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

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

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

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

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

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