Natural Disulfide Bond-disrupted Mutants of AVR4 of the Tomato Pathogen Cladosporium fulvum Are Sensitive to Proteolysis, Circumvent Cf-4-mediated Resistance, but Retain Their Chitin Binding Ability
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m212196200
ISSN1083-351X
AutoresHarrold A. van den Burg, Nienke Westerink, Kees‐Jan Françoijs, Ronelle Roth, Esmeralda Woestenenk, Sjef Boeren, P.J.G.M. de Wit, M.H.A.J. Joosten, Jacques Vervoort,
Tópico(s)Plant Pathogenic Bacteria Studies
ResumoThe extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. Here, the four disulfide bonds of AVR4 were identified as Cys-11-41, Cys-21-27, Cys-35-80, and Cys-57-72 by partial reduction with Tris-(2-carboxyethyl)-phosphine hydrochloride, subsequent cyanylation, and base-catalyzed chain cleavage. The resulting peptide fragments were analyzed by mass spectrometry. Sequence homology and the disulfide bond pattern revealed that AVR4 contains an invertebrate (inv) chitin-binding domain (ChBD). Binding of AVR4 to chitin was confirmed experimentally. The three disulfide bonds encompassing the inv ChBD motif are also required for protein stability of AVR4. Independent disruption of each of the three conserved disulfide bonds in AVR4 resulted in a protease-sensitive protein, whereas the fourth disulfide bond appeared not to be required for protein stability. Most strains of C. fulvum virulent on Cf-4 tomato contain Cys to Tyr substitutions in AVR4 involving two (Cys-11-41, Cys-35-80) of the three disulfide bonds present in the inv ChBD motif. These natural Cys to Tyr mutant AVR4 proteins did retain their chitin binding ability and when bound to chitin were less sensitive to proteases. Thus, the widely applied tomato Cf-4 resistance gene is circumvented by C. fulvum by amino acid substitutions affecting two disulfide bonds in AVR4 resulting in the absence of the corresponding AVR4 isoforms in apoplastic fluid. However, these natural isoforms of AVR4 appear to have retained their intrinsic function, i.e. binding to chitin present in the cell wall of C. fulvum, most likely to protect it against the deleterious effects of plant chitinases. The extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. Here, the four disulfide bonds of AVR4 were identified as Cys-11-41, Cys-21-27, Cys-35-80, and Cys-57-72 by partial reduction with Tris-(2-carboxyethyl)-phosphine hydrochloride, subsequent cyanylation, and base-catalyzed chain cleavage. The resulting peptide fragments were analyzed by mass spectrometry. Sequence homology and the disulfide bond pattern revealed that AVR4 contains an invertebrate (inv) chitin-binding domain (ChBD). Binding of AVR4 to chitin was confirmed experimentally. The three disulfide bonds encompassing the inv ChBD motif are also required for protein stability of AVR4. Independent disruption of each of the three conserved disulfide bonds in AVR4 resulted in a protease-sensitive protein, whereas the fourth disulfide bond appeared not to be required for protein stability. Most strains of C. fulvum virulent on Cf-4 tomato contain Cys to Tyr substitutions in AVR4 involving two (Cys-11-41, Cys-35-80) of the three disulfide bonds present in the inv ChBD motif. These natural Cys to Tyr mutant AVR4 proteins did retain their chitin binding ability and when bound to chitin were less sensitive to proteases. Thus, the widely applied tomato Cf-4 resistance gene is circumvented by C. fulvum by amino acid substitutions affecting two disulfide bonds in AVR4 resulting in the absence of the corresponding AVR4 isoforms in apoplastic fluid. However, these natural isoforms of AVR4 appear to have retained their intrinsic function, i.e. binding to chitin present in the cell wall of C. fulvum, most likely to protect it against the deleterious effects of plant chitinases. Gene-for-gene-based disease resistance in plants commonly requires two complementary genes, an avirulence (Avr) 1The abbreviations used are: AVR, avirulence gene product; AF, apoplastic fluid; des-(Cys-x—Cys-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.1The abbreviations used are: AVR, avirulence gene product; AF, apoplastic fluid; des-(Cys-x—Cys-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. gene in the pathogen and a matching resistance gene in the host (1Flor H.H. 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Although some Avr genes in these virulent C. fulvum strains were found to be absent (11van Kan J.A.L. van den Ackerveken G.F.J.M. de Wit P.J.G.M. Mol. Plant-Microbe Interact. 1991; 4: 52-59Crossref PubMed Scopus (262) Google Scholar), others contained point mutations (12Joosten M.H.A.J. Cozijnsen T.J. de Wit P.J.G.M. Nature. 1994; 367: 384-386Crossref PubMed Scopus (295) Google Scholar) or transposon insertions (13Luderer R. Takken F.L. de Wit P.J.G.M. Joosten M.H.A.J. Mol. Microbiol. 2002; 45: 875-884Crossref PubMed Scopus (122) Google Scholar). The natural strains of C. fulvum carrying these mutated Avr genes did not exhibit significantly reduced virulence under laboratory conditions (11van Kan J.A.L. van den Ackerveken G.F.J.M. de Wit P.J.G.M. Mol. Plant-Microbe Interact. 1991; 4: 52-59Crossref PubMed Scopus (262) Google Scholar, 12Joosten M.H.A.J. Cozijnsen T.J. de Wit P.J.G.M. Nature. 1994; 367: 384-386Crossref PubMed Scopus (295) Google Scholar, 13Luderer R. Takken F.L. de Wit P.J.G.M. Joosten M.H.A.J. Mol. Microbiol. 2002; 45: 875-884Crossref PubMed Scopus (122) Google Scholar, 14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar), suggesting that AVR proteins are not essential for virulence or that the modified isoforms of AVRs still contribute to virulence of C. fulvum. The genetic variation is so far strictly limited to the race-specific Avrs and is absent in genes that encode other extracellular elicitor proteins of C. fulvum (15Luderer R. de Kock M.J.D. Dees R.H.L. de Wit P.J.G.M. Joosten M.H.A.J. Mol. Plant Pathol. 2002; 3: 91-95Crossref PubMed Scopus (32) Google Scholar). 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 (16Kjemtrup S. Nimchuk Z. Dangl J.L. Curr. Opin. Microbiol. 2000; 3: 73-78Crossref PubMed Scopus (120) Google Scholar, 17White F.F. Yang B. Johnson L.B. Curr. Opin. Plant Biol. 2000; 3: 291-298Crossref PubMed Scopus (148) Google Scholar, 18Bonas U. Lahaye T. Curr. Opin. Microbiol. 2002; 5: 44-50Crossref PubMed Scopus (138) Google Scholar). 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 (12Joosten M.H.A.J. Cozijnsen T.J. de Wit P.J.G.M. Nature. 1994; 367: 384-386Crossref PubMed Scopus (295) Google Scholar, 14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). 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. 3H. A. van den Burg, S. Harrison, M. H. A. J. Joosten, J. Vervoort, and P. J. G. M. de Wit, submitted for publication.3H. A. van den Burg, S. Harrison, M. H. A. J. Joosten, J. Vervoort, and P. J. G. M. de Wit, submitted for publication. 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) (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). Some of these avr4 alleles still exhibited necrosis inducing activity when transiently expressed in Cf-4 tomato using potato virus X (PVX) (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). However, none of the AVR4 mutant isoforms could be detected in apoplastic fluid isolated from tomato leaves inoculated with C. fulvum (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). Mass spectrometry revealed that all Cys residues in AVR4 are involved in disulfide bonding (20van den Burg H.A. de Wit P.J.G.M. Vervoort J. J. Biomol. NMR. 2001; 20: 251-261Crossref PubMed Scopus (32) Google Scholar). Disulfide bond patterns and the sequential spacing between Cys residues define to a large extent the protein fold of secreted small proteins (21Harrison P.M. Sternberg M.J.E. J. Mol. Biol. 1996; 264: 603-623Crossref PubMed Scopus (112) Google Scholar, 22Mas J.M. Aloy P. Marti R.M.A. Oliva B. Blanco A.C. Molina M.A. De Llorens R. Querol E. Aviles F.X. J. Mol. Biol. 1998; 284: 541-548Crossref PubMed Scopus (34) Google Scholar). 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) (23Shen Z. Jacobs-Lorena M. J. Mol. Evol. 1999; 48: 341-347Crossref PubMed Scopus (135) Google Scholar), 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. Construction of PVX Derivatives and Transcription—Avr4 mutants encoding various Cys to Ala substitutions were generated by PCR-based primer-directed mutagenesis on the plasmid pTXΔGC3a, containing the native Avr4 sequence (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). PCR amplification was carried out using mutagenic primers (see Supplementary Table for primers) designated to generate two overlapping PCR fragments. PCR was used to combine the overlapping PCR fragments using the primers OX10 and N31, and the PCR product was cloned into the ClaI site of the vector pTXΔGC3a (24Chapman S. Kavanagh T. Baulcombe D. Plant J. 1992; 2: 549-557PubMed Google Scholar) and sequenced. In vitro transcription of the plasmids and subsequent inoculation on Nicotiana clevelandii and tomato was performed as described (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). N. clevelandii and the tomato (Lycopersicon esculentum) cultivars Moneymaker (MM) and the near-isogenic line MM-Cf4 were grown as described previously (25de Wit P.J.G.M. Flach W. Physiol. Plant Pathol. 1979; 15: 257-267Crossref Scopus (49) Google Scholar). Partial Reduction and Cyanylation of the AVR4 Protein—Expression of heterologous AVR4 was achieved in the methylotrophic yeast Pichia pastoris, and AVR4 was purified from culture fluid (20van den Burg H.A. de Wit P.J.G.M. Vervoort J. J. Biomol. NMR. 2001; 20: 251-261Crossref PubMed Scopus (32) Google Scholar). The disulfide bonds of AVR4 were partially reduced with TCEP (Sigma) (26Wu J. Watson J.T. Protein Sci. 1997; 6: 391-398Crossref PubMed Scopus (157) Google Scholar, 27Wu J. Watson J.T. Anal. Biochem. 1998; 258: 268-276Crossref PubMed Scopus (68) Google Scholar). 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 Mixture—The TCEP/CDAP reaction mixtures were separated by analytical reverse-phase high performance liquid chromatography (RP-HPLC) using a 150 × 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 Analysis—Lyophilized 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 (α-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 (28Karas M. Hillenkamp F. Anal. Chem. 1988; 60: 2299-2301Crossref PubMed Scopus (4803) Google Scholar, 29Kussmann M. Nordhoff E. Rahbek-Nielsen H. Haebel S. Rossel-Larsen M. Jakobsen L. Gobom J. Mirgorodskaya E. Kroll-Kristensen A. Palm L. Roepstorff P. J. Mass Spectrom. 1997; 32: 593-601Crossref Scopus (432) Google Scholar). Depicted spectra were averages of 100–256 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. 30Primus J.L. Boersma M.G. Mandon D. Boeren S. Veeger C. Weiss R. Rietjens I.M. J. Biol. Inorg. Chem. 1999; 4: 274-283Crossref PubMed Scopus (46) Google Scholar). Incubation of the AVR4 Des-species with Apoplastic Fluid—Apoplastic fluids (AFs) were isolated from intercellular spaces of near isogenic tomato genotypes Cf-4 and Cf-0 (31de Wit P.J.G.M. Spikman G. Physiol. Plant Pathol. 1982; 21: 1-11Crossref Scopus (224) Google Scholar) 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 (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). 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 (32Schägger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10457) Google Scholar). Polysaccharide Substrate Binding Assay—Native 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 (19Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (2994) Google Scholar). The insoluble material was pelleted by centrifugation (13.000 × 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) was used as template structure to model the structure of AVR4 using Modeler 6.1 (33Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10447) Google Scholar, 34Sali A. Potterton L. Yuan F. Van Vlijmen H. Karplus M. Proteins. 1995; 23: 318-326Crossref PubMed Scopus (945) Google Scholar, 35Fiser A. Do R.K.G. Sali A. Protein Sci. 2000; 9: 1753-1773Crossref PubMed Scopus (1613) Google Scholar). 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 (36Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1754) Google Scholar, 37Sanchez R. Sali A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13597-13602Crossref PubMed Scopus (313) Google Scholar). Four Cys Residues Are Required in AVR4 to Induce Cf-4-specific Defense Responses in Tomato—By using transient PVX-mediated expression some AVR4 alleles, i.e. C35Y, Y38H, and C80Y, were identified that exhibited reduced necrosis-inducing activity (NIA) in the tomato genotype Cf-4, whereas the other natural Avr4 alleles induced no NIA (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). To determine whether other Cys residues, for which no mutations were found in strains of C. fulvum, are also required for NIA of AVR4, we independently replaced all individual Cys residues by Ala in PVX::Avr4. Four-week-old tomato plants were inoculated with these PVX::Avr4 derivatives, and NIA was scored (Fig. 1) as described (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). The introduction of an Ala residue at the positions 35, 41, and 80 gave similar results as previously reported for the corresponding Tyr mutations (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar). Interestingly, the mutations C21A and C27A were found to result in reduced NIA on Cf-4 tomato similar to the mutations C35A and C80A. The PVX::Avr4 constructs carrying a Cys to Ala mutation in either Cys-11, -41, -57, or -72 induced no NIA on Cf-4 tomato plants. These finding suggest that the latter four Cys residues have interrelated disulfide bonds in AVR4. For Cys-21, -27, -35, and -80, of which the single mutations showed reduced NIA, double Cys to Ala mutations were constructed. Four of the six double mutants no longer induced hypersensitive response on Cf-4 tomato, whereas mutants carrying the mutations C21A+C27A and C35A+C80A were as active as the corresponding single Cys to Ala mutants (Table I). These data suggest that disulfide bonds connect Cys-21 with Cys-27 and Cys-35 with Cys-80.Table INecrosis inducing activity (NIA) on tomato cv. moneymaker-Cf4 of mutant PVX::Avr4 NIA was scored as described (14Joosten M.H.A.J. Vogelsang R. Cozijnsen T.J. Verberne M.C. de Wit P.J.G.M. Plant Cell. 1997; 9: 367-379PubMed Google Scholar).Single Cys to Ala mutationNecrosis-inducing activityDouble Cys to Ala mutationNecrosis inducing activityWild type+++++C21A + C27A++C11A-C21A + C35A-C21A++C21A + C80A-C27A++C27A + C35A-C35A++C27A + C80A-C41A-C35A + C80A++C57A-C72A-C80A++ Open table in a new tab 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 (38Gray W.R. Protein Sci. 1993; 2: 1732-1748Crossref PubMed Scopus (235) Google Scholar, 39van den Hooven H.W. van den Burg H.A. Vossen P. Boeren S. de Wit P.J.G.M. Vervoort J. Biochemistry. 2001; 40: 3458-3466Crossref PubMed Scopus (65) Google Scholar). 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. Assignment of the Disulfide Bonds with Mass Mapping—To 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) (27Wu J. Watson J.T. Anal. Biochem. 1998; 258: 268-276Crossref PubMed Scopus (68) Google Scholar). 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 β-elimination (26Wu J. Watson J.T. Protein Sci. 1997; 6: 391-398Crossref PubMed Scopus (157) Google Scholar, 40Degani Y. Patchornik A. Biochemistry. 1974; 13: 1-11Crossref PubMed Scopus (183) Google Scholar). The latter is a side reaction that occurs at either one of the two half-cystines, thereby preventing cleavage at this half-cystine. 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 1–20 and 27–86; 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 β-eliminations, i.e. m/z 2882.3 and 7271.3 Da (the peptide fragment 1–26 with a β-elimination at Cys-21 and 21–86 with a β-elimination at Cys-27, respectively). The remaining fragment (iminothiazolidine 21–26) 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).Table IITheoretical mass per charge ([M + H]) of the peptide fragments obtained after base-catalyzed cleavage of the peptide bond as calculated for the AVR4 des-speciesdes-(11-41)des-(21-27)des-(35-80)des-(57-72)Fragmentm/zFragmentm/zFragmentm/zFragmentm/z1-101144.31-202285.61-343805.41-566160.2itz-(11-40)3415.9itz-(21-26)676.8itz-(35-79)5031.8itz-(57-71)1800.0itz-(11-86)5075.9itz-(27-86)6673.7itz-(80-86)798.9itz-(72-86)1675.9β-(1-40)4482.2β-(1-26)2884.4β-(1-79)8759.2β-(1-71)7882.2β-(11-86)8413.8β-(21-86)7272.4β-(35-86)5752.7β-(57-86)3397.9 Open table in a new tab Table IIISummary of the observed phenotypes per disrupted disulfide bond in AVR4Disrupted disulfide bondaDisruption of one disulfide bond by either a cysteine substitution in PVX::Avr4 or by partial reduction.Cys residue pairbSuccessive numbering of the Cys residues as in Fig. 5.Necrosis inducing activitycNecrosis inducing activity was assayed on tomato cv. moneymaker-Cf4 using PVX::Avr4 (Fig. 1).Stability in apoplastic fluiddAVR4 des-species is present (+) or absent (-) after incubation with apoplastic fluid (Fig. 4).Binding to chitineAffinity of AVR4 des-species for chitin; +, binds to chitin; +/- decreased affinity for chitin (Fig. 6).Stability of the AVR4 chitin complexfStability of AVR4 des-species when bound to chitin in the presence of AF; +, stable; +/-, partially stable in the presence of chitin (Fig. 7).Number of strains of racegNumber of strains of Cladosporium fulvum identified so far with a single Cys substitution (12,14).Native-++++++++Cys-21-272-3++++++0Cys-57-726-7--+/-+(+/-)0Cys-11-411-5--++(+/-)2Cys-35-804-8+++-++6a Disruption of one disulfide bond by either a cyst
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