Affinity of Avr2 for tomato cysteine protease Rcr3 correlates with the Avr2-triggered Cf-2-mediated hypersensitive response.
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.
Wageningen University, Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands.
John W
JW
Van't Klooster
Marc W
MW
Van der Kamp
Jacques
J
Vervoort
Jules
J
Beekwilder
Sjef
S
Boeren
Matthieu H A J
MH
Joosten
Bart P H J
BP
Thomma
Pierre J G M
PJ
De Wit
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002963:Cladosporium / Q000235:genetics / Q000472:pathogenicity*; D057056:Cysteine Proteases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D017931:DNA Primers / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D005800:Genes, Fungal; D054884:Host-Pathogen Interactions; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000382:microbiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D010935:Plant Diseases / Q000382:microbiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity
D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D004220:Disulfides; D005656:Fungal Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D057056:Cysteine Proteases