Abstract
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.
Highlights
The intrinsic role of the AVR proteins of C. fulvum during infection remains obscure, they are anticipated to contribute to virulence in susceptible hosts (16 –18)
These findings argue that mutant AVR4 isoforms are fully functional and can associate with chitin upon release, whereas excess of secreted protein is degraded before triggering host defense responses
To determine whether other Cys residues, for which no mutations were found in strains of C. fulvum, are required for necrosisinducing activity (NIA) of AVR4, we independently replaced all individual Cys residues by Ala in potato virus X (PVX)::Avr4
Summary
Construction of PVX Derivatives and Transcription—Avr mutants encoding various Cys to Ala substitutions were generated by PCR-based primer-directed mutagenesis on the plasmid pTX⌬GC3a, containing the native Avr sequence [14]. The reaction was initiated by adding a 6-fold molar excess of TCEP to AVR4, followed by incubation at 20 °C for 15 min. Peptide Cleavage and Full Reduction of the Disulfide Bonds/Peptide Mass Analysis—Lyophilized HPLC fractions containing the AVR4 desspecies were dissolved in two consecutive steps: first, 2 l in 1 M NH4OH, 6 M guanidine-HCl, and second, 5 l of 1 M NH4OH, and incubated at 20 °C for 1 h. The remaining disulfide bonds were reduced by adding an excess of TCEP (10 l of 0.1 M TCEP stock), and the mixture was incubated at 37 °C for 30 min. Polysaccharide Substrate Binding Assay—Native AVR4 and AVR4 des-species (4 g) 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 l of final volume) as described [19]. The models were found reliable using standard algorithms [36, 37]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
