Abstract: Three α-elicitin isoforms, named Hα1, Hα2 and Hα3, were isolated by reverse phase-low pressure liquid chromatography (RP-LPLC) steps from culture filtrates of Phytophthora hybernalis, the causal agent of citrus lemon brown rot. Hα1, that we also named hybernalin for the first time, proved to be identical to syringicin, an α-elicitin, previously isolated from culture filtrates of Phytophthora syringae. Hα2 and Hα3, shared the same primary structure as Hα1, but showed to be oxidized at Met50 as sulphoxide and sulphone derivatives, respectively. By SDS-PAGE, the three proteins showed the same electrophoretic mobility corresponding to ca 10 kDa. Exact Mr values were obtained by MALDI-TOF mass spectrometry (10,194.82 for Hα1, 10,209.33 for Hα2 and 10,223.80 for Hα3), while ESI-MS gave a Mr value of 10,194.90 for Hα1 and no results for Hα2 and Hα3, due to extensive polymerization in the instrument capillary. In addition, the three isoforms showed a high propensity to self-association, as dimeric and trimeric forms were present in aq buffer and even higher oligomers after exposure to acetonitrile. The three elicitins could also be distinguished on the basis of their biological activities. Hα1 showed to be active both in the hypersensitivity response (HR) and electrolytes leakage assay (ELA); Hα2 was only weakly active in the HR and inactive in the ELA, while Hα3 proved to be inactive in both tests. Correspondingly, the different activity of the three elicitin isoforms was very likely attributed to both the oxidation at Met50 and the formation of oligomers.
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