Actin and tubulin cytoskeletons in germlings of the oomycete fungus Phytophthora infestans

Abstract

Actin and tubulins of Phytophthora infestans germlings were detected with monoclonal antibodies on Western blots of crude extracts separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Mr of actin was approximately 43,000, whereas alpha- and beta-tubulin, which migrated as a single band, had an Mr of 53,000. Rhodamine-phalloin revealed peripheral patches of actin in ungerminated cysts. In young germlings, actin fibers were visible in the conversion zone between cyst and germ tube and as connections between actin patches and the incipient germ tube. Actin patches also occurred throughout the peripheral cytoplasm of longer germ tubes, except for the hyphal apex, which commonly contained actin fibers, but actin patches only exceptionally. Associations between patches and fibers were frequent. A monoclonal antibody specific for actin also stained fibers, but in addition it revealed diffuse staining of the apex and fine granular structures, indicative of the presence of G-actin or of single actin filaments. Cysts incubated with a monoclonal antibody against tubulin contained an array of cytoplasmic microtubules (MTs) that arise from a nucleus-associated center. Some of these MTs circumflexed the nucleus, whereas others extended to the cyst periphery. In germ tubes, axially oriented MT bundles extended from the nucleus-associated center into the proximal and distal cytoplasm. Their density was highest near the nucleus, and their number decreased towards the tip, with only a few remaining at the extreme apex. Bundles of MTs were continuous from the nucleus to the subapical region, reaching lengths of up to 20 microns. Ultrastructurally the bundles consisted of as many as 10 MTs. The architecture of the actin and tubulin cytoskeletons in germ tubes of P. infestans bolsters the hypothesis that they maintain the spatial organization of the hyphal protoplast and support or accomplish intrahyphal movements.