previously for
native Rs-AFP2 (20). Briefly, 250 ml of culture supernatant (minimal
selective SD medium: 0.8 g/liter CSM-URA from BIO 101, La Jolla, CA;
6.5 g/liter yeast nitrogen base from Difco; 20 g/liter glucose (Merck); 5 g/liter casamino acids from Difco) was passed over an anion-exchange chromatography column (Q-Sepharose Fast Flow, Pharmacia) connected on-line with a disposable reversed phase C8 silica column
(Bond Elut, 500 mg solid phase, Varian, Harbor City, CA). The
C8 silica column was subsequently rinsed with 6 ml of 10%
(v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid. The
Rs-AFP2 variants were eluted from the latter column with 4 ml of 30%
(v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid. After
drying in a rotating vacuum concentrator, the eluted fractions were
purified by reversed-phase chromatography on a
C2/C18 silica column (Pep-S, 5-µm beads,
0.4 × 25 cm, Pharmacia). Fractions were collected manually, and
the elution position of the Rs-AFP2 variants was determined by a
combination of antifungal activity analysis on F. culmorum
in SMF (synthetic medium fungi; Ref. 17) and SDS-PAGE analysis. In
all cases, elution positions could be determined unambiguously.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was carried out according to Ref. 22 using a 15% (w/v)
acrylamide, 0.5% (w/v) bisacrylamide separating gel and a 5% (w/v)
acrylamide, 0.1% (w/v) bisacrylamide stacking gel. Gels were either
stained with Coomassie Brilliant Blue R250 or immunoblotted using
anti-Rs-AFP1 antibodies as described previously (8). Protein
concentrations were determined by the bicinchoninic acid method (23)
using authentic Rs-AFP2 as a standard. Free cysteine thiol groups were determined by the Ellman assay on both reduced and unreduced protein samples as described previously (17). Circular dichroism spectra were
obtained on a Jasco 600 spectropolarimeter with a cell path of 0.02 cm.
Proteins were dissolved at 0.5 mg/ml in distilled water. The spectra
were acquired in a single scan mode (10 nm/min) in the ultraviolet
region of 265-185 nm. Circular dichroism data were base line-corrected
and are presented in units of (M
1
cm
1) (24).
Recombinant yeast (S. cerevisiae) cells containing vectors for expression of Rs-AFP2(Y38G) and Rs-AFP2(V39R), respectively, were grown for 7 days in a fermentor (Biostat E 15 liter) at 25 °C as a batch-fed culture and harvested at a final OD of approximately 80. Cells were pelleted by centrifugation at 3000 × g for 20 min. The supernatants were passed directly over a Biopilot S-Sepharose column (15 × 10 cm, Pharmacia) pre-equilibrated in 20 mM ammonium acetate, pH 6. A flow rate of approximately 100 ml/min was maintained using gravity feed. The bound fraction was eluted with a single wash of 1 liter of 500 mM ammonium acetate (pH 6) and freeze-dried for 3 days to completely remove the ammonium acetate salt. The freeze-dried fraction was dissolved in 20 mM ammonium acetate and refractionated by cation exchange chromatography on a S-Sepharose Fast Flow column (10 × 2.6 cm, Pharmacia) equilibrated in 20 mM ammonium acetate, pH 6. The bound fraction was eluted with a linear gradient of 20-500 mM ammonium acetate (pH 6) over 325 min at 3 ml/min. Proteins were monitored by on-line measurement of absorbance at 280 nm. Fractions containing Rs-AFP2 variants were identified either by using a standard in vitro antifungal bioassay (see below) or by Western blot (see above). Fractions containing the expressed peptide were pooled, freeze-dried, and further purified by reversed phase high performance liquid chromatography on a Pep-S column (C2/C18 silica, 25 × 0.93 cm, Pharmacia). Peptides were eluted with a linear gradient of 0.1% (v/v) trifluoroacetic acid to 99.9% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid over 100 min at a flow rate of 3 ml/min. A single predominant peak of absorbance at 280 nm containing the Rs-AFP2 variants was eluted at approximately 20% (v/v) acetonitrile.
Antifungal Activity AssaysThe antifungal activity assays
were carried out in microplates as described in Ref. 25. A 2-fold
dilution series of the protein in sterile water was prepared, and 20 µl of the serial dilutions were added to 80 µl of synthetic low
ionic strength medium (SMF, Ref. 17) containing 104
spores/ml of the test fungus without or with the addition of extra
salts as indicated under "Results." The plates were incubated at
room temperature. Growth of the fungi was monitored microscopically after 24 h and by microspectrophotometry after 72 h unless
otherwise indicated. The protein concentration required for 50% growth
inhibition (IC50 value) was calculated as described in Ref.
17. The specific antifungal activity was defined as
1/IC50.
F. culmorum was grown at an inoculum density of 5 × 104 spores/ml in a 100-ml Erlenmeyer flask placed on a rotary shaker (200 rpm). The medium consisted of half-strength potato dextrose broth supplemented with 0.5 µCi of [3H]N-acetyl-D-glucosamine/ml (ICN Radiochemicals, Costa Mesa, CA), which merely incorporates into the chitin fraction of the fungal cell wall. The 3H label was used as a measure for biomass, allowing to correct for sample to sample variations in biomass. After 20 h of incubation at 22 °C, 2 µCi/ml 45CaCl2 (ICN Radiochemicals) was added, together with the antifungal proteins. After appropriate incubation times, 250-µl samples were taken (in quadruplicate) and transferred to wells of a MultiScreen Durapore 96-well filtration plate (Millipore, Bedford, MA), placed on a MultiScreen vacuum filtration manifold (Millipore). After filtration, harvested hyphae were washed four times with 250 µl of 10 mM CaCl2. Membranes with the hyphae were punched out manually with MultiScreen punch tips (Millipore), and counted for 3H and 45Ca in a liquid scintillation counter (Wallac 1410, Pharmacia).
Preliminary Three-dimensional Solution Structure Determination by 1H NMRAll 51 residues of Rs-AFP1 have been sequence
specifically assigned following the strategy of Wüthrich (26)
using a combination of double quantum filtered correlation spectroscopy
(DQF-COSY) (27), nuclear Overhauser effect spectroscopy (NOESY) (28), and homonuclear Hartmann-Hahn spectroscopy (HOHAHA) (29) spectra recorded on a Bruker AM-500 (Bruker Analytische Messtechnik, Karlsruhe, Germany). These were recorded at a protein concentration of 1.3 mM and pH 4.2, in both 9/1 H2O/D2O
and D2O solutions at two different temperatures (304.5 and
313.2 K). The data set at 312.2 K was used to extract 775 nuclear
Overhauser effect cross-peaks, and 44 3JNH
and 62 3J
coupling constants. Using the
programs CALIBA, HABAS, and GLOMSA (30), 775 upper limit constraints,
94 angle constraints, and 19 stereospecific assignments were generated.
These data, together with 13 upper and 13 lower limits for the
disulfide bridges and the pyroglutamate ring, were used to calculate
500 structures with the program DIANA using the REDAC protocol (30) on
a SG Crimson (Crimson, Mountain View, CA). The 25 best structures, with
a root mean square deviation of the backbone atoms of all the 51 residues of 1.15 ± 0.22 Å and a root mean square deviation of
all the heavy atoms of 1.80 ± 0.24 Å, were optimized by
simulated annealing (31, 32) using DISCOVER (AMBER forcefield) and
INSIGHT II for visualization (Biosym Technologies, Inc., San Diego,
CA). The root mean square deviation of these final structures is 1.33 ± 0.26 Å for the backbone atoms and 1.84 ± 0.28 Å for all the heavy
atoms.
Proteins
belonging to the family of plant defensins have been purified and
sequenced from a range of taxonomically divergent plant species, while
others have been identified via cDNA sequencing (9). Fig.
1 represents a comparison of the complete amino acid sequences of 12 different plant defensins whose antifungal activity against F. culmorum has been assessed in our laboratories
(8, 14, 17, 33). In terms of biological activity, three groups of plant
defensins can be discerned: group I, those who are inhibitory to
F. culmorum and cause increased hyphal branching; group II, those inhibitory to F. culmorum without causing hyphal
deformations; and group III, those not affecting growth of F. culmorum at concentrations below 100 µg/ml. Rs-AFP2, the protein
studied in this work, belongs to the first group.
As can be seen from the alignment of the sequences in Fig. 1, the
pattern of cysteines is totally conserved in all the sequences, as is
the glycine residue at position 34 (numbering relative to the studied
protein Rs-AFP2). Those residues are important secondary structure
elements and are part of the cysteine-stabilized motif
characterized by the sequences CXXXC, GXC, and
CXC (X stands for any amino acid) (34). Other
well conserved residues are the serine at position 8, the glycine at
position 13, and the glutamate at position 29. Those conserved residues
were not considered for substitution in the present study, since it is
likely that they play a role in determining the structure of the
peptide.
A number of amino acid residues were found to be fully conserved among the antifungal plant defensins (group I and II) but subject to non-conservative changes in plant defensins devoid of antifungal activity (group III). Those residues, namely Gln-5, Thr-10, Gly-16, and Ala-31, were considered to be suitable candidate residues for site specific mutational analysis. Lys-44 and Tyr-48, which are conserved in all group I and II plant defensins, except Dm-AMP1 and Hs-AFP1, respectively, were also retained for mutational analysis. In addition, amino acids that are conserved in group I but not in group II could be important for causing the typical morphological deformation of fungal hyphae, which is characteristic for group I plant defensins. These residues comprise Tyr-38, Phe-40, and Pro-41 and were likewise selected for mutational analysis.
A first series of Rs-AFP2 variants was conceived such that the amino
acid residues selected as discussed above were substituted by the
corresponding residue of SI2, a group III plant defensin devoid of
antifungal activity. Residue Pro-41 was deleted rather than substituted
as the loop between the
-strand 2 and
-strand 3 comprising Pro-41
is shorter in group III plant defensins than in group I plant defensins
and, furthermore, contains no proline residue in SI
2. It was
expected that some of these substitution variants would have a reduced
antifungal activity versus wild-type Rs-AFP2.
A second series of Rs-AFP2 variants was conceived in such a way that amino acids at selected positions were replaced by the basic amino acid residue arginine. The underlying rationale for these substitutions is that Rs-AFP1, the near-identical but less basic natural analogue of Rs-AFP2, has a lower antifungal activity than Rs-AFP2, especially when assessed in media with a high ionic strength (17). Rs-AFP1 and Rs-AFP2 only differ at two residues (Gln-5 is Glu and Arg-27 is Asn in Rs-AFP1), both of which substitutions result in a higher net positive charge of Rs-AFP2 versus Rs-AFP1. This suggests that an increase in the net charge of Rs-AFP2 by replacement of certain residues with arginine might further increase its antifungal activity. The positions selected for the arginine substitutions were those that show weak conservation among the different plant defensins or that are occupied by basic residues in plant defensins other than Rs-AFP2 (see Fig. 1).
For the production of the different Rs-AFP2 variants with the desired
amino acid substitution, the Rs-AFP2 coding sequence was mutated
site-specifically by PCR, fused in frame to the yeast mating factor
1 (MF
1) promoter and prepro-sequence (20, 21) and subsequently
transferred to yeast via a yeast shuttle vector. The different Rs-AFP2
analogues were purified from the yeast culture supernatant by a
combination of ion-exchange chromatography and reversed-phase
chromatography. Using this approach, we have previously shown that
wild-type Rs-AFP2 can be produced in a correctly processed and
bioactive form in yeast (20). In total, 19 Rs-AFP2 variants were
produced and purified in this way (see Table II). The purity of the
preparations was assessed by SDS-PAGE analysis. All Rs-AFP2 variants
migrated essentially as single bands which had the same electrophoretic
mobility as wild-type Rs-AFP2 (Fig. 2). In addition, all
purified proteins were recognized by anti-Rs-AFP1 antiserum on
immunoblots prepared from SDS-PAGE gels, confirming their identity as
variants of Rs-AFP2 (results not shown).
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Two Rs-AFP2 variants, Rs-AFP2(Y38G) and Rs-AFP2(V39R) with a
substitution of the tyrosine at position 38 by glycine and of valine at
position 39 by arginine, respectively, were purified on a large scale
from 15-liter fermentation cultures of the appropriate recombinant
yeast strains. Circular dichroism spectroscopic studies were performed
on these variants as well as on authentic Rs-AFP2. The circular
dichroism spectrum of Rs-AFP2(V39R) was virtually identical to that of
Rs-AFP2, indicating that neither the substitution itself nor the way
the variant was synthesized in yeast had imposed alterations of
backbone secondary structure elements (Fig. 3). Rs-AFP2(Y38G) had a circular dichroism spectrum which was almost identical to that of Rs-AFP2, except for a slightly decreased steepness
of the drop in the 190-208 nm region (Fig. 3).
Antifungal Activity of Rs-AFP2 Variants
The purified Rs-AFP2
substitution variants were assessed for their antifungal activity
against F. culmorum in two different media: a low ionic
strength medium called SMF (17), and the same medium supplemented
with 1 mM CaCl2 and 50 mM KCl,
called SMF+. The presence of salts in the test medium, especially salts with divalent cations, is known to reduce the specific antifungal activity of Rs-AFP2 (17). Seed-purified as well as yeast-expressed wild-type Rs-AFP2 served as controls in the assays. The results of
these comparative tests, expressed as IC50 values, are
presented in Table II. Most of the variants of the first series
(substitutions by corresponding SI
2 residues) showed no or only a
minor decrease of their antifungal activity in medium SMF
, except
Rs-AFP2(A31W), Rs-AFP2(Y38G), and Rs-AFP2(P41
), which showed a
substantial decrease in antifungal potency. In SMF+, the medium with
added salts, a significant decrease in antifungal activity was observed
for the following Rs-AFP2 analogues of the first series:
Rs-AFP2(T10G), Rs-AFP2(A31W), Rs-AFP2(Y38G), Rs-AFP2(F40M),
Rs-AFP2(P41
), and Rs-AFP2(K44Q). On the other hand, the
substitutions Q5M and G16M resulted in a slight but significant
increase in antifungal potency, especially noticeable in medium SMF+,
whereas the substitution Y48I had little or no effect on the antifungal
activity.
In contrast to what was expected, most of the Rs-AFP2 variants of the second series (arginine substitutions) did not show an enhanced antifungal activity compared to Rs-AFP2. In some cases, an even lower antifungal activity was observed, possibly caused by the unfavorable presence of a positive charge at that position or by the absence of a residue necessary for interaction with the fungal target. The largest decrease in antifungal activity was observed for substitution variants Rs-AFP2(L28R) and Rs-AFP2(I46R), whereas variants Rs-AFP2(S12R), Rs-AFP2(I42R), and Rs-AFP2(I49R) showed only a modest reduction in antifungal activity. However, two Rs-AFP2 variants, namely Rs-AFP2(G9R) and Rs-AFP2(V39R), were about 2-fold more active than wild-type Rs-AFP2 when assessed in SMF+.
The antifungal activity of Rs-AFP2(V39R) purified from a large scale
culture of recombinant yeast was further characterized in SMF with
increasing Ca2+ or K+ concentrations and
compared with that of authentic Rs-AFP2 (isolated from seed) as well as
yeast-purified Rs-AFP2. As is shown in Fig. 4, the
antifungal activity against F. culmorum of Rs-AFP2(V39R) was
less reduced by the presence of cations in the growth medium in
comparison with wild-type Rs-AFP2. Indeed, in the presence of 5 mM CaCl2 and at a concentration of 10 µg/ml,
Rs-AFP2(V39R) caused complete inhibition of the growth of F. culmorum, whereas wild-type Rs-AFP2 was basically inactive under
the same conditions. At 10 µg/ml, wild-type Rs-AFP2 was fully active
against F. culmorum only when the CaCl2
concentration was equal or lower than 1.25 mM. Likewise,
the activity of wild-type Rs-AFP2 was drastically reduced in the
presence of 100 mM KCl, whereas Rs-AFP2(V39R) was still
fully inhibitory to F. culmorum at this KCl
concentration.
The potency of Rs-AFP2(V39R) relative to authentic Rs-AFP2 was also
assessed on a set of seven different phytopatogenic fungi in three
media differing in ionic strength: SMF, SMF including 1 mM CaCl2 and 50 mM KCl (SMF+), and
SMF including 5 mM CaCl2 and 50 mM
KCl. As can be seen from the data presented in Table III, the relative
antifungal activity of the variant was dependent on the test organism.
On three fungi (F. culmorum, N. hematococca, and
V. dahliae), Rs-AFP2(V39R) was more active than
Rs-AFP2. In the medium SMF+, for instance, Rs-AFP2(V39R) was about 2-, 5-, and 5-fold more potent than Rs-AFP2 against F. culmorum, N. hematococca and V. dahliae, respectively. As in this
medium neither Rs-AFP2 nor Rs-AFP2(V39R) inhibited growth of A. brassicicola, A. pisi, or B. cinerea at
concentrations below 50 µg/ml, the highest concentration tested, no
difference in antifungal potency could be observed for these fungi.
However, on P. betae, Rs-AFP2(V39R) was less potent than
Rs-AFP2. The differences in antifungal potency between Rs-AFP2(V39R)
and Rs-AFP2 were always more pronounced in the SMF media with added
salts than in the low ionic strength medium SMF
.
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Although the precise molecular target of Rs-AFP2 on fungal
hyphae is not yet known, recent work in our laboratory has shown that
Rs-AFP2 causes very rapid ion fluxes, including increased Ca2+ uptake, when added to fungal hyphae (15). To
investigate whether the ability of Rs-AFP2 to stimulate
Ca2+ uptake in fungi is linked to its antifungal effect,
45Ca2+ uptake was measured in F. culmorum treated with different concentrations of either Rs-AFP2,
the virtually inactive variant Rs-AFP2(Y38G), and the variant with
increased antifungal potency, Rs-AFP2(V39R). The medium used for this
test consisted of half-strength potato dextrose broth supplemented with
1 mM MgCl2 and 50 mM KCl. As shown
in Fig. 5, Rs-AFP2 caused a dose-dependent
increase of 45Ca2+ uptake, which at a dose of
100 µg/ml reached a level that was about 20-fold higher relative to
water-treated controls. At the same dose, Rs-AFP2(V39R) stimulated
45Ca2+ uptake by over 50-fold, and the higher
45Ca2+ uptake stimulation of Rs-AFP2(V39R)
versus wild-type Rs-AFP2 was observed over the whole
concentration range tested. In marked contrast, however, addition of
the variant Rs-AFP2(Y38G) with impaired antifungal properties resulted
in 45Ca2+ uptake rates that fluctuated around
the levels observed for water-treated control cultures.
A structure-activity analysis of Rs-AFP2, a plant defensin from
radish causing growth inhibition of fungal hyphae (17), was carried out
in order to investigate which residues are important for antifungal
activity of the peptide. Candidate amino acid residues were considered
to be those conserved among plant defensins exhibiting antifungal
activity but not among those devoid of antifungal activity as outlined
in Fig. 1. Following this rationale, we have chosen to produce a series
of nine Rs-AFP2 analogues in which particular amino acid residues were
changed to the corresponding residue of the plant defensin SI2,
which does not display antifungal activity. Residue Pro-41 was deleted
rather than substituted as the loop encompassing this residue is
shorter in SI
2 than in Rs-AFP2. A second series of Rs-AFP2 variants
was aimed at increasing the net positive charge (at physiological pH)
of Rs-AFP2 by substituting particular residues by an arginine at
various non-conserved positions along the Rs-AFP2 sequence. This
approach was inspired by the fact that Rs-AFP2, which has a higher net
positive charge than Rs-AFP1, has a 2-30-fold higher activity relative
to Rs-AFP1 (17).
Wild-type and variant peptides were produced in yeast and purified
using identical chromatographic procedures. After the last purification
step consisting of reversed phase chromatography, the different peaks
were assayed for antifungal activity in order to identify the elution
position of the Rs-AFP2 variant. All peptides showed similar retention
times. When analyzed by SDS-PAGE, all variant peptides showed the same
electrophoretic mobility as wild-type Rs-AFP2, indicating that they
have approximately the same size. The structural conformation was
studied into more detail by circular dichroism spectroscopy for the
variants Rs-AFP2(V39R) and Rs-AFP2(Y38G). In the case of Rs-AFP2(V39R),
the circular dichroism spectrum was virtually identical to that of
Rs-AFP2, whereas the spectrum of Rs-AFP2(Y38G) showed a slightly
altered spectrum in the 190-280 nm region. This alteration is most
probably due to the conformational flexibility of glycine within a
polypeptide chain. The presence of glycine in the type VI -turn
connecting
-strand 2 and
-strand 3 may entail some relaxation of
this region and loosen the packing of the
-sheet. The conformation
of the other variants was not verified, but the absence of free thiol
groups indicated that the disulfide bridges had formed.
Within the first substitution series, variants that showed a clearly
reduced activity on F. culmorum were Rs-AFP2(T10G),
Rs-AFP2(A31W), Rs-AFP2(Y38G), Rs-AFP2(F40M), and Rs-AFP2(P41). The
importance of the residues at positions 10, 38, and 40 is underscored
by our observation that substitution variants in which those residues were replaced by an alanine showed a similar drop in antifungal activity.2 In the second series, consisting
of arginine substitution variants, additional variants were identified
that displayed reduced antifungal activity, namely Rs-AFP2(S12R),
Rs-AFP2(L28R), Rs-AFP2(A42R), and Rs-AFP2(I46R). It is not clear
whether the reduction in antifungal activity of these variants was due
to the unfavorable presence of an extra charge or to the replacement of
an amino acid essential for the antifungal activity.
Remarkably, the loss in antifungal potency in all these cases was less noticeable in the low ionic strength medium than in the medium supplemented with 1 mM CaCl2 and 50 mM KCl. This may be explained by assuming that the interaction between Rs-AFP2 and its putative receptor on fungal hyphae is based both on ionic interactions and non-ionic stereospecific interactions. Upon increasing the ionic strength of the medium, the ionic interactions with the putative receptor are weakened due to competition between Rs-AFP2 and inorganic cations. In the case where non-ionic stereospecific interactions are weakened due to an unfavorable substitution, the overall interaction is also expected to become more susceptible to ionic competition.
The two most interesting Rs-AFP2 analogues of the arginine substitution series are Rs-AFP2(G9R) and Rs-AFP2(V39R). Although these variants show no significantly increased activity on F. culmorum in the low ionic strength medium, their activity on this fungus is much less influenced by the presence of cations in comparison with wild-type Rs-AFP2. This is again consistent with our model, which predicts that the interaction between Rs-AFP2 and its putative receptor is based both on ionic and non-ionic interactions. Introducing an extra charged residue at positions 9 or 39 may reinforce the ionic interactions, leading to variants that are at an advantage in competing with cations for binding at the putative receptor site.
The relative antifungal potency of the arginine substitution variant Rs-AFP2(V39R) compared to Rs-AFP2 appeared to be dependent on the test fungus. Rs-AFP2(V39R) was more active on F. culmorum, N. hematococca, and V. dahliae (three taxonomically related fungi belonging to the family Nectriaceae), but less active on P. betae. This suggests that the putative receptor on hyphae of different fungal species may reveal conformational or compositional differences.
As relatively high ionic strength conditions occur in all plant cell compartments (17), Rs-AFP2 variants such as Rs-AFP2(G9R) and Rs-AFP2(V39R) displaying a decreased cation antagonism in their activity against some phytopatogenic fungi could be useful for plant transformation experiments aimed at obtaining disease-resistant crops. We have previously shown that transgenic tobacco plants expressing wild-type Rs-AFP2 are more resistant to the fungal pathogen Alternaria longipes than untransformed plants (8). Further enhancement of the resistance level may be achieved through the expression of either Rs-AFP2(G9R) or Rs-AFP2(V39R) in transgenic plants.
We have previously shown that Rs-AFP2 stimulates Ca2+ uptake by fungal hyphae, an effect that can be observed within minutes after addition of the peptide (15). This stimulation of Ca2+ uptake may be part of the responses triggered by the interaction of Rs-AFP2 with its putative receptor. Our results now seem to indicate that antifungal activity and ability to trigger enhanced Ca2+ uptake are correlated. Indeed, the variant Rs-AFP2(Y38G), which is virtually devoid of antifungal activity in presence of inorganic salts, was unable to stimulate Ca2+ uptake in F. culmorum. On the other hand, the arginine substitution variant Rs-AFP2(V39R) displaying enhanced antifungal potency caused about 2.5-fold higher Ca2+ uptake than Rs-AFP2. Controlled Ca2+ influx is believed to be essential for directing polar growth at the tip of fungal hyphae (35). For pollen tubes, which like fungal hyphae grow at their tip, it has been documented that various treatments resulting in elevated cytosolic Ca2+ levels invariably lead to growth arrest (36).
The three-dimensional structure of Rs-AFP1 has been studied by
two-dimensional 1H NMR, which has revealed that Rs-AFP1
consists of an -helix (Asn-18-Leu-28) and a triple-stranded
antiparallel
-sheet (
-strand 1: Lys-2-Arg-6;
-strand 2:
His-33-Tyr-38;
-strand 3: His-43-Pro-50) (Fig. 6;
Ref. 37). Meanwhile, the structure of Rs-AFP1 has been refined down to
a root mean square deviation of 1.60 Å for all heavy atoms of the
backbone, and the results of this refinement will be presented
elsewhere. Since Rs-AFP1 is near-identical to Rs-AFP2, it is assumed
that it adopts the same conformation. The spatial orientation of the
residues affecting the antifungal activity of Rs-AFP2 upon substitution
was analyzed using the high resolution structure of Rs-AFP1. According
to the Rs-AFP1 model, all residues substituted in the present study do
face outwards of the peptide backbone and are therefore unlikely to be
essential for structure stabilization. The only exception is residue
Ala-31, which is positioned at the interior face of the hairpin loop
connecting the
-helix to
-strand 2 (Fig. 6). Substitution of
Ala-31 by a bulky tryptophan residue in Rs-AFP2(A31W) most probably
results in a conformational distortion, which might explain the drastic reduction of the antifungal activity of this variant. In addition, deletion of Pro-41, which adopts a cis-configuration in Rs-AFP1 as part
of a type VI
-turn, is also likely to entail a distortion of at
least the domain encompassing the second and third
-strand and the
interconnecting type VI
-turn.
A graphical overview of the specific antifungal activity determined on
F. culmorum of the different amino acid substitution variants when assayed in high ionic strength medium is provided in Fig.
7. When those residues affecting the antifungal activity are visualized on a three-dimensional model (Fig. 8), it
becomes apparent that they all cluster into two adjacent sites. A first site is formed by the residues Tyr-38, Phe-40, Pro-41, Ala-42, Lys-44,
and Ile-46. Except for Pro-41 and Lys-44, all those residues are highly
hydrophobic. When Lys-44 was substituted by the neutral residue Gln, a
substantial decrease of the antifungal potency was observed, suggesting
that a positive charge within this predominantly hydrophobic cluster is
important for the antifungal activity. This is further substantiated by
the observation that the introduction of an additional positive charge
within this site at position 39 resulted in enhanced antifungal
activity in the presence of inorganic salts. The second site is formed
by Thr-10, Ser-12, Leu-28, and Phe-49, which form a patch of contiguous
residues despite their scattered positions along the Rs-AFP2 sequence
(Fig. 7). Here again, introducing a positive charge within the cluster, namely at position 9, resulted in an enhanced antifungal potency in the
high ionic strength medium.
The two regions important for the antifungal activity of Rs-AFP2 might constitute two sites contacting a single putative receptor. Alternatively, the presence of two sites could be indicative of two binding sites on each of two receptor molecules. The latter possibility has been proposed in a model for the interaction between the human growth hormone and its receptor (38). In the case of the human growth hormone, a mutational analysis has also revealed two domains that are involved in the interaction with the human growth hormone receptor. Each of the two domains interacts with a receptor molecule, entailing receptor dimerization, the initial trigger in the signal transduction pathway (39). The physiological meaning of the two functional sites of Rs-AFP2 will remain an open question until its putative receptor has been identified and characterized.