A single amino acid mutation affects elicitor and expansins-like activities of cerato-platanin, a non-catalytic fungal protein

<div><p>Cerato-platanin (CP) is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response. Indeed, CP is an elicitor of primary defences acting as a Pathogen-Associated Molecular Patterns (PAMP). So far, structure-function relationship study has been mainly performed on the bacterial BsEXLX1 expansin, probably due to difficulties in expressing plant expansins in heterologous systems. Here, we report a subcloning and purification method of CP in the engineered <i>E</i>. <i>coli</i> SHuffle cells, which proved to be suitable to obtain the properly folded and biologically active protein. The method also enabled the production of the mutant D77A, rationally designed to be inactive. The wild-type and the mutated CP were characterized for cellulose weakening activity and for PAMP activity (i.e. induction of Reactive Oxygen Species synthesis and phytoalexins production). Our analysis reveals that the carboxyl group of D77 is crucial for expansin-like and PAMP activities, thus permitting to establish a correlation between the ability to weaken cellulose and the capacity to induce defence responses in plants. Our results enable the structural and functional characterization of a mono-domain eukaryotic expansin and identify the essential role of a specific aspartic residue in cellulose weakening.</p></div

MALDI–TOF mass spectrometry.

<p>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.</p

3D model of CP structure.

<p>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.</p

Purification yield of wild type and mutated CP starting from 1L of induced culture.

<p>Values are the means of data from four independent experiments ± s.d.</p

Structural characterisation of CP.

<p>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.</p

Weakening activity of CP on filter paper.

<p>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 <i>t-test</i>.</p

Stomatal closure induced by CP.

<p>(A) Open and closed stomata in <i>Arabidopsis</i> epidermal peels. Bar = 10 µm. (B) Measurement of stomatal aperture after 30 min, 1 and 2 h of treatment with 150 µM CP or MES buffer (control). Results are the mean of 100 measurements ± SEM. Statistical analysis was performed by unpaired <i>t</i>-test (treated vs. control). Asterisks indicate statistically significant difference at <i>P</i><0.05. The experiment was repeated with similar results.</p

Phosphorylation of <i>Arabidopsis</i> MPK6 and MPK3 after treatment with 150 µM CP.

<p>(A) Analysis carried out with human phospho-p44/42 antibodies (pERK1/2) on protein extracts obtained after 0, 5, 15, 30 and 60 min of treatment. pMPK6 and pMPK3 indicate phosphorylation. (B) Image analysis showing the phosphorylation level normalized to the respective protein amount. Error bars indicate SD of three biological replicates. Statistical analysis was performed by unpaired <i>t</i>-test by comparing the phosphorylation level to the appropriate background level (0 h). Asterisks indicate statistically significant difference at <i>P</i>≤0.05.</p

H₂O₂ production at the level of stomata.

<p>Epidermal peels from <i>Arabidopsis</i> leaves were first loaded with the fluorescent probe H₂DCF-DA and then treated either on the cuticle side (A and B) or on the underside (C) with 150 µM CP to analyse the origin of the ROS signalling. (A) Single guard cells photographed at 2-minute intervals after treatment with CP (same peel). Bar = 30 µm. (B) Photographs of representative regions of three different peels treated with CP for 5, 10 or 30 min respectively, or 30 min with MES buffer (control). Fluorescence microscopy (H₂DCF-DA), light microscopy (bright field) and merged pictures (merge) are shown in order to facilitate the localisation of stomata. The bar is 40 µm and applies to all photographs. (C) Peels treated on the underside (the side devoid of cuticle) with CP. Bars = 100 µm (5 min) and 30 µm (10 and 30 min).</p

Resistance induction assays against <i>Botrytis cinerea</i> and <i>Pseudomonas syringae</i> pv. <i>tomato</i> (<i>Pst</i>).

<p><i>Arabidopsis</i> detached leaves were treated on the lower surface with 150 µM CP, sterile distilled water (control) or 0.1% chitosan for 24 h before pathogen inoculation. (A) <i>B. cinerea</i> strain PM10 was inoculated by placing a single 10-µl drop of a suspension 2×10⁵ conidia ml⁻¹ in 1% Sabouraud Maltose Broth on a side of the middle vein. Infected leaves were incubated for 3 days at 22°C before taking photos and measuring the lesion size. (B) <i>Pst</i> strain DC3000 was inoculated by placing a single 10-µl drop of a suspension 10⁸ colony-forming units (CFU) ml⁻¹ (OD₆₀₀ = 0.2) in sterile distilled water containing 0.02% Silwet L-77 on a side of the middle vein. Infected leaves were incubated for 3 days at 28°C before taking photos and determining bacterial titer. (C) Measurement of the lesion diameter (mm) ± SD after 3 days of incubation with <i>B. cinerea</i> (n = 8). (D) Counting of CFU mg⁻¹±SD after 3 days of incubation with <i>Pst</i> DC3000 (n = 8). Statistically significant differences among treatments are indicated at <i>P</i>≤0.05.</p