From the
Laboratory of Biochemistry, Wageningen
University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands and the
Laboratory of Phytopathology, Wageningen
University, Binnenhaven 5, NL-6709 PD Wageningen, The Netherlands
Received for publication, December 2, 2002 , and in revised form, May 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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Although the intrinsic role of the AVR proteins of C. fulvum during infection remains obscure, they are anticipated to contribute to virulence in susceptible hosts (1618). This implies that evasion of Cf-mediated resistance by modification of Avr genes might be associated with a reduction or loss in virulence unless a functional gene remains. A candidate protein to investigate the latter idea is the race-specific elicitor AVR4 because Cf-4-mediated resistance is overcome in all but one case by single amino acid substitutions in the Avr4 gene (12, 14). Moreover, for AVR4 a virulence function is proposed in association with its ability to bind to chitin. AVR4 was found to protect fungi against degradation by plant chitinases by association with their hyphal wall.3 Mutations in the Avr4 gene, as found in natural C. fulvum isolates virulent on the tomato genotype Cf-4, encode mostly single Cys to Tyr substitutions. In addition, two other mutations were found, i.e. Thr-66 to Ile and Tyr-67 to His. The Cys to Tyr substitutions involved the positions 64, 70, or 109 (which corresponds with Cys-35, -41, and -80 in the mature protein, respectively) (14). Some of these avr4 alleles still exhibited necrosis inducing activity when transiently expressed in Cf-4 tomato using potato virus X (PVX) (14). However, none of the AVR4 mutant isoforms could be detected in apoplastic fluid isolated from tomato leaves inoculated with C. fulvum (14).
Mass spectrometry revealed that all Cys residues in AVR4 are involved in disulfide bonding (20). Disulfide bond patterns and the sequential spacing between Cys residues define to a large extent the protein fold of secreted small proteins (21, 22). Here, the disulfide bond connectivities of AVR4 are elucidated. The disulfide bond pattern of AVR4 shows homologies with the disulfide bond pattern found in the recently identified invertebrate chitin-binding domain (inv ChBD) (23), i.e. three of the four disulfide bonds of AVR4 (Cys-11-41, Cys-35-80, and Cys-57-72) are represented in the inv ChBD motif. Independent disruption of each of these three disulfide bonds in AVR4 results in a protein that is sensitive to proteases present in the apoplast, which suggests that these disulfide bridges are required for conformational stability of AVR4. The Cys to Tyr mutations identified in natural strains of C. fulvum involve two (Cys-11-41 and Cys-35-80) of these three conserved disulfide bonds. AVR4 isoforms with a disruption in one of these two disulfide bonds are still able to bind chitin. Our data support a model where evasion of Cf-4-mediated resistance appears to be based on decreased conformational stability of the AVR4 isoform, leading to protein degradation upon release in the tomato apoplast. Noteworthy, the AVR4 isoforms were found to be more resistant to proteases when bound to chitin. These findings argue that mutant AVR4 isoforms are fully functional and can associate with chitin upon release, whereas excess of secreted (and unbound) protein is degraded before triggering host defense responses.
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EXPERIMENTAL PROCEDURES |
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Partial Reduction and Cyanylation of the AVR4 ProteinExpression of heterologous AVR4 was achieved in the methylotrophic yeast Pichia pastoris, and AVR4 was purified from culture fluid (20). The disulfide bonds of AVR4 were partially reduced with TCEP (Sigma) (26, 27). A stock solution of 0.1 M TCEP was prepared in 6 M guanidine-HCl in 0.1 M citrate buffer (pH 3) and stored at 20 °C (without any deterioration for up to six months). For each reduction reaction, 100 µg of native AVR4 was dissolved in 10 µlof6 M guanidine-HCl in 0.1 M citrate buffer (pH 3). The reaction was initiated by adding a 6-fold molar excess of TCEP to AVR4, followed by incubation at 20 °C for 15 min. Subsequently, an 80-fold molar excess of CDAP (Sigma) was added to cyanylate the freed thiol groups (15 min, 20 °C, in the dark). The 0.1 M CDAP stock solution in 6 M guanidine-HCl in 0.1 M citrate buffer (pH 3) was freshly prepared prior to each reaction.
Reverse-phase High Performance Liquid Chromatography of the Peptide
MixtureThe TCEP/CDAP reaction mixtures were separated by
analytical reverse-phase high performance liquid chromatography (RP-HPLC)
using a 150 x 3.9 mm Delta-Pak C18 column (300 Å, 5 µm;
Waters). The separation was monitored at 215 nm, and predominant peaks were
manually collected. HPLC elution solvents consisted of 0.1% (v/v)
trifluoroacetic acid in water (solvent A), and 0.1% (v/v) trifluoroacetic acid
in acetonitrile (solvent B). The HPLC was operated at a flow rate of
1 ml/min. The applied gradient was 5% 20%B (percentage B in solvent A)
in 2 min, 20%
30% in 40 min, and 30%
60% in 3 min. AVR4 eluted
at
25% B. Integration of the HPLC profile was achieved using the supplied
Waters software. Appropriate fractions (containing the AVR4
des-species) were lyophilized for storage. All solvents used were
HPLC grade.
Peptide Cleavage and Full Reduction of the Disulfide
Bonds/Peptide Mass AnalysisLyophilized HPLC fractions
containing the AVR4 des-species were dissolved in two consecutive
steps: first, 2 µl in 1 M NH4OH, 6 M
guanidine-HCl, and second, 5 µl of 1 M NH4OH, and
incubated at 20 °C for 1 h. The excess of NH4OH was evaporated
in a Speed-Vac system in 30 min (to almost complete dryness). Subsequently,
the remaining disulfide bonds were reduced by adding an excess of TCEP (10
µl of 0.1 M TCEP stock), and the mixture was incubated at 37
°C for 30 min. The peptide mixtures were analyzed by mass spectrometry
using a MALDI-TOF MS (Perseptive Biosystems Voyager DE-RP). Small aliquots of
the peptide samples were applied to a saturated matrix solution that was
freshly prepared (-cyano-4-hydroxycinnamic acid; Sigma; 10 mg/ml in
acetonitrile/water/trifluoroacetic acid (50/50/1, v/v/v)); one µl was
deposited on a sample plate
(28,
29). Depicted spectra were
averages of 100256 consecutive laser pulses. The instrument was
generally operated in the positive mode at an acceleration voltage of 23 kV
combined with delayed extraction. Spectra were externally calibrated with
bovine cytochrome c (12,230.9 Da), bovine insulin (5,734.6 Da) (both
Sigma), and Microperoxidase 8 (MP8, 1,506.5 Da; Ref.
30).
Incubation of the AVR4 Des-species with Apoplastic
FluidApoplastic fluids (AFs) were isolated from intercellular
spaces of near isogenic tomato genotypes Cf-4 and Cf-0
(31) that had been inoculated
with race 4 of C. fulvum (strain 38, a non-AVR4 producing strain) and
race 5 (an AVR4-producing strain), respectively
(14). Native AVR4 and AVR4
des-species (4 µg) were incubated at 30 °C for 1 h in the
presence of 0.1 µl of AF (0.1 µg of total protein). Protease
inhibitors used were from a standard protein inhibitor mixture with EDTA
(Roche Applied Science; 1 tablet/ml AF). Protein samples were separated on
Tricine SDS-PAGE gels
(32).
Polysaccharide Substrate Binding AssayNative AVR4 and AVR4 des-species (4 µg) were incubated at ambient temperature for 1 h (unless stated otherwise) with an excess of 5 mg of insoluble chitin beads (New England Biolabs) or chitosan (Sigma) in 50 mM Tris-HCl (pH 8) and 150 mM NaCl (500 µl of final volume) as described (19). The insoluble material was pelleted by centrifugation (13.000 x g for 3 min). Supernatants were recovered and lyophilized. The pellet fraction was boiled in 200 µl of 1% SDS to release bound protein and centrifuged. The retrieved supernatant (containing bound AVR4) as well as the lyophilized supernatant fraction (containing unbound AVR4) were examined for protein content by Tricine SDS-PAGE.
Molecular Modeling of AVR4 with the Tachycitin NMR Structure The mean NMR structure of tachycitin (Protein Data Bank code 1DQC [PDB] ) was used as template structure to model the structure of AVR4 using Modeler 6.1 (3335). Additional loop refinement was used to model the two relatively large gaps (12 and 6 amino acids). The disulfide bridges were fixed during the calculations. One thousand models were constructed, of which the ten lowest scoring structures were further examined. The models were found reliable using standard algorithms (36, 37).
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RESULTS |
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Chemical Reduction and Cyanylation of the Disulfide Bonds in AVR4
To determine the disulfide bond connectivities in AVR4 by a direct
chemical approach, we partially reduced the disulfide bridges with TCEP at pH
3.0, thereby minimizing intramolecular rearrangements of the disulfide bridges
(38,
39). The formed cysteine thiol
groups were directly modified by alkylation with CDAP under acidic conditions,
and the resulting peptides were separated by reverse-phase HPLC
(Fig. 2). In the presence of 6
molar equivalents of TCEP/AVR4, 50% of native AVR4 was reduced, as
indicated by an increased HPLC retention time of the newly formed species
(Fig. 2). Subsequent MALDI-TOF
mass spectrometry identified four product peaks containing AVR4 species with
one disulfide bond reduced (hereafter denoted as des-species)
(Fig. 2C). The peaks
eluting at 30.8 and 30.9 min could not be separated by one HPLC run, but after
an additional run both species appeared more than 85% pure
(Fig. 2B). The peaks
containing the des-species together constituted
70% of the
reduced AVR4 species, whereas peaks that eluted at higher acetonitrile
concentrations contained AVR4 species with more than one disulfide bond
reduced (as detected by mass spectrometry). The increased retention time
reflects the more unfolded state of these species, as the results of increased
hydrophobicity of the protein species.
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Assignment of the Disulfide Bonds with Mass MappingTo
determine which disulfide bond was reduced in each des-species, the
HPLC fractions were lyophilized and redissolved in 1 M
NH4OH, which induces base-catalyzed cleavage at the peptide bond
that precedes the modified half-cystines (converting them to iminothiazolidine
derivatives) (27). After
complete reduction, the reaction mixtures were analyzed by MALDI-TOF MS
(Fig. 3). Theoretically, the
reaction should yield five peptide fragments per des-species,
i.e. three peptide fragments originating from the double chain
cleavage reaction and two fragments originating from a -elimination
(26,
40). The latter is a side
reaction that occurs at either one of the two half-cystines, thereby
preventing cleavage at this half-cystine.
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Assignment of the disulfide bonds was performed in a two-step approach.
Mass peaks that correspond with peptide fragments from the N and C terminus up
to the reduced half-cystines were first assigned. In
Fig. 3A, the mass
peaks m/z 2286.6 and 6673.9 Da correspond to the peptide
fragments encompassing the residues 120 and 2786; Cys-27 is
converted to an iminothiazolidine derivative in the latter fragment
(Table II). This assignment
could subsequently be confirmed by other mass peaks that originate from
-eliminations, i.e. m/z 2882.3 and 7271.3 Da (the
peptide fragment 126 with a
-elimination at Cys-21 and
2186 with a
-elimination at Cys-27, respectively). The remaining
fragment (iminothiazolidine 2126) was too small to be detected because
of the settings of the lower mass detection limit (1000 Da). Together, these
data establish the disulfide bond Cys-21-27. The relative mass deviations
between measured and calculated mass were less than 0.05% for the majority of
the peptide fragments. Comparable analyses of the other reaction mixtures
resulted in the assignment of the other disulfide bridges, i.e.
Cys-11-41, Cys-57-72, and Cys-35-80 (Fig.
3, B, C, and D, respectively). The intensity of
the reoccurring mass peaks at 3805.4 and 5031.8 Da in fraction 3
(Fig. 3C) would
suggest more overlap between fractions 3 and 4 than shown in the HPLC elution
profile in Fig. 2B.
However, these two mass peaks were consistently readily observed in multiple
independent replicate experiments, which suggests that these two peptides are
easily ionized by MALDI. The intensity of the mass peaks, therefore, does not
correspond to the actual concentration of the purified des-species.
In conclusion, we established the connectivities of the disulfide bonds.
Moreover, these are consistent with the PVX data (Tables
I and
III).
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AVR4 Contains a Single Invertebrate Chitin-binding DomainThe identified disulfide pattern of AVR4 was further exploited to perform a query (motif.genome.ad.jp). This search identified a homologous sequence stretch in genes of the invertebrates Manduca sexta, Brugia malayi, and Penaeus japonicus. These genes encode for chitinases. The homology is restricted to the C-terminal domain, which contains six conserved Cys residues. This C-terminal domain was recently identified as a chitin-binding domain designated the inv ChBD (23). For one family member, tachycitin of Japanese horseshoe crab (Tachypleus tridentatus) (41), the disulfide bond pattern has been solved. Unlike AVR4, tachycitin contains five disulfide bonds, of which three reflect the conserved disulfide bonds. Sequence alignment helped to appoint the conserved disulfide bridges, i.e. Cys-11-41, Cys-35-80, and Cys-57-72 of AVR4 (Fig. 5). The additional disulfide bond Cys-21-27 within AVR4 does not share homology with the disulfide bonds in tachycitin. In contrast to tachycitin and AVR4, the other inv ChBD family members do not contain additional disulfide bonds (Fig. 5). A three-dimensional model of AVR4 was constructed using the three-dimensional structure of tachycitin as a template structure (42) (data not shown). The modeled structure of AVR4 contains the secondary structure elements as found in tachycitin, but the sequence insertion encompassing the disulfide bridge Cys-21-27 was too large to construct a reliable model for this part of the protein.
Disulfide Bond-disrupted Mutants of AVR4 Display Affinity for ChitinIn a concurrent paper,3 it is demonstrated that native AVR4 binds specifically to chitin but not to other cell-wall polysaccharides. Moreover, for human chitinase, it has been shown that the six conserved Cys residues that belong to the inv ChBD need to be intact for chitin binding (43). To examine whether AVR4 des-species still exhibited chitin binding activity, we incubated the des-species with chitin (Fig. 6). Following incubation, each of the des-species was detected together with chitin. This suggests that absence of one disulfide bridge in AVR4 does not abolish chitin binding. However, for des-(Cys-57-72), binding was repeatedly less complete than for the other three des-species (as more protein remained in solution) (Fig. 6), but after prolonged incubation (from 1 to 4 h) all of the des-(Cys-57-72) protein was found in the pellet. This finding suggests a decreased affinity of this AVR4 isoform for chitin. It is noted that des-(Cys-57-72) is potentially contaminated with des-(Cys-35-80), which could have interfered with the chitin binding assay. Because des-(Cys-35-80) has a comparable chitin binding affinity as native AVR4, the des-(Cys-57-72) might even exhibit a lower affinity than observed in our assay.
Binding to Chitin Extends the Lifetime of the Des-species in the Presence of Apoplastic FluidDuring growth in tomato, C. fulvum remains confined to the intercellular spaces of tomato (44). It has been proposed that after release AVR4 associates directly to regions of the hyphal walls of C. fulvum where chitin is exposed (19). When chitin is saturated, excess of AVR4 is thought to be distributed throughout the apoplast. We investigated whether the various des-species were less sensitive to proteolytic degradation after binding to chitin. Therefore, we incubated the des-species with chitin for 4 h, rather than 1 h, to ensure complete association between these proteins and chitin. After incubation with chitin, the solutions were supplied with AF and stability of the AVR4 isoforms was followed in time. Over the period of 4 h, native AVR4 and des-(Cys-21-27) bound to chitin remained fully stable in the presence of AF (Fig. 7). Moreover, association with chitin resulted in an increased half-life time of des-(Cys-11-41), des-(Cys-35-80), and des-(Cys-57-72) in the presence of AF. Thus, when bound to chitin, normally unstable des-species are protected against proteases present in AF.
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DISCUSSION |
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Strains of C. fulvum were found to evade Cf-4-mediated resistance by producing AVR4 mutant proteins. The majority of these modifications involved substitution of Cys residues at positions 35, 41, and 80 by Tyr (14), indicating that disruption of only two of the three conserved disulfide bonds has, so far, contributed to evasion of AVR4 recognition (Table III). AVR4 mutant proteins carrying a disruption in one of these conserved disulfide bonds could not be detected in AF from tomato leaves inoculated with C. fulvum race 4 carrying these alleles, which suggested degradation of these isoforms. It was previously reported (14, 45) that both AVR4 and AVR9 are processed by extracellular proteases as an integral part of their maturation to elicitors. We have now established that AVR4 isoforms are indeed rapidly degraded as a result of protease activity present in AF, as suggested by Joosten et al. (14). In wild-type AVR4, the presence of the conserved disulfide bonds prevents further degradation of mature AVR4 protein. Although Cf-4-mediated resistance is evaded by the production of protease-sensitive AVR4 isoforms, we noticed that the unstable AVR4 isoforms are still able to bind to chitin.
As mentioned before, disruption of the disulfide bond Cys-57-72 appeared to
reduce the affinity for chitin. Supporting data for a role of Cys-57-72 in
chitin binding comes from the three-dimensional-structure of tachycitin. Part
of the three-dimensional structure of tachycitin can be superimposed on the
structure of hevein, a plant chitin-binding lectin
(42). This part of the
tachycitin was, therefore, proposed to act as the chitin-binding domain. The
shared structural motif encompasses the second -sheet in tachycitin, a
short helical turn, and the third disulfide bridge conserved in the ChBD motif
(Cys-57-72 in AVR4). An NMR study of
AVR4 recently showed
that AVR4 and tachycitin seem to adopt a similar protein fold and that the
residues important for the interaction with chitin could be superimposed on
the structure of hevein. Hence, our findings predict that strains of C.
fulvum producing isoforms of AVR4 lacking this specific disulfide bond
Cys-57-72 will evade Cf-4-mediated resistance. However, strains of
C. fulvum producing such a disulfide bond-disrupted mutant have not
yet been identified. A possible explanation for the absence of such a strain
could be that selection pressure exists associated with maintaining the chitin
binding ability of AVR4. Modification of this disulfide bond Cys-57-72 could,
therefore, come with a virulence penalty for C. fulvum.
Different from the three conserved disulfide bonds, our data indicate that
the disulfide bond Cys-21-27 does not contribute to conformational stability
of AVR4. Possibly, this disulfide bond is required for local conformational
stability around the sequence insertion. However, preliminary NMR data
indicate that the sequence insertion surrounding Cys-21 adopts an
-helix in AVR.5
Because the
-helix apparently increases the stability of this part of
the structure, the disulfide bond Cys-21-27 itself would not be essential for
the conformational stability of AVR4. Similar observations were previously
reported for the cystine-knot fold
(46).
When the NIA was tested of the AVR4 isoform lacking the disulfide bond Cys-21-27 in Cf-4 tomato using PVX, the NIA was found to be similar to the NIA of AVR4 isoforms lacking Cys-35-80. NIA of both mutant proteins, however, is less than that of native AVR4. However, in contrast to disulfide bond Cys-35-80, disulfide bond Cys-21-27 does not contribute to protein stability, suggesting that the mechanisms underlying the reduced NIA of both AVR4 des-species are different. When the disulfide bond Cys-35-80 is disrupted, a certain fraction of the resulting protein will be degraded by proteases present in the intercellular space, whereas the remaining fraction of protein triggers to some degree the Cf-4-mediated defense responses. The reduced NIA of AVR4 mutant carrying a disrupted disulfide bond Cys-21-27 cannot be explained by sensitivity to proteases but is most likely because of the amino acid substitution itself or because of a conformation change of the protein. The remaining NIA could be a reason why, despite the fact that disruption of disulfide bond Cys-21-27 does not affect chitin binding activity of AVR4, no natural C. fulvum strains have thus far been found that carry such a mutation.
In addition to the three Cys substitutions found in natural isolates, two
other natural amino acid mutations have been found in AVR4 (T37I and Y38H).
Both modifications are thought to affect the conformational stability of AVR4,
based on a report by Zhu and Braun
(47). The latter two mutations
affect the strand 2 of the first anti-parallel
-sheet, which is in
the core of the protein structure
(42). In AVR4, Thr-37 and
Tyr-38 are putatively paired with Pro-30 and Ile-29 in strand
1,
respectively. These four residues are well conserved in the inv
ChBD.3 The report of Zhu and Braun indicates that Pro-Ile
cross-strand contact pairs in
-strands are virtually absent, whereas
Pro-Thr cross-strand contact pairs are allowed
(47). Similarly, aromatic
residues are exclusively found at position 38 (Tyr/Phe/Trp>98%) of the inv
ChBD. Hydrophobic residues are found to be the favorite cross-strand contact
partners, whereas His residues are disfavored. Based on these statistical
analyses, we propose that the mutations T37I and Y38H both will lead to a
partially destabilized first anti-parallel
-sheet, which could decrease
the overall conformational stability of AVR4 and increase the sensitivity to
proteases.
Overall, the disulfide bond-disrupted AVR4 isoforms as they are produced by natural strains of C. fulvum show increased sensitivity toward proteases present in the apoplast as compared with native AVR4. However, after binding to chitin in the cell wall of C. fulvum, the mutant isoforms seem to escape degradation by proteases present in the apoplast. Thus, although Cf-4-mediated recognition is evaded, the chitin binding activity of natural AVR4 mutants of C. fulvum remains, thereby contributing to the protection of C. fulvum against plant chitinases.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains a supplementary table.
¶ To whom correspondence should be addressed. Tel.: 31-317-484466; Fax: 31-317-484801; E-mail: Jacques.vervoort{at}wur.nl.
1 The abbreviations used are: AVR, avirulence gene product; AF, apoplastic
fluid; des-(Cys-xCys-y), AVR4 species
lacking a specific disulfide bond (the involved half-cystines are reduced and
the sulhydryl group is cyanylated); CDAP, 1-cyano-4-diethylamino-pyridinium;
ChBD, chitin-binding domain; HPLC, high performance liquid chromatography;
inv, invertebrate; MALDI-TOF MS, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry; NIA, necrosis-inducing activity; PVX, potato
virus X; TCEP, Tris-(2-carboxyethyl)-phosphine hydrochloride.
2 C. F. de Jong, A. M. Laxalt, W. Ligterink, P. J. G. M. de Wit, M. H. A. J.
Joosten, and T. Munnik, submitted for publication.
3 H. A. van den Burg, S. Harrison, M. H. A. J. Joosten, J. Vervoort, and P.
J. G. M. de Wit, submitted for publication.
4 H. A. van den Burg, C. A. E. M. Spronk, M. A. Kennedy, J. P. C. Visser, G.
W. Vuister, P. J. G. M. de Wit, and J. Vervoort, manuscript in
preparation.
5 C. A. E. M. Spronk and H. A. van den Burg, unpublished data.
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REFERENCES |
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