Abstract
Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ψβ-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the β-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ψβ-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.
Highlights
Among the members of the CP family
The first line provides a basal defense against all potential pathogens and relies on the recognition either of conserved pathogen-associated molecular patterns (PAMPs),7 including flagellin, peptidoglycans, chitin, and -glucan, some of the proteins relevant for the life cycle of the pathogen [2], or of an array of cell wall-degrading enzymes not necessarily involved in pathogenicity [3]
CP Presents a Double -Barrel Fold—The three-dimensional structure of CP was solved by triple resonance NMR methods using Ͼ3400 distance and dihedral angle restraints together with 324 Residual Dipolar Constant (RDC) in iterative cycles of structure calculations
Summary
The recombinant CP protein was expressed in Pichia pastoris and purified by reverse-phase liquid chromatography as described previously [29]. The recombinant CP protein used in this work presents an additional 9-residue N-terminal stretch (EEGVSLEKR) because, under our experimental conditions, the signal peptide used for extracellular expression turned out not to be cut at the expected site. The protein shows the same biological activity and the same CD and 1H NMR spectra of native CP [29]. CP was expressed in minimal medium supplemented with [15N]ammonium sulfate and, in the case of double-labeled proteins, with [13C]glycerol and [13C]methanol as described previously [30]. The purified protein was estimated to be ϳ95% pure by SDS-PAGE, and MALDI-TOF mass spectrometry confirmed that it was 100% 13C- and 15N-isotopically labeled
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