Fig 1.
A) Gel filtration chromatography. Purification of wtCP on 100 x 3.5 cm Superdex G75 column; 50 mM Tris-HCl buffer pH 8.0 containing 150 mM NaCl was used. Peak 1: GST-wtCP; Peak 2: GST, Peak 3: wtCP. B) RP-HPLC purification of mutCP. The column was Phenomenex, 4,6 ×250 mm; 5 μm;Solvent A, 10 mM TFA in water; Solvent B, 10 mM TFA in acetonitrile. Flow rate was 0.8 ml/min. The elution was performed by an acetonitrile gradient as indicated by the dotted line (……). Peak 1: mutCP; Peak 2: GST-mutCP. C) 15% SDS-PAGE of the pure proteins. Lane St: Prestained Protein SHARPMASS VI marker (Euroclone); Lane 1: native CP; Lane 2: recombinant wtCP; Lane 3: D77A mutCP. 2μg of each protein were applied; gel was stained with Comassie Brilliant Blue.
Table 1.
Purification yield of wild type and mutated CP starting from 1L of induced culture.
Values are the means of data from four independent experiments ± s.d.
Fig 2.
Twenty picomoles of proteins were dissolved in 50% acetonitrile containing 0.05% TFA, diluted 1:1 in saturated sinapinic acid matrix and analysed on a MALDI–TOF mass spectrometer. A) nCP. B) wtCP. C) mutCP.
Fig 3.
Structural characterisation of CP.
A) Far-UV CD spectra of 6.25 μM nCP, wtCP and mutCP. B) Near-UV CD spectra of 1.5 mg/mL wtCP and mutCP. All samples were dissolved in 10 mM Na-phosphate buffer.C) equilibrium denaturation experiment of 0.04 mg/ml wtCP and mutCP in the presence of GndHCl concentrations ranging from 0 to 5.6 M.
Fig 4.
A) Surface representation. Coloured residues are located in the putative oligosaccharide binding region. Colours shown conserved amino acid along all CPF members. Red for acid residues, blue for basic residues and yellow for-polar residues. B) H bonds distances in wtCP. The bonds between D77-Y9, D77-S78 and D77-A19 are indicated. Yellow rectangle indicates the zoomed region in C. C) H bonds in mut CP. Lack of H bonds in mutCP with A77 instead D77.
Fig 5.
Eliciting activities of CP on Arabidopsis leaves.
A) Phytoalexins production. Fluorescence emitted by 150 μM of nCP, wtCP and mutCP was assayed after 24h of incubation. Data are the mean ± s.d. of values obtained by six independent experiments performed in duplicate. Values marked with different letters are significantly different at p< 0.01 according to the t-test. B) ROS production. A. thaliana leaves treated for 24h with H2O (or with150 μM wtCP (C) and mutCP (D). H2O2 was visualized in situ by the fluorescent probe DCFH2-DA.
Fig 6.
Weakening activity of CP on filter paper.
A) Releasing of paper fragments from the paper disc Each tube contains a filter paper disc in 0.5 mL of 50 mM sodium acetate buffer and 30 μM of wtCP or mutCP. Buffer only or buffer containing 30μM BSA at the same concentration were used as negative controls. Weakening activity was visible as paper fragments released in suspension from the paper disc after 48h, at 38°C, with shaking at 700 rpm. B) Quantification of the paper fragments produced. Absorbance at 500 nm was measured on a Ultraspec 2000 (Pharmacia biotech) spectrophotometer. Error bars indicate the standard deviation of measurements from three separated experiments. Values marked with different letters are significantly different at p< 0.05 according to the t-test.