PCR 40 pmol of the primer Q was used as
downstream primer and the upstream primer was 150 pmol of the degenerated primer P1 5-
GCACGTTGCTACGACGTTGCTATG- AA: position 315 – 331 in the IWF5 cDNA se-
quence. In the second nested PCR 50 pmol of the primer Q
1
was used as downstream primer and the upstream primer was 50 pmol of the degenerated
primer P2
5-GGACGTATACTAACT- CACTAAGATA: position 354 – 370 in the
IWF5 cDNA sequence. The PCR conditions were: 1 min at 94°C, 2 min at 42°C, 1 min at 50°C
and 5 min at 72°C for 1 cycle followed by 1 min at 94°C and 2 min at 42°C and 3 min at 72°C for 35
cycles followed by 10 min at 72°C. After the second PCR a single DNA product of 390 bp was
obtained. The DNA product was cloned into the pT7Blue vector Novagen, Madison, WI and se-
quenced using a termo sequenase fluorescent cycle sequencing kit Amersham Pharmacia Biotech,
Uppsala, Sweden and an ALF DNA sequencer Amersham Pharmacia Biotech, Uppsala, Swe-
den.
2
.
8
.
5
RACE The sequence of the 5 end of IWF5 cDNA was
obtained by 5 RACE using the 5 RACE system from Gibco BRL MD, USA with 3 gene specific
primers constructed from the partial cDNA se- quence obtained by 3 RACE. Briefly, 1 mg of the
same total RNA as used for 3 RACE and 2.5 pmol of a gene specific primer GSP5-1 5-TG-
GAATTGGAGATTATGTAAG: position 619 – 643 in the IWF5 cDNA sequence was incubated
at 70°C for 10 min followed by the addition of reverse transcriptase and incubating at 42°C for 30
min, 70°C for 15 min and the addition of RNaseH and incubating further 10 min at 55°C. The cDNA
was dC-tailed according to the protocol of Gibco BRL. The tailed cDNA was subjected to two
rounds of PCR. In the first PCR 20 pmol of the 5-Anchor primer was used as upstream primer
and the downstream primer was 20 pmol of the gene specific primer GSP5-2 5-TCACTTTA-
GATGTAAGAAGCACACATG: position 596 – 622 in the IWF5 cDNA sequence. In the second
PCR 50 pmol of the 5-UNI primer was used as upstream primer and the downstream primer was
50 pmol of the gene specific primer GSP5-3 5- TAAGCAGAAAGTTCCAGAAAGCATG: posi-
tion 548 – 572 in the IWF5 cDNA sequence. The condition for the first PCR was: 1 min at 94°C and
1 min at 51°C and 2 min at 72°C for 35 cycles followed by 10 min at 72°C. The condition for the
second PCR was: 1 min at 94°C and 1 min at 55°C and 2 min at 72°C for 35 cycles followed by 10
min at 72°C. The single 600 bp DNA product was cloned into the pT7Blue vector Novagen and
sequenced using a Termo Sequenase fluorescent cycle sequencing kit Amersham and an ALF
DNA sequencer Pharmacia.
2
.
9
. Southern hybridization Genomic DNA was isolated from sugar beet
leaves according to Ref. [31], digested with appro- priate restriction enzymes and separated on a 0.8
agarose gel. A DNA probe of IWF5 cDNA was
32
P-labeled by random priming using the Ready- To-Go Labeling Kit from Pharmacia. Southern
transfer and hybridization was performed accord- ing to [32] using Hybond N + membranes Amer-
sham following the recommendations of the Manufacturer.
3. Results
3
.
1
. Purification and characterization of IWF
5
The IWF isolated from sugar beet leaves was subjected to cation exchange chromatography and
bound proteins eluted by stepwise increasing the salt concentration in the starting buffer 0.1, 0.3
and 0.5 M NaCl. The 0.3 M NaCl eluate dis- played strong antifungal activity and was further
purified on a Mono S FPLC column using a salt gradient. Five major and a number of smaller
protein peaks were eluted data not shown. One of the major protein peaks, showing antifungal
activity, separated into five major peaks and a number of minor peaks when subjected to rpH-
PLC Fig. 1. Mass spectrometric analysis of the individual fractions revealed the presence of two
previously identified proteins in fraction 2 AX2 and 3 IWF2 Table 1, while fraction 4 contained
a protein with a MW of 9319.07, in the following named IWF5 Fig. 2. In Fig. 2, both the doubly
charged MH
2 2 +
molecular ion at mz 4660.98 and singly charged MH
+
molecular ion at mz
9319.07 are observed as well as the molecular ion of the dimer M
2
H
+
at mz 18634.1. In fraction 5, a number of smaller proteins were present, none of
which could be identified on basis of their molecu- lar weight. Analysis by SDS-PAGE of fraction 4
using a high-density gel, revealed that this fraction contained a protein with an apparent MW of ca. 9
kDa as well as a minor component with a MW of ca. 8.5 kDa Fig. 3. The fraction was further
analyzed by tricine SDS-PAGE performed with and without reducing agent and silver stained
data not shown, and no shift in MW was ob- served when adding the reducing agent. Thus, the
protein exists as a monomer. The N-terminal se- quence of fraction 4 was determined after reduc-
tion and carboxymethylation and the first 38
Fig. 2. Mass spectrometric analysis of fraction 4 from the high performance liquid chromatography HPLC purifica-
tion shown in Fig. 1. In addition to the molecular ion at mz 9319.07, the doubly charged molecular ion at mz 4660.98
and the molecular ion of the dimer at mz 18634.1 are observed.
Fig. 1. High performance liquid chromatography HPLC elution profile of one of the major fractions from the CM-
Sepharose purification. IWF5 was present in peak 4. Proteins were eluted with a linear gradient from 5 to 80 of the
B-buffer as indicated dotted line. The flow rate was 1 ml min
− 1
. Fig. 3. Sodium dodecyl polyacrylamide gel electrophoresis
SDS-PAGE analysis of the purified sugar beet non specific lipid-transfer protein using high-density gels Pharmacia.
Molecular mass markers were CNBr-derived myoglobin frag- ments.
residues were determined Fig. 6, sequence in italic. The complete sequence was obtained by
cleavage of the reduced and alkylated protein with endoproteinase Lys-C, resulting in six peptides
Fig. 6, peptides derived from cleavage at Lys- residues bold are underlined. All peptides were
completely sequenced after mass spectrometric analysis. For all peptides, the determined sequence
could be verified by the observed mass Table 2. Alignment of the cysteines present in the IWF5-
derived peptides with those of IWF1 a nsLTP previously isolated from sugar beet, made it pos-
sible to deduce the entire sequence of IWF5 from this single digest Fig. 6. The deduced sequence of
IWF5 was confirmed by mass analysis of overlap- ping peptides generated by a second digestion with
endoproteinase Asp-N data not shown.
Table 1 Mass spectrometric analysis of the different fractions Fig. 1
from the rpHPLC purification of the active fraction from the fast protein liquid chromatography FPLC cation exchange
chromatography of the fraction eluting at 0.3 M NaCl from a crude cation exchange purification step
a
Mr obs. Fraction
Mr calc. Protein
4015.6 N.I.
1 N.I.
5178.8 2
5176.9 AX2
b
8937.7 3
8948.3 IWF2
c
9319.1 4
9317.7 IWF5
N.I. 5
2–4000 N.I.
a
Mr obs. and Mr calc. are the observed and calculated molecular masses of the fractions. N.I. not identified.
b
Ref. [23];
c
Ref. [16].
The MWs determined by SDS-PAGE and mass spectrometry 9319.1 are in good agreement with
that deduced from the amino acid sequence data 9317.7. In summary, IWF5 is a 92 amino acid
protein, eight of which are cysteines. A potential site for N-linked glycosylation Asn-Xxx-SerThr
at position 37 – 39 was found not to be glycosy- lated as verified by the good agreement between
the measured molecular mass and that deduced from the sequence.
3
.
2
. Characterization of the IWF
5
cDNA The nucleotide sequence of the full-length IWF5
cDNA clone, obtained by a combination of 3 and 5 RACE, and the deduced amino acid sequence is
shown in Fig. 4. The IWF5 cDNA contains 722 nucleotides including a polyA tail of 20 nucle-
otides. The sequence contains an open reading frame encoding 118 amino acid residues with the
first in frame ATG at position 96 and a stop codon at position 450. In comparison with the
amino acid sequence of IWF5, the cDNA seems to encode a precursor protein with an N-terminal
signal sequence of 26 amino acid residues followed by a functional domain of 92 amino acid residues,
identical to the amino acid sequence of the iso- lated IWF5.
3
.
3
. Antifungal acti6ity The in vitro antifungal activity of IWF5 was
tested against spore cultures of C. beticola grown in liquid growth medium in microtiter plates Fig.
5. The protein showed a strong growth inhibiting effect against C. beticola and the antifungal activ-
ity increased with increasing protein concentra- tions.
In control
cultures, growth
started approximately 30 h after incubation start Fig. 5,
triangles, while the addition of IWF5 caused a
Table 2 N-terminal sequence and observed and calculated Mr of the endoproteinase Lys-C derived peptides from IWF5
HPLC fraction Mr obs.
Mr calc. N-terminal sequence
1 RTACRCMK
1178.3 1178.5
2 YSAALPGK
805.9 805.9
1388.0 3
1387.5 TLRNLTPTTQDK
4 1164.9
1165.3 SAASAIPGINHK
CGVSIPGPVGPQAD 2075.3
2075.4 5
3644.3 AINCGLVSQSLAACLGFLENGQPNAACCNGVK
3647.0 6
Fig. 4. cDNA sequence and deduced amino acid sequence of IWF5. The amino acid sequence of the putative N-terminal signal sequence is in italic. The position of the primers see Section 2 used to generate the RACE cDNA clones are indicated with
arrows.
Fig. 5. Antifungal activity of IWF5 against spore cultures of C. beticola tested in the microtiter plate assay. The fungal
growth was measured as increase in absorbance at 620 nm. Two different concentrations of IWF5 were used in the wells:
16 mg ml
− 1
circles and 40 mg ml
− 1
squares. Growth of the control culture is indicated by the triangles.
Microscopical analysis of spore cultures of C. beticola grown in microtiter plates in the presence
of 16 mg ml
− 1
IWF5 shows that the hyphae were shorter and less branched as compared to control
culture data not shown.
3
.
4
. Homology to non-specific lipid transfer proteins
The amino acid sequence of IWF5 shows ho- mology to the family of nsLTPs. In Fig. 6 the
sequence of IWF5 is compared to the seven plant nsLTPs to which the highest degree of similarity
was found. The sequences were aligned [26] and gaps introduced to optimize the alignment. IWF5
shows 49 identity to an nsLTP isolated from spinach [33] and is 47 identical to IWF1 Fig. 6.
IWF1 has previously been isolated from sugar beet leaves [17] and comprises part of a small gene
family in sugar beet. When the amino acid se- quences of nsLTPs from different plant species are
compared, only few residues are conserved [34]. Among these are the eight cysteines and 12 posi-
tions occupied by hydrophobic or aromatic residues [35]. The amino acid sequence of IWF5
corresponds largely to this sequence homology including the presence of eight cysteines at con-
served positions at positions 4, 14, 28, 29, 49, 51, 75 and 89, numbering as in Fig. 6. However, two
of the positions normally occupied by aromatic or hydrophobic residues, have been exchanged with a
threonine at position 39 and a glycine at position 81.
concentration dependent delay in growth start Fig. 5, circles and squares. At 16 mg ml
− 1
, the growth was markedly delayed and strongly inhib-
ited Fig. 5, filled circles. At 40 mg ml
− 1
no fungal growth could be detected even at 160 h after assay start Fig. 5,
filled squares. The concentration required for 50 growth inhibition IC
50
after 100 – 120 h incuba- tion time was 2 – 4 mg ml
− 1
. The amount of protein needed for 50 growth inhibition is simi-
lar to the level found for the antifungal proteins IWF1 and IWF2 [17].
Fig. 6. Comparison of the amino acid sequences of antifungal sugar beet proteins IWF5, IWF1 [16], an non-specific lipid transfer protein nsLTP from spinach [30] and five nsLTPs isolated from rice or and almond pr. Identical residues are indicated by an
asterisk. Gaps marked by dashes, have been introduced to optimize the alignment. The N-terminal sequence obtained for IWF5 is shown in italic. Cleavage sites of endoproteinase Lys-C are indicated in bold, and the obtained peptides are underlined.
Consensus symbols: is any amino acid, is I or V, is L or M, is F or Y. Residues in small letters indicates the most probable amino acid, while capital letters in bold are conserved residues.
3
.
5
. Southern hybridization Genomic DNA from sugar beet was digested
by the restriction enzymes BamHI or XbaI and subjected to Southern hybridization analysis us-
ing IWF5 cDNA as probe. Each restriction en- zyme digest resulted in only one hybridizing
band using stringent conditions, indicating that only one gene or a small number of genes for
IWF5 is present in the genome of sugar beet.
4. Discussion
