Identification of cytoskeletal components in pine ectomycorrhizas

Ectomycorrhizal associations were synthesized between pine seedlings and the fungi Suillus bovinus (L.) ex Fr. or Paxillus involutus (Batsch ex Fr.) Fr. Immunoblotting of polypeptides separated electrophoretically from crude tissue extracts revealed the abundant presence of tubulin and actin in ectomycorrhiza and lower amounts in the fungal strands surrounding the ectomycorrhizal roots. In ectomycorrhiza the alpha-tubulins from fungal hyphae and plant cells were clearly distinguishable but such discrimination was not possible for beta-tubulin or actin due to the similar mobility of proteins originating from the conifer and fungal tissues. Young ectomycorrhizal short roots were fixed while still attached to the seedlings and, using indirect immunofluorescence microscopy with tubulin antibodies, microtubules were detected in both the conifer cells and in fungal hyphae. In the host plant cytoplasmic and spindle microtubules were visualized in meristem cells and in differentiating vascular tissue but not in the cortical cells. In symbiotic hyphae the microtubule tracks and spindles of dividing nuclei were clearly distinguished in the mantle hyphae in the tip region of the short roots. In the Hartig net hyphae microtubule tracks changed to a less clear, reticulate structure. Actin was visualized as long filaments in vascular tissue cells and as small microfilament bundles in mantle hyphae. Short microtubules and actin dots were detected in cytoplasm-containing hyphae on the strand surface. The possible role of the cytoskeletal elements in the maintenance of the ectomycorrhizal association is discussed