Affinity of Avr2 for tomato cysteine protease Rcr3 correlates with the Avr2-triggered Cf-2-mediated hypersensitive response

John W Van't Klooster, Marc W Van der Kamp, Jacques Vervoort, Jules Beekwilder, Sjef Boeren, Matthieu H A J Joosten, Bart P H J Thomma, Pierre J G M De Wit

Research output: Contribution to journalArticle (Academic Journal)peer-review

16 Citations (Scopus)

Abstract

The Cladosporium fulvum Avr2 effector is a novel type of cysteine protease inhibitor with eight cysteine residues that are all involved in disulphide bonds. We have produced wild-type Avr2 protein in Pichia pastoris and determined its disulphide bond pattern. By site-directed mutagenesis of all eight cysteine residues, we show that three of the four disulphide bonds are required for Avr2 stability. The six C-terminal amino acid residues of Avr2 contain one disulphide bond that is not embedded in its overall structure. Avr2 is not processed by the tomato cysteine protease Rcr3 and is an uncompetitive inhibitor of Rcr3. We also produced mutant Avr2 proteins in which selected amino acid residues were individually replaced by alanine, and, in one mutant, all six C-terminal amino acid residues were deleted. We determined the inhibitory constant (K(i) ) of these mutants for Rcr3 and their ability to trigger a Cf-2-mediated hypersensitive response (HR) in tomato. We found that the two C-terminal cysteine residues and the six amino acid C-terminal tail of Avr2 are required for both Rcr3 inhibitory activity and the ability to trigger a Cf-2-mediated HR. Individual replacement of the lysine-17, lysine-20 or tyrosine-21 residue by alanine did not affect significantly the biological activity of Avr2. Overall, our data suggest that the affinity of the Avr2 mutants for Rcr3 correlates with their ability to trigger a Cf-2-mediated HR.
Original languageEnglish
Pages (from-to)21-30
Number of pages10
JournalMolecular Plant Pathology
Volume12
Issue number1
DOIs
Publication statusPublished - Jan 2011

Bibliographical note

© 2010 The Authors. Molecular Plant Pathology © 2010 BSPP and Blackwell Publishing Ltd.

Keywords

  • Mutant Proteins
  • Genes, Fungal
  • Recombinant Proteins
  • Amino Acid Sequence
  • Disulfides
  • Fungal Proteins
  • Plant Diseases
  • Host-Pathogen Interactions
  • Mutagenesis, Site-Directed
  • Lycopersicon esculentum
  • Base Sequence
  • DNA Primers
  • Molecular Sequence Data
  • Cysteine Proteinase Inhibitors
  • Cladosporium
  • Cysteine Proteases
  • Amino Acid Substitution

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