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Vacuolar localization of phosphorus in hyphae of Phialocephala fortinii, a dark septate fungal root endophyte.

Phialocephala fortinii is a dark septate fungal endophyte that colonizes roots of many host species. Its effect on plant growth varies from being pathogenic to beneficial. The basic biology of this species has received little research, and thus the main objectives of this study were to determine cytological features of hyphae, including the nature of the vacuolar system, and whether polyphosphate was present in vacuoles. Both living hyphae and hyphae that had been rapidly frozen and freeze substituted before embedding were studied. A complex system of vacuoles, including a motile tubular vacuolar system, elongated vacuoles, and spherical vacuoles, was demonstrated in living hyphae by the fluorescent probe Oregon Green 488 carboxylic acid diacetate, using laser scanning confocal microscopy. The motile tubular vacuolar system was more prevalent at the hyphal tip than in more distal regions, whereas elongated vacuoles and spherical vacuoles were more abundant distal to the tip. All vacuoles contained polyphosphate as shown by labelling embedded samples with recombinant polyphosphate binding domain of Escherichia coli exopolyphosphatase, containing Xpress tag at the N-terminal end, followed by anti-Xpress antibody and a secondary antibody conjugated either to a fluorescent probe for laser scanning confocal microscopy or colloidal gold for transmission electron microscopy. The polyphosphate was dispersed in vacuoles. This was confirmed by staining embedded samples with 4',6-diamidino-2-phenylindole and viewing with UV light using epifluorescence microscopy. These cytological methods showed that the tubular vacuolar system had lower concentrations of polyphosphate than the spherical vacuoles. Lipid bodies were present around vacuoles.