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.

Detection of an elicitor on infection structures of Puccinia graminis using monoclonal antibodies.

The basidiomycetous fungus Puccinia graminis f. sp. tritici causes the stem rust disease of wheat. Resistance of wheat to the fungus is often associated with the hypersensitive reaction of infected host cells. A glycoprotein isolated from germ tube cell walls of the pathogen elicits a hypersensitive-like response when injected into wheat leaves. Infection structures morphologically identical to those grown on wheat were induced in the absence of the host plant, and indirect immunofluorescence together with specific monoclonal antibodies to the elicitor was employed to locate the antigen at fungal infection structures. No binding occurred to germ tubes or appressoria. The antibodies located the antigen only at that part of the fungal infection structure that develops endophytically in nature and, moreover, only at the youngest part of this structure. In rust-infected wheat leaves, the immunolabel appeared only at haustoria, the structures thought to be involved in specific recognition between host and parasite.

Isolation and characterization of the EF-1 alpha gene of the filamentous fungus Puccinia graminis f. sp. tritici.

A gene of Puccinia graminis f. sp. tritici, coding for the translation elongation factor 1 alpha (EF-1 alpha), was isolated from a P. graminis genomic library using the EF-1 alpha gene sequence of Absidia glauca. The coding region of 1389 nucleotides encodes a polypeptide of 463 amino acids and is interrupted by eight introns. An additional intron is located in the 5' untranslated region. A single transcription start point (tsp) was mapped by primer extension. A cDNA fragment corresponding to P. graminis EF-1 alpha mRNA hybridized with a 1.9-kb-long poly(A+)RNA, sufficient to encode the EF-1 alpha protein. Southern hybridization of digested genomic DNA revealed that two copies of the EF-1 alpha gene exist in the genome of P. graminis.

Transient transformation of the rust fungus Puccinia graminis f. sp. tritici.

The biotrophic rust fungus Puccinia graminis f. sp. tritici (Pgt) was transformed by particle bombardment. The promoter from the Pgt translation elongation factor 1alpha (EF-1alpha) gene was fused to the bacterial marker genes hygromycin B phosphotransferase (hpt) and beta-glucuronidase (GUS). Transformation constructs were introduced into uredospores of Pgt, an obligate pathogen of wheat, by biolistic bombardment. Uredospores transformed with the construct containing the hpt gene germinated and initiated branching on selective medium, indicating that they had acquired resistance to hygromycin B. However, transformants stopped growing 5 days after bombardment. GUS activity in uredospores and germlings was histochemically detected 4-16 h after bombardment. GUS expression was also obtained using the INF24 promoter from the bean rust fungus Uromyces appendiculatus, demonstrating that heterologous genes can be expressed in P. graminis under the control of regulatory sequences from closely related organisms.