Molecular identification and functional analysis of HrpZ2, a new member of harpin superfamily from Pseudomonas syringae inducing hypersensitive response in tobacco

Harpins belong to a group of proteins with distinctive features such as heat stability, glycine richness, and absence of cysteine. Harpins are secreted by many gram-negative phytopathogens via the type III secretion system and are known for their ability to trigger hypersensitive response followed by induction of systemic acquired resistance in non-host plants. However, there is lack of studies on exploring the mechanism of action of harpins. In this study, we report cloning of a novel harpin gene (HrpZ2) from Pseudomonas syringae strain MTCC-11950, belonging to harpin superfamily. In silico analysis revealed that about 50.29% of the protein consists of alpha-helices, 48.53% are random coils, and only 1.16% are beta-sheets, and nearly half (42%) of the protein consists of intrinsically disordered regions. Based on a prokaryotic predictive model and the presence of a signal peptide on its N-terminus, the subcellular localization of harpin is predicted as extracellular. Determination of protein sequence and structure are fundamental to understanding its biological function. To date, no experimentally determined crystal structure of any harpin protein is available. Using this information, we built and validated an ab initio model (with 99% of residues in allowed/additionally allowed regions and a Z-score of −5.3) of harpin. Phylogenetic analysis and functional domain studies revealed that this novel harpin belongs to the harpin superfamily. Infiltration of harpin in tobacco leaves resulted in hypersensitive response, which was associated with oxidative burst, callose deposition, localized cell death, and increased activity of defense-related enzymes such as phenylalanine ammonia-lyase and polyphenol oxidase. Furthermore, infiltration of harpin in non-host plants from different angiosperm families induced hypersensitive response, indicating broad-spectrum agricultural applicability of this novel harpin protein. Current study sheds light on the molecular and functional analysis of novel harpin protein and its ability to induce HR response in broad range of non-host plants.