Transient expression and insertional mutagenesis of Puccinia triticina using biolistics

The fungal genus Puccinia contains more than 4,000 species. Puccinia triticina, causal agent of wheat leaf rust, is an economically significant, biotrophic basidiomycete. Little is known about the molecular biology of this group, and tools for understanding gene function have not yet been established. A set of parameters was established for the transient transformation of urediniospores. The expression of three heterologous promoters (actin, elongation factor 1-α, and Hss1, Heat Shock 70 protein), derived from Puccinia graminis, was evaluated along with the potential for insertional mutagenesis. The UidA (GUS) gene was used as a marker for transient expression. When transferred into P. triticina urediniospores, transient expression was observed across four helium pressures using one size of gold and three sizes of tungsten microprojectiles. Each of the three promoters displayed strong transient expression in germinated urediniospores; however, higher numbers of GUS-positive urediniospores were observed when either the actin or Hss1 promoters were used. Possible concomitant insertional mutagenesis of several avirulence genes was selected in wheat cultivars harboring the cognate resistance genes. Using a linearized cloning plasmid, stable integration into the genome was achieved as demonstrated by PCR and sequencing analysis.