Isolation and identification of actin from the phytopathogenic filamentous fungus uromyces appendiculatus

Crude protein extracts of Uromyces appendiculatus contain a polypeptide that resembles actin in several ways. This protein eluates from DEAE-cellulose with concentrations of KCl known to release actin of other species from the cation. The polypeptide is recognized by polyclonal antibodies directed to sodium dodecyl sulfate-denatured actin of chicken gizzard as well as by a monoclonal antibody also made to gizzard actin from chicken, but not by antibodies made against rabbit skeletal muscle actin. Western blot analysis after electrophoresis of the protein on polyacrylamide revealed that the protein has an electrophoretic mobility very similar to that of rabbit skeletal muscle actin. We were unable either to isolate actin by affinity chromatography using immobilized DNase-I, or to identify bean rust actin using DNase-I inhibition assays. Nevertheless, large quantities of the protein sedimented by high speed centrifugation. The sedimented protein resisted attempts to solubilize it under conditions normally used to depolymerize actin filaments. Both of the latter findings indicate unusual features of bean rust actin. Immunocytochemical studies of actin localization in germlings of the fungus using two chicken gizzard actin antibodies revealed actin-containing sites which were similar to those previously observed with fluorescently tagged phallotoxin derivatives.