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, and are secreted by many Gram-negative phytopathogens via the type III secretion system. Harpins are known to trigger hypersensitive response followed by induction of systemic acquired resistance in non-host plants. However, the molecular mechanism of harpin-induced hypersensitive response remained largely unexplored, mainly because of lack of structural information. In this study, we report the cloning of a new harpin gene ( hrpZ2 ) from Pseudomonas syringae strain MTCC-11950, belonging to the 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. To date, no experimentally determined crystal structure of any harpin protein is available. Therefore, we built and validated a three-dimentional 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 new 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 new harpin protein. This study elucidates the molecular and functional properties of the new harpin protein and its ability to induce hypersensitive response across a broad range of non-host plants.