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

NMR investigation of random coil regions: clues to assess the different biological activity of two cerato platanin family members

Plant pathogenic fungi secrete several non-catalytic proteins involved in various aspects of the pathogenesis process. Amongst these, cerato-platanin (CP) was first identified and characterized as a PAMP (pathogen-associated molecular pattern) [1,2]. A sequence homology search revealed a set of fungal Cys-rich secreted proteins that have been grouped in the CP family. They induce synthesis of phytoalexins, overexpression of defense-related genes, H2O2 and NO production, markers of defense activation [3]. The core member of this family, CP (from C. platani) shows a double beta barrel fold [2]. Here we present the results of a CP orthologue with 73% of similarity, cerato-populin (Pop1), produced by C. populicola. Though both CP and Pop1 are host defense inducers, Pop1 shows a slower and weaker defense induction capacity than CP [4]. The aim of the present investigation is to define the basis of the different biological activity of the two proteins at a molecular level. Pop1 15N, 13C and 1H resonances have been assigned [5]. The analysis of Pop1 structure obtained by homology modelling, in comparison with the CP NMR structure, will be presented. A detailed analysis of the NMR-derived protein dynamics (fast and slow regime) and NOE data indicated differences between the two proteins, mainly located in the random coil region. Interestingly, this region was proposed to have an important role in oligosaccharides binding and in necrosis induction of leaves’ cells [6]; therefore, the different pattern of residues’ interactions might be the leading cause of their diverse biological activity. To address this hypothesis we have performed NMR experiments in the course of a titration of both Pop1 and CP with oligosaccharides. The high dependence on chemical pesticides in Europe poses large risks to both the environmental and human health; thus, reducing the use of those chemicals in crop production is one of the major objectives in sustainable agriculture. We expect that our results, besides providing new hints on the molecular mechanisms operating in plants induced resistance, will contribute to reach the major goal of environment protection. Besides that, from a basic science perspective, this work illustrates a time saving approach for the investigation of a protein structure-function relationship when the high-resolution structure of an orthologous protein is already available. [1] Pazzagli et al., Cell Biochem Biophys., 44 (2006), 512. [2] de Oliveira et al., J. Biol. Chem., 286 (2011), 17560. [3] Comparini et al., Appl. Microbiol. Biotechnol., 84 (2009), 309. [4] Lombardi et al., Phys. Plant. 149 (2013), 408. [5] Baroni et al., Biomol NMR Assign., (2013), epub. [6] Frias et al., Mol. Plant Pathology (2013), epub

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

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

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