Isolation, characterization and structure-elicitor activity relationships of hibernalin and its two oxidized forms from Phytophthora hibernalis Carne 1925

Three a-elicitins, named hibernalin1, hibernalin2 and hibernalin3 (hib1, hib2 and hib3, respectively), were isolated by reverse phase-low-pressure liquid chromatography from culture filtrates of Phytophthora hibernalis Carne 1925, the causal agent 20 of citrus lemon brown rot. Hib1 proved to be identical to syringicin previously isolated from culture filtrates of Phytophthora syringae. Hib2 and hib3 shared the same primary structure with hib1, but contained, at position 50, Met sulphoxide or sulphone, respectively. By SDS–PAGE, the three proteins showed the same electrophoretic mobility, corresponding to about 10 kDa. Exact Mr values were obtained by 25 MALDI-TOF-MS (10,194.82 for hib1, 10,209.33 for hib2 and 10,223.80 for hib3), while by ESI-MS an Mr value of 10,194.90 was found for hib1 and no results for hib2 and hib3. The hibernalin forms showed a high propensity to self-association, after exposure to acetonitrile. Hib1 showed to be active in both the hypersensitivity response and electrolytes leakage assays; the sample containing hib1 and hib2 was only weakly 30 active in the first assay and inactive in the second assay, while the sample containing all three hibernalin forms proved to be inactive in both tests. It is proposed that the different activities of the three hibernalin samples could be very likely attributed to both Met50 oxidation and aggregation.