CONICET | Buscador de Institutos y Recursos Humanos

Hpa1 is a harpin protein encoded in the hrp (hypersensitive response (HR) and pathogenicity) cluster of Xanthomonas axonopodis pv. citri (Xac), the phytopathogenic bacteria that causes citrus canker disease. This cluster encodes the Type III Secretion System (TTSS), which is essential for pathogenicity in host plants and induction of HR in non-host plants since it mediates the translocation of effector proteins for pathogenicity. HR is characterized by a local rapid programmed cell death that is induced after recognition of the pathogen and slows the spread of infection. Harpin proteins are glycine-rich heat stable proteins that can form pores in membranes, induce HR in some plants and also form fibrils rich in b-sheet secondary structures, typical of amyloid proteins. In order to biophysically study the protein Hpa1, it was expressed using the vector pET28a and the strain Escherichia coli BL21pLysS, and highly purified using a Ni-NTA agarose matrix and a gel filtration column on a FPLC equipment, showing a tetrameric structure. Using the Congo Red dye binding assay and polarized light microscopy, it was observed the formation of amyloid-like fibrils. This b-sheet-rich fibrils formation was consistent to the observation made by circular dichroism, analyzed over time. By using this technique, the stability to heat of both ?fresh? and ?fibril? forms of Hpa1 was also determined, as well as the behavior against pH. The kinetics of amyloid-like fibrils formation by Hpa1 under different conditions using the specific dye Thioflavin T was performed, showing different rates of fibrils formation. In agreement to our previous results about the participation of Hpa1 in plant-pathogen interaction, this fibril structure may be involved in the elicitation of HR in non-host plants as well as in virulence in host plants.