Abstract
Harpins are heat-stable, glycine-rich type III-secreted proteins produced by plant pathogenic bacteria, which cause a hypersensitive response (HR) when infiltrated into the intercellular space of tobacco leaves; however, the biochemical mechanisms by which harpins cause plant cell death remain unclear. In this study, we determined the biochemical characteristics of HpaG, the first harpin identified from a Xanthomonas species, under plant apoplast-like conditions using electron microscopy and circular dichroism spectroscopy. We found that His(6)-HpaG formed biologically active spherical oligomers, protofibrils, and beta-sheet-rich fibrils, whereas the null HR mutant His(6)-HpaG(L50P) did not. Biochemical analysis and HR assay of various forms of HpaG demonstrated that the transition from an alpha-helix to beta-sheet-rich fibrils is important for the biological activity of protein. The fibrillar form of His(6)-HpaG is an amyloid protein based on positive staining with Congo red to produce green birefringence under polarized light, increased protease resistance, and beta-sheet fibril structure. Other harpins, such as HrpN from Erwinia amylovora and HrpZ from Pseudomonas syringae pv. syringae, also formed curvilinear protofibrils or fibrils under plant apoplast-like conditions, suggesting that amyloidogenesis is a common feature of harpins. Missense and deletion mutagenesis of HpaG indicated that the rate of HpaG fibril formation is modulated by a motif present in the C terminus. The plant cytotoxicity of HpaG is unique among the amyloid-forming proteins that occur in several microorganisms. Structural and morphological analogies between HpaG and disease-related amyloidogenic proteins, such as Abeta protein, suggest possible common biochemical characteristics in the induction of plant and animal cell death.
Highlights
Mostly encode proteins necessary for type III protein secretion systems, are involved in the secretion of harpins [1, 2]
Harpins are heat-stable, glycine-rich type III-secreted proteins that cause a hypersensitive response (HR)2 when filtrated into the intercellular space of tobacco leaves [1, 3, 4]
The plant HR is similar to programmed cell death, or apoptosis, in animal cells, and the biochemical changes that occur during the HR in plant cells have been well documented [5,6,7,8]
Summary
Mostly encode proteins necessary for type III protein secretion systems, are involved in the secretion of harpins [1, 2]. To study HpaG aggregation under plant apoplast-like conditions, we examined fibril formation in XVM2 and apoplastic fluid.
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