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.
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.
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
Harrold A
HA
van den Burg
Nienke
N
Westerink
Kees-Jan
KJ
Francoijs
Ronelle
R
Roth
Esmeralda
E
Woestenenk
Sjef
S
Boeren
Pierre J G M
PJ
de Wit
Matthieu H A J
MH
Joosten
Jacques
J
Vervoort
D000409:Alanine / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002963:Cladosporium / Q000235:genetics*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005838:Genotype; D018551:Solanum lycopersicum / Q000378:metabolism / Q000382:microbiology; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D009336:Necrosis; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014158:Transcription, Genetic; D014443:Tyrosine / Q000737:chemistry
C085987:AVR4 protein, Cladosporium fulvum; D000596:Amino Acids; D001798:Blood Proteins; D002352:Carrier Proteins; D004220:Disulfides; D005656:Fungal Proteins; D010455:Peptides; D011134:Polysaccharides; D020033:Protein Isoforms; C104989:tachycitin protein, Tachypleus tridentatus; D002686:Chitin; D014443:Tyrosine; D003545:Cysteine; D000409:Alanine