From the Dipartimento di Biotecnologie Agrarie ed Ambientali,
Facoltà di Agraria and the Istituto di Biochimica,
Facoltà di Medicina e Chirurgia, University of Ancona, Via Brecce
Bianche, 60100, Ancona, Italy
Received for publication, February 13, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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A novel protein factor, named PcF, has been
isolated from the culture filtrate of Phytophthora cactorum
strain P381 using a highly sensitive leaf necrosis bioassay with tomato
seedlings. Isolated PcF protein alone induced leaf necrosis on
its host strawberry plant. The primary structure and cDNA sequence
of this novel phytotoxic protein was determined, and BLAST searches of
Swiss-Prot, EMBL, and GenBankTM/EBI data banks showed that
PcF shared no significant homology with other known sequences. The
52-residue PcF protein, which contains a 4-hydroxyproline residue along
with three S-S bridges, exhibits a high content of acidic sidechains,
accounting for its isoelectric point of 4.4. The molecular mass of
isolated PcF is 5,622 ± 0.5 Da as determined by mass spectrometry
and matches that calculated from the deduced amino acid sequence with
cDNA sequencing. The cDNA sequence indicates that PcF is first
produced as a larger precursor, comprising an additional N-terminal,
21-residue secretory signal peptide. Maturation of this protein
involves the hydroxylation of proline 49, a feature that is unique
among other known secreted fungal phytopathogenic proteins.
In modern agriculture, the selection of pathogen-resistant
cultivars remains of the utmost importance. Conventional breeding selection protocols are relatively inefficient as a consequence of the
general lack of genetic variability in cultivated plants as well as the
inability to keep pace with the rapid adaptation of pathogen genotypes.
New strategies, aimed at achieving resistant plants through gene
engineering, require an in depth knowledge of the mechanism of
pathogenesis at the molecular level. Host-pathogen interactions can
result in either "incompatibility" (resistance) or
"susceptibility" (pathogenesis). In either case, mounting evidence suggests that the process is mediated by the production of so-called elicitor- and toxin-signaling molecules (1-3). Signal recognition at
the plant cell surface triggers an ordered cascade of downstream events, leading to a range of host-cell responses (4-7). Because elicitor and toxic mediators play a central role, elucidation of the
mechanism of plant-pathogen interactions promises to provide insights
about strategies for incorporating pathogen resistance in cultivated plants.
The European cultivated strawberry plants (Fragaria vesca × ananassa Duch.) are mostly susceptible to attack by
Phytophthora cactorum (8, 9), a pathogenic oomycete for many
herbaceous and woody plants because of its wide range host specificity.
This pathogen is the causal agent of the "leather rot" and "root
rot" diseases in strawberry plants, whose morphological symptoms are recognized by rotting of root, crown, and fruit tissues (10). Plich and
Rudnicki (11) first reported that culture filtrates of P. cactorum possessed phytotoxins with action on tomato and involvement in the development of apple tree diseases. Most of these
metabolites, however, have not been purified and characterized. The
exception is "cactorein" from P. cactorum, a secreted
protein that elicits an incompatibility reaction when applied to
non-host tobacco, that is reportedly a classical avirulence gene
product (12, 13). This elicitor belongs to the 10-kDa elicitin family, a highly conserved protein group from Phytophthora spp.
showing structural and functional similarities (14, 15).
We recently identified a small cysteine-rich protein endowed with toxic
activity on both strawberry and tomato plants that is present in
culture filtrates of P. cactorum (P381 strain) previously isolated from infected strawberry plants (16). The molecular characteristics of this protein do not possess the properties of known
members of elicitins from Phytophthora spp. This phytotoxic protein also appears to be different from other agents involved in
plant-pathogen interaction. In this report, we describe the isolation,
characterization, and molecular cloning of this novel phytotoxic
protein, hereby named
PcF.1
Materials--
All chemicals were of analytical grade and were
obtained from Sigma, except where indicated. HPLC and FPLC
chromatographic procedures were performed on an AKTA Purifier system
(Amersham Pharmacia Biotech).
Fungal Culture--
P. cactorum strain P381, isolated
from infected strawberry plants, was kindly provided by Prof. G. Cristinzio (University of Naples "Federico II", Italy) to Prof. P. Rosati. They were routinely grown on a solid medium (13.5 g/liter Difco
Bacto-agar, 250 ml/liter tomato juice, and 2.7 g/liter
CaCO3, pH 6.25) in the dark at 25 °C. Maintenance
subculture was carried out every 10-15 days. For growth in liquid
culture, a medium deprived of yeast extract and peptone, adapted after
Hall et al. (17) was used. Growth was carried out in a
rotary shaker over 20 days at 25 °C in the dark. Other
Phytophthora species used, namely Phytophthora nicotianae, Phytophthora cinnamomi, Phytophthora
cryptogea, Phytophthora capsici, Phytophthora
citrophthora, and Phytophthora infestans were kindly
provided by Prof. A. Scala (University of Florence, Italy).
Bioassays--
Phytotoxic activity of either crude culture
filtrate or purified fractions, was routinely assayed on tomato
seedlings (18). Tomato seedlings (Lycopersicon esculentum
cv. Marmande) were grown in a moisture chamber until they had produced
only the two cotyledon leaflets. They were then resected near the roots
and incubated for 24-36 h in Eppendorf tubes containing 100 µl of
the solution to be assayed. Each assay was made in duplicate and
controls were performed to test the effect of the various buffers on
the viability of the seedlings. The activity was evaluated by the
ability of inducing distal necrosis on tomato leaves, and it was scored
using an arbitrary scale, ranging from 0 (symptomless) to 5 (complete leaf necrosis and wilting). When appropriate, the toxic activity was
assayed directly on Fragaria vesca × ananassa host plant leaves. The leaves were detached from a
mature plant of a strawberry cultivar susceptible to the P381 isolate
of P. cactorum. 100-µl aliquots of each solution to be
assayed were appropriately infiltrated into the lower leaf surface and
incubated at room temperature in a moisture chamber. The formation of a
necrotic area, localized overleaf to the infiltration site, was then
evaluated at intervals after infiltration. Appropriate controls were
carried out in parallel to test for the effect of the solvent buffers.
PcF Protein Purification--
PcF protein was purified starting
from 2 liters of P. cactorum culture. Mycelia and spores
were removed from the exhausted medium by filtration through a 3MM
paper (Whatman), followed by centrifugation at 13,000 × g for 30 min at 4 °C. The 13,000 × g
supernatant is referred as the culture filtrate. All the subsequent purification steps were carried out at room temperature.
DEAE-Sepharose Fast Flow Chromatography--
The P. cactorum culture filtrate was adjusted to pH 5.5 with 1 M NH4OH, and loaded onto a DEAE-Sepharose Fast
Flow column (2.6 × 9 cm, Amersham Pharmacia Biotech) previously
equilibrated with 10 mM ammonium acetate, pH 5.5 (buffer
A). After washing the column with buffer A, the elution was carried out
isocratically with buffer A containing 1 M KCl (buffer B).
A 5 ml/min flow rate was maintained. Aliquots of the eluted fractions
were assayed for toxicity on tomato seedlings as described above.
Active fractions were combined and added to trifluoroacetic acid and
acetonitrile to 0.1 and 7.5% final concentrations, respectively
(DEAE-Sepharose pool).
Resource RPC FPLC Chromatography--
The DEAE-Sepharose pool
was filtered through a 0.22-µm membrane (Millipore) and loaded onto a
FPLC Resource RPC column (3 ml, Amersham Pharmacia Biotech), previously
equilibrated with 0.1% trifluoroacetic acid, 7.5%
acetonitrile. The column was eluted at a flow rate of 2.5 ml/min with a
discontinuous gradient of acetonitrile obtained with buffers A (0.1%
trifluoroacetic acid) and B (0.1% trifluoroacetic acid, 65%
acetonitrile). The gradient conditions were: 11.5% buffer B for 16 min; 11.5-60% B in 112 min; 60-100% B in 16 min, and then hold at
100% B for 24 min. Because of the interference of both trifluoroacetic
acid and acetonitrile on the tomato bioassay, 100-µl aliquots of each
fraction were evaporated under vacuum (Speed-Vac, Savant) and
resuspended in distilled water before performing the bioassay. Active
fractions were pooled and added to 10 mM ammonium acetate,
pH 5.5, 20 mM NaCl. The pH was adjusted to 5.5 with 1 M NH4OH (Resource RPC pool).
TSK-DEAE FPLC Chromatography--
The Resource RPC pool was
loaded onto a FPLC Spherogel TSK-DEAE column (4 × 300 mm, Altex),
previously equilibrated with 10 mM ammonium acetate buffer,
pH 5.5, 20 mM NaCl. The elution of the column was carried
out with a discontinuous gradient of NaCl obtained with buffers A (10 mM ammonium acetate buffer, pH 5.5, 20 mM NaCl)
and B (10 mM ammonium acetate, pH 5.5, 0.5 M
NaCl). A flow rate of 1 ml/min was maintained. The gradient conditions were: buffer A from 0 to 5 min; 0-27% buffer B in 50 min; 27-100% B
in 5 min, and then hold at 100% B for 5 min.
LC-18 FPLC Chromatography--
The pooled biologically active
fractions from the previous step were directly applied to an FPLC
Supelcosil LC-18-DB column (4.6 × 250 mm, Supelco), equilibrated
with 0.1% trifluoroacetic acid buffer. The column was eluted at a flow
rate of 1.3 ml/min, with a discontinuous gradient of acetonitrile
obtained with buffers A (0.1% trifluoroacetic acid) and B (0.1%
trifluoroacetic acid, 65% acetonitrile). The gradient conditions were:
buffer A from 0 to 10 min; 0-60% buffer B in 80 min; 60-100% B in 4 min, and then hold at 100% B for 15 min. The active pool was dried
under vacuum (Speed-Vac, Savant), dissolved in distilled water, and stored at Protein Determination--
Protein concentration was routinely
evaluated according to Bradford (19), using bovine serum albumin as the
standard. The protein concentration of pure PcF protein was determined
spectrophotometrically using a molar Evaluation of Molecular Size--
Relative molecular mass of
pure PcF was evaluated by tricine-SDS-PAGE as described by
Schägger and Von Jagow (22), with minor modifications. Before
loading the gel, the pure PcF protein sample was heated to 100 °C
for 5 min in a denaturating mixture containing 2% SDS, 5%
Determination of Isoelectric Point--
The isoelectric pH value
of PcF was determined by FPLC chromatofocusing. A 5-µg aliquot of
pure PcF, dissolved in 25 mM Bis-Tris buffer, pH 6.5, was
loaded onto a Mono P HR 5/5 FPLC column (1 ml, Amersham Pharmacia
Biotech) previously equilibrated with the same buffer. Elution was
performed with 10-fold diluted Polybuffer 74 (Amersham Pharmacia
Biotech), pH 4.0.
Amino Acid Analysis--
4-µg samples of pure PcF protein were
heated to 155 °C in evacuated sealed tubes for 90 min in the
presence of 6 N HCl. Amino acid analysis was carried out on
a Cromakon 500 (Kontron) automated analyzer using an
o-phthalaldehyde postcolumn derivatization procedure (23).
Alternatively, for the determination of the hydroxyproline residues
(Hyp), amino acid analysis was performed by HPLC on LC-18 column after
a PITC precolumn derivatization, according to Heinrikson and Meredith
(24). Cysteine was determined as cysteic acid after performic acid
oxidation (25). Tryptophan content was investigated spectrophotometrically, according to Edelhoch (26).
SH Groups Determination--
Free thiol groups were determined
through mass spectrometry analysis by comparison of the native PcF
protein molecular mass with the mass of the protein after
N-terminal Sequencing--
After spotting the protein (10 nmol)
onto a polyvinylidene difluoride membrane, N-terminal sequencing was
carried out on both native PcF protein and Pam-PcF protein by automated
Edman degradation on a Procise Model 491 gas-phase sequencer, connected
with an online phenyl thiohydantoin (PTH) amino acid analyser, model
140C (Applied Biosystems, Foster City, CA). Amino acids were assessed, as their PTH derivatives, on the basis of coelution with appropriate standards (20 PTH-amino acid standard solution, PerkinElmer Life Sciences), with the exception of the Fungal RNA Preparation--
Fresh P381 P. cactorum
mycelia (100-mg aliquots, wet weight), harvested by filtration from the
liquid culture, were collected in DEPC-treated tubes and directly
pestle-homogenized in liquid nitrogen. Subsequently, total RNA and
poly(A+) RNA extractions were carried out with the High
Pure RNA Isolation kit (Roche Molecular Biochemicals) and the mRNA
Capture kit (Roche Molecular Biochemicals), respectively.
RACE Experiments--
A cDNA partial sequence encoding for
the PcF protein was obtained from the fungal poly(A+) RNA
after a one-step cDNA synthesis and amplification (RT-PCR). RT-PCR
was directly performed in the oligo(dT)-coated PCR tubes, saturated
with poly(A+) RNA by the TitanTM One Tube
RT-PCR System kit (Roche Molecular Biochemicals). The degenerate
oligonucleotide primers used, PcF 1 and PcF 2, are represented in Fig.
3. The RT-PCR conditions were: 30 min at 50 °C; 2 min at 94 °C;
10 cycles (30 s at 94 °C; 30 s at 45 °C; 1 min at 68 °C);
25 cycles (30 s at 94 °C; 30 s at 45 °C; 1 min at 68 °C,
with 5-s elongation increase at each cycle); 7 min at 68 °C.
Furthermore, both 3'- and 5'-end extensions of the cDNA partial
sequence were performed with the 5'/3'-RACE kit (Roche Molecular
Biochemicals), according to the instructions and reagents provided by
the manufacturer (29). The high fidelity DyNAzymeTM EXT DNA
polymerase was obtained from Finnzymes (Finland). The oligonucleotide
primers used, designed from the cDNA partial sequence, were as
depicted in Fig. 3. The oligo(dT) anchor primer, V1, and the anchor primer, V2, were part of the kit. cDNA
synthesis was performed by AMV reverse transcription from 2 µg of
total RNA in the presence of either the PcF 3 primer, for 5'-end
extension, or the V1 primer, for 3'-end extension.
Thereafter, for 5'-RACE, the single strand cDNA was purified (High
PureTM PCR Products Purification kit, Roche Molecular
Biochemicals), and poly(A) tailed at the 3'-end by terminal
dTransferase. Tailed cDNA was then used as the template for PCR in
the presence of the V1 and PcF 3 primers. A nested PCR was
carried out by diluting an aliquot of the sample in a second PCR
mixture containing the V2 and PcF 5 primers. A second
nested PCR was further performed, in the same manner with the
V2 and PcF 7 primers. Similarly, for 3'-RACE, the single
strand cDNA was utilized as the template in the subsequent PCR and
two nested PCRs. The anchor primer V2 was used in all
amplifications, either together with the PcF 4 primer (first
amplification) or with the PcF 6 primer (nested PCR) or with the PcF 8 primer (second nested PCR). Throughout, PCR conditions were as follows:
2 min at 94 °C; 10 cycles (15 s at 94 °C; 30 s at 65 °C;
40 s at 72 °C); 25 cycles (15 s at 94 °C; 30 s at 65 °C; 40 s at 68 °C, with 20-s elongation increase at each
cycle); 7 min at 72 °C.
Cloning and Sequencing--
The cDNA fragments obtained from
RACE experiments were re-amplified by PCR in 50-µl reaction mixtures
containing 10 mM Tris-HCl buffer, pH 8.3, 50 mM
KCl, 0.2 mM each dNTP, 1.5 mM
MgCl2, 1 unit of AmpliTaq DNA polymerase (Perkin Elmer), 10 pmol of each primer, and an appropriate amount of the template. PCR
conditions were as follows: 96 °C for 7 min; 30 cycles (30 s at
96 °C; 30 s at the appropriate annealing temperature, either
45 °C or 65 °C; 30 s at 72 °C); 72 °C for 30 min. The
annealing temperature was either 45 °C, for re-amplification of the
first cDNA fragment, or 65 °C, for re-amplifications of both 3'-
and 5'-end extension fragments. The amplified cDNA fragments were
then purified from the agarose gel and ligated by a standard TA cloning
procedure, in the pGEM-5Zf(+) vector (2× Rapid Ligation Buffer kit,
Promega). After transformation of the Escherichia coli JM109
strain, the recombinant plasmid clones were purified and subjected to
automated dideoxy chain-termination sequencing (30).
Purification of the PcF Protein--
The presence of a phytotoxic
activity in the culture filtrates of P. cactorum P381
strain, was determined through bioassays on tomato seedlings (31). We
found that about 3 µg of culture filtrate protein was the minimum
required to attain a toxicity score of 5 within 17 h. During
subsequent purification, toxic activity was only observed in those
chromatographic fractions that yielded the pure PcF protein (Fig.
1). Because there were no other active
fractions, the tomato bioassay appears to be highly sensitive and
specific for PcF. Furthermore, although the activity resisted to
heating at 100 °C for 5 min (32), the proteinaceous nature of the
secreted P. cactorum toxin(s) responsible for tomato seedling necrosis was indicated by loss of toxicity upon combined Pronase and heat treatments (32).
The protein factor, named PcF, with reference to the organisms P. cactorum and Fragaria that are involved in the
interaction, was purified to homogeneity. The four chromatographic
treatments comprising a typical purification procedure are illustrated
in Fig. 1. From an initial 2 liters of culture filtrate containing about 50 mg of total protein, the procedure yielded about 200 µg of
pure PcF protein, corresponding to an enrichment factor of 250. The
active final preparation exhibited a single protein band on SDS-PAGE
(not shown). The absence of protein contaminants was also evidenced by
automated Edman degradation sequencing, which resulted in one single
N-terminal sequence (see below). The final preparation was used for the
subsequent characterization of the PcF protein.
Biological Activity--
We found that 3-5 µg of pure PcF
protein were sufficient to induce a complete leaf necrosis on the
tomato seedling bioassay within 20 h. Under the same bioassay
conditions, similar amounts of the
By applying the very same PcF purification scheme on the culture
filtrates of two other P. cactorum strains isolated
independently from infected strawberry plants harvested from different
Italian fields, we obtained a protein that had six N-terminal residues that were identical to PcF (not shown). In addition, with the specific
bioassay on tomato seedlings, the toxic activity was only found in the
culture filtrates from P. cactorum and P. nicotianae; none was found from P. cinnamomi, P. cryptogea, P. capsici, P. citrophthora, or
P. infestans. Furthermore, P. nicotianae, a well known tomato pathogen, from which other phytotoxic proteins have been
characterized (18, 35), lacked the PcF protein based on the PcF
purification protocol. Therefore, the observed constitutive PcF protein
expression and secretion from P. cactorum RACE, but not from
other Phytophthora spp., leads us to hypothesize that PcF
might be either unique for the P. cactorum, or, possibly, for those P. cactorum strains that are pathogenic to strawberry.
Molecular Characterization--
Chromatofocusing of pure PcF
protein revealed an acidic isoelectric point of 4.4 ± 0.2. ISMS
analysis of the native protein showed a molecular mass of 5,622 ± 0.5 Da. Following
A partial N-terminal sequence, corresponding to the first 50 amino
acids, was directly determined by automated Edman degradation of the
pure PcF protein, demonstrating that the N terminus is unblocked. In
addition, automated sequencing did not reveal any heterogeneity,
confirming the protein homogeneity. The first residue is represented by
a glutamic acid, instead of methionine. The 6 cysteine residues were
assigned, as Pam-Cys, at positions 6, 11, 26, 39, 40, and 44. Proline
residues were found at positions 3 and 12. The 49th Edman cycle did not
yield any signal that would match with any of the standard PTH-derived
amino acids, rather showing two unidentified peaks eluting just before
PTH-derived histidine and after PTH-derived alanine, respectively.
These two peaks were subsequently identified as trans-4-Hyp
and cis-4-Hyp, both on the basis of co-elution with the
corresponding genuine standards, and by comparison with the retention
times of expected peaks from 4-Hyp-containing polypeptides, as
described in the "Experimental Procedures." The presence of
trans-4-Hyp in the PcF protein native structure was also
confirmed by amino acid analysis of the PTC-amino acid derivatives from
the hydrolyzed PcF protein, by comparison with trans-4-Hyp
standard (Sigma). Even though our results do not enable a clarification
of whether the cis, trans, or both isomers are
present, these findings appear to be consistent with the presence of a
4-Hyp amino acid residue at position 49 in the mature PcF protein.
The presence of a 4-hydroxyproline residue represents a unique feature
of the PcF protein with respect to known fungal phytotoxic proteins
(36-41). The involvement of a prolyl 4-hydroxylase (EC 1.14.11.2) in a
post-translational hydroxylation of proline 49 is suggested by
(a) the striking specificity for only one of the three
proline residues found in the PcF protein sequence, and (b)
modification at the C-4 position in the proline ring. Although the
function of this post-translational modification of the PcF protein
remains to be investigated, several hydroxyproline-rich proteins have
been described in the cell wall of pseudo-fungal oomycetes (42).
Moreover, 4-hydroxyproline is known to be involved in the-folding and
stabilizing of the collagen triple helix (43). This hydroxyimino acid
also plays a role in linking arabinose units of carbohydrate moieties
attached to the plant cell wall extensins (44).
Even after accounting for the changes in mass resulting from
hydroxylation of proline 49 and the formation of three disulfide cross-links, the 50 residues that were sequenced yielded a molecular mass of 5,464 Da, a value that is 158 mass units lower than the ISMS-determined mass of 5,622 Da. This discrepancy suggested that one
or two additional residues are present at the C terminus. Because PcF
protein lacks methionine residues, thereby precluding CNBr digestion,
and because PcF resists digestion by trypsin, Asp-N endoproteinase, and
Glu-C endoproteinase (not shown), no additional C-terminal fragments
could be sequenced. We also found that the PcF protein is not an
inhibitor of any of the above proteases (not shown). To investigate the
upstream N-terminal and the downstream C-terminal sequences, we
resorted to cDNA cloning and sequencing.
cDNA Cloning--
Knowledge of the partial N-terminal PcF
sequence allowed us to design oligonucleotide primers to identify the
PcF coding cDNA by means of RT-PCR. Degenerate primers PcF 1 and
PcF 2 correspond to amino acid residues 1-8 and 41-48, respectively
(Fig. 3). After RT-PCR, a single 144-bp
cDNA was isolated, and the sequence of this cDNA yielded a
deduced amino acid sequence that exactly matched amino acid residues
1-48 of the chemically determined protein sequence (Fig.
3A). RACE experiments were undertaken to obtain information
on both the 3'- and 5'-end of this original fragment, and thus on the
complete PcF-coding cDNA sequence. Our hypothesis that PcF protein
is likely to be synthesized as a larger precursor is based on the
consideration that (i) PcF is a secreted protein, and (ii) that the
N-terminal amino acid is a glutamate, as demonstrated by chemical
sequencing, and not a methionine. RACE experiments yielded single
fragments for both 3'- and 5'-end extensions (Fig. 3, B and
C), as indicated by agarose gel electrophoresis. After cloning and sequencing, we obtained a single 5'-RACE clone of 179 bp
and two 3'-RACE clones of 211 bp and 220 bp. Both 3'-RACE clone
sequences proved to be identical; however, the larger sequence showed
an additional 9-bp sequence at its 3'-end, presumably arising from a
different polyadenylation site on the mRNA. All three RACE clones
overlapped with the corresponding cDNA ends of the original fragment isolated by RT-PCR, confirming their authenticity. The discrepancies because of the third-base degeneration of the primers originally utilized, were accordingly corrected. Fig.
4 shows the complete cDNA sequence
encoding for the PcF protein. A data bank (Swiss-Prot, EMBL, and
GenBankTM/EBI) survey using the BLAST program failed to
yield any significant similarity score with any other known sequences.
Furthermore, the PcF protein appears to be unique among other known
phytotoxic proteins from Phytophthora spp. such as the
10-kDa elicitin family (15, 39) or the 42-kDa glycoprotein elicitor
(41).
Upon inspection of the N-terminal region of the PcF-coding cDNA,
one finds an ATG start codon 63-bp upstream from the first amino acid
in the mature PcF protein. Therefore, the PcF protein appears to be
first synthesized as a 73-residue pre-protein precursor, which then
undergoes proteolytic removal of a 21-amino acid signal peptide.
Indeed, the SignalP program at the Expasy database server predicted
that the cleavage site should occur between residues 21 and 22 of the
full-length pre-protein sequence, in excellent agreement with
experiment. Moreover, the cleaved peptide sequence strikingly conformed
to all the proposed criteria for a signal peptide that designates the
processed protein for extracellular secretion (45). The C-terminal
region was predicted to encode for proline 49, alanine 50, serine 51, and a terminal alanine-52 residue, after which a TAG stop-codon is
present. Therefore, the secreted mature PcF protein sequence must
comprise 52 amino acid residues. Taking into account the hydroxylation
of proline 49 and the occurrence of 3 disulfide bridges, the
PeptideMass program at the Expasy server yielded a predicted 5,622.18 molecular mass and a predicted 4.4 isoelectric point, which agree with
the experimental data reported above.
The computer-generated hydropathy profile (Fig. 4, bottom)
indicated the presence of two distinct domains, a predominantly hydrophilic C terminus and a clustered hydrophobic region at the N-terminal. This amphipathic structure has a grand average of hydropathy (GRAVY) score of
In conclusion, we have purified and characterized a novel secreted
phytotoxic protein that was assigned the name PcF to indicate the first
letters of its organism-of-origin, namely the pathogenic oomycete
P. cactorum, and its host plant Fragaria, the
strawberry. Our findings suggest that PcF is most likely a tightly
bound structure in its native state, consistent with its observed small
size and high, intramolecular S-S bridge content. The phytotoxic PcF
protein can be considered to be a new member of the low molecular
weight extracellular fungal proteins responsible for pathogenesis in certain host plants (3).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
20 °C.
280 nm of 3,355 M
1 cm
1, as calculated from the
amino acid composition according to Pace et al. (20).
-Propionamidation--
Pure PcF protein (2 nmol) was
subjected to cysteine alkylation with acrylamide, after protein
denaturation-reduction in 2% SDS, 0.1 M DTT, according to
Brune (21). The resulting
-propionamidated protein (Pam-PcF) was
purified by gel filtration on an FPLC Superdex Peptide PE 7.5/300
column (Amersham Pharmacia Biotech), equilibrated and eluted with 0.1%
trifluoroacetic acid, 30% acetonitrile.
-mercaptoethanol, and 10 mM DTT. Alternatively, precise
determinations of the molecular mass of pure native PcF and Pam-PcF
proteins were performed by ion spray mass spectrometry (ISMS) on a
Navigator apparatus (Finnigan, Manchester, UK).
-propionamidation in the absence of 0.1 M DTT. Following
-propionamidation, the expected mass increase corresponds to 71 Da
per free SH group.
-propionamidated cysteine (Pam-Cys) and the hydroxyproline residues that did not yield any signal
matching the above standards. The cysteine residues were identified as
PTH-2-propionamide cysteine (21). The 4-hydroxyproline (4-Hyp) residue
was identified, as a double peak by comparison with the retention times
of expected peaks obtained during the automated sequencing of both
[Hyp3]bradykynin (Sigma) and tryptic fragments from human
collagen, type IV (Sigma). Both latter proteins are known to contain
4-Hyp at known positions in their primary structure (27, 28). The double-peak yield of PTH-4-Hyp, resulting from the automated sequencing process of the above 4-hydroxyproline-containing proteins, was further
investigated, and the two peaks were subsequently identified as
trans-4-Hyp and cis-4-Hyp, respectively, on the
basis of coelution with the corresponding genuine standards (Sigma),
directly loaded onto the glass fiber disc of the sequencer cartridge,
followed by a single Edman cycle.
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ABSTRACT
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RESULTS AND DISCUSSION
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Fig. 1.
PcF protein purification procedure from the
culture filtrate of P. cactorum strain P381. The
dotted lines in the four chromatography profiles indicate
the gradient elution conditions (see text for the corresponding
buffers), whereas the hatched areas indicate the toxic
eluted fractions able to induce distal leaf necrosis on tomato
seedlings. A, DEAE-Sepharose Fast Flow chromatography of the
culture filtrate. B, Resource RPC FPLC chromatography of the
pooled biologically active fractions eluted from the DEAE-Sepharose
column. C, TSK-DEAE FPLC chromatography of the pooled
biologically active fractions eluted from the Resource RPC column.
D, LC-18 FPLC chromatography of the pooled biologically
active fractions eluted from the TSK-DEAE column. The samples were
loaded onto the HPLC columns where indicated by arrows,
either by HPLC pump (B and C) or by a 1-ml loop
HPLC injection valve (D).
-propionamidated PcF protein
(Pam-PcF) were completely inactive. To evaluate the activity on its
strawberry host, purified PcF was also assayed for its ability to
induce localized leaf necrosis on a Fragaria vesca × ananassa cultivar that is susceptible to the P. cactorum P381 strain. As shown in Fig.
2, the purified PcF (80 µg) induced the
formation of a marked necrotic area within 24 h after leaf
infiltration. Such a level of activity is comparable with that of
previously reported phytotoxic fungal proteins toward their hosts (33,
34). Our findings indicate an involvement of the PcF protein in this
P. cactorum-Fragaria interaction as an
host-specific factor. The mode of action of PcF appeared to be
consistent with an hypersensitivity response (HR), a distinctive behavior caused by fungal pathogens and their phytotoxic factors (4-7). However, further experimental evidence appears to be needed to
establish the specific virulence or avirulence role of PcF protein
(e.g. either in the P. cactorum-strawberry
incompatible interaction or in its interaction with other plants).
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Fig. 2.
Leaf necrosis induced by PcF on host
strawberry plant. Infiltration with 80 µg of pure PcF
protein, dissolved in distilled water, induces localized necrosis on
host strawberry (Fragaria vesca × ananassa)
plant leaf (circle). Control solution was distilled water
(C, arrow). The necrotic effect was evaluated and
recorded at 24 h after infiltration.
-propionamidation of the denatured PcF protein in
the absence of reducing agents, the same molecular mass of 5,622 Da was
obtained, a finding that supports the absence of free thiol groups in
the protein. Furthermore, ISMS analysis of Pam-PcF protein, obtained
after
-propionamidation under strongly reducing conditions, revealed
a molecular mass of 6,054 ± 0.35 Da, consistent with the presence
of 6 oxidized cysteine residues per mol of protein, putatively involved
in 3 disulfide bridges. The presence of 6 cysteine residues was
confirmed by amino acid analysis of the pure protein. Amino acid
analysis also revealed the absence of methionine residues and a
relative excess of both hydrophobic and acidic residues, consistent
with the observed protein behavior in solution. In addition,
spectrophotometric determinations revealed the absence of tryptophan
residues. SDS-PAGE of pure PcF protein under standard
denaturating-reducing conditions (22) shows one single, 15-kDa band
(not shown), which is not consistent with the mass value of 5,622 Da
obtained by ISMS mass spectrometry. However, the 15-kDa SDS-PAGE band
was shifted to an estimated 6-kDa band in the presence of 10 mM DTT in the sample buffer (not shown). Because the ISMS
mass value of 5,622 Da excludes the possibility of intermolecular
disulfide bridges, the 15-kDa band observed on SDS-PAGE under mild
reducing conditions, represents an artifact because of suboptimal
SDS-protein binding, arising from inefficient reduction of
intramolecular disulfide bridges (48).
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Fig. 3.
Isolation scheme for PcF-coding
cDNA. Upper, cDNA encoding for the PcF protein,
which has been obtained by overlapping the nucleotide fragments A, B,
and C, identified after RACE experiments (see details in the text).
A, the 144-bp RT-PCR fragment isolated from
poly(A+) RNA with the PcF 1 and PcF 2 degenerate primers.
B, 5'-RACE fragment of 179 bp amplified with the PcF 3, PcF
5, and PcF 7 nested primers. C, 3'-RACE fragment of 220 bp
amplified with the PcF 4, PcF 6, and PcF 8 nested primers. The
oligonucleotide primers utilized were: PcF 1, 5'-GARGAYCCICTITACTGYCARGC-3'; PcF 2, 5'-GTIGTIGAICCRCAYTGYTCYTC-3';
PcF 3, 5'-GGTGGTGGAGCCACACTGCT-3'; PcF 4, 5'-GAGGATCCGCTGTACTGTCA-3';
PcF 5, 5'-CCTGGTCACGGCACTCTTTGC-3'; PcF 6, 5'-AGCAAAGAGTGCCGTGACCAGG-3'; PcF 7, 5'- CGGCAAGGTTAGCCTCAGA-3'; PcF
8, 5'-TGGGCGATGATTTCCACAGG-3'. V1 and
V2 primers were the oligo(dT) anchor primer and
the anchor primer, respectively, supplied by the 3'/5' RACE kit (Roche
Molecular Biochemicals).
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Fig. 4.
PcF-coding cDNA sequence and deduced
protein sequence. Top, 254, amino acid sequence
obtained by Edman degradation from the whole Pam-PcF protein.
P*, corresponds to the 4-hydroxyproline in the mature
protein. The termination codon is marked END. The
dotted lines correspond to a 21-amino acid signal peptide
for extracellular secretion. Two different polyadenylation sites of the
mRNA are depicted. Bottom, the hydropathy profile from
the proposed protein sequence was computed with the method of Kyte and
Doolittle (46) by the ProtScale program at the Expasy database server
(window width of 7). For computing, the 4-hydroxyproline at position 49 was replaced with proline.
0.56, a value that appears to rule out
any ability of the phytotoxic PcF protein to bind to the lipid-rich components of membranes (46). Furthermore, whereas analysis using
NetPhos 2.0 strongly suggested serine 44 as a potential phosphorylation
site (47), our Edman sequencing gave no indication of any unusual
repetitive yield decrease that is characteristic of such modified residues.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. G. Cristinzio and Prof. A. Scala for kindly providing the fungal isolates. We also thank J. M. Berjeaud and P. Pucci for their help with early mass spectrometry determinations, and Prof. Y. Cenatiempo and Prof. G. Magni for helpful discussion throughout.
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FOOTNOTES |
---|
* This work was supported by a grant from the Regione Marche 5b.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work is dedicated to the memory of Professor Pasquale Rosati.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF354650.
§ Present Address: Instituto Nacional de Investigacion Agropecuaria, Montevideo, Uruguay.
¶ To whom correspondence should be addressed: Dipartimento di Biotecnologie Agrarie ed Ambientali, Università di Ancona Via Brecce Bianche, 60131 Ancona, Italy. Tel.: 71-2204395; Fax: 71-2802117; E-mail: ruggieri@popcsi.unian.it.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101377200
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ABBREVIATIONS |
---|
The abbreviations used are:
PcF, derived from
the P. cactorum-Fragaria interaction system;
Pam-PcF, -propionamidated PcF protein;
Pam-Cys,
-propionamidated
cysteine;
PTH, phenylthiohydantoin;
HPLC, high pressure liquid
chromatography;
FPLC, fast protein liquid chromatography;
PAGE, polyacrylamide gel electrophoresis;
ISMS, ion spray mass spectrometry;
PITC, phenylisothiocyanate;
PTC, phenylthiocarbamyl;
bp, base pair;
DEPC, diethyl pyrocarbonate;
RACE, rapid amplification of complementary
DNA ends;
PCR, polymerase chain reaction;
Hyp, hydroxyproline;
DTT, dithiothreitol.
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