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

• Published in: PloS one Vol. 12; no. 5; p. e0178337
• Main Authors: Luti, Simone, Martellini, Federica, Bemporad, Francesco, Mazzoli, Lorenzo, Paoli, Paolo, Pazzagli, Luigia
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 25.05.2017; Public Library of Science (PLoS)
• Subjects: Acids; Aggregates; Amino acid sequence; Amino Acid Substitution; Amino acids; Bacteria; Bacterial proteins; Batch culture; Binding energy; Biochemistry; Biocompatibility; Biological activity; Biological control; Biology and Life Sciences; Carbohydrates; Catalysis; Cell culture; Cell walls; Cellulose; Cellulose - metabolism; Chemical bonds; Chemical properties; Chitin; Chymotrypsin; Circular dichroism; Cloning; Colonization; Crystal structure; Defensive behavior; Deoxyribonucleic acid; DNA; E coli; Engineering and Technology; Entropy; Enzymes; Escherichia coli; Escherichia coli - genetics; Fragmentation; Fungal Proteins - genetics; Fungal Proteins - physiology; Fungi; Gene expression; Growth hormones; Health care; Homeostasis; Homology; Host-Pathogen Interactions - genetics; Immunoglobulins; Kinases; Metabolism; Metabolites; Methods; Molecular biology; Morphogenesis; Mutants; Mutation; Nitric oxide; Oxygen; Pathogen-Associated Molecular Pattern Molecules - metabolism; Pathogens; Pattern recognition; Photosynthesis; Physical Sciences; Physiological aspects; Plant Proteins - genetics; Plant Proteins - physiology; Polysaccharides; Protein kinase; Proteins; Purification; Quality; Reactive oxygen species; Research and Analysis Methods; Residues; Rot; Saccharides; Signal transduction; Surgical implants
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0178337

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 E. coli 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.