| Abstract |
syringae pv. phaseolicola, are responsible both for their ability to cause disease on susceprible plants and to elicit the hypersensitive reaction (HR) on resistant plants. The HR is one of the major defence mechanisms of plants to pathogen attack and its phenotypic manifestation depends both on bacterial genes (hrp and avr) and plant genes (R). Certain hrp genes encode components of a specialized protein secretion pathway, called "type III" secretion system, similar to the synonymous pathway used by animal pathogenic bacteria, such as Salmonella, Yersinia, Shigella to secrete proteinaceous pathogenic determinants. In phytopathogenic bacteria, this pathway controls the secretion of several proteins, among them are the harpins, which are able to elicit the hypersensitive reaction, upon injection, in purified form, into the leaves of resistant plants. In order to study the role of harpin, HrpZPsp, of the bacterium Pseudomonas syringae pv. phaseolicola, in hypersensitive reaction, the hrpZPsp gene was expressed in tobacco plants. For this purpose, two expression systems were used: the tetracycline regulated system, where stable transfosmant plants were made and the transient viral expression system, which is based on the PVX202 virus. In both cases, constructs designed to express either the canonical form of harpin or a N-terminally extended version of the protein carrying the signal peptide portion of the tobacco pathogenesis related protein PR1a were used, so that the produced protein could be secreted into intercellular space of leaves. The production of HrpZ, in either form in transgenic plants, did not lead to necrosis, as expected, probably because of the low expression level. In contrast, the accumulation of secreted form of the harpin by the viral expression vector PVX202 induced the appearance of necrotic lesions, characteristic of HR, in tobacco leaves. These results indicate that the harpin acts of a site(s) external to the plant cell. In the present work, it was observed that the expression of the harpin in plant and bacterial cells leads to the formation of two electrophoritically different forms. The N-terminal sequencing indicated that the faster-moving form is a N-terminally truncated version of the harpin. Site-directed mutagenesis and in vitro translation further suggested that this form may originate from alternative initiation of translation, at an internal codon AUG16. The Hrp secretion pathway is also capable of delivering Avr proteins into the plant cells. Based on indications that both harpins and Avr proteins must be expressed by the same bacterial cell in order to trigger the hypersensitive reaction, we examined whether these proteins interact with each other. Similar experiments were carried out to identify possible interactions among Hrp/Hrc proteins to provide a basis for understanding the functional architecture of the Hrp secretion apparatus. Two experimental approaches were followed: the yeast two hybrid system and the Far western technique. In the case of the AvrPphB and AvrPto proteins, no interaction with harpin was detected, indicating that their translocation, through the type III apparatus, into the plant cells does not need physical contact between the Avr proteins and harpin. In respect to other Hrp proteins, interaction was detected between HrcQa and HrcQb. Moreover, it was showed that the HrcQb protein interacts with the carboxyl-terminal portion of HrcQa,. Based on these results and on other evidence, a model for the probable topology of HrcQa protein in the bacterial cell is proposed.
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Μοριακή και βιοχημική μελέτη πρωτεϊνων του εκκριτικού συστήματος τύπου III στο φυτοπαθογόνο βακτήριο PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA και διερεύνηση του ρόλου τους στην αντίδραση υπερευαισθησίας των φυτών
