Transformation of Gaeumannomyces graminis with β-Glucuronidase Reporter and Hygromycin Resistance Genes

Abstract

Take-all disease is caused by Gaeumannomyces graminis, (Sacc.) Arx & D. Olivier, a soil-borne fungus, which colonizes the root and crown tissue of many members of the Poaceae plant family. This fungus is able to grow along the surface of roots as darkly pigmented runner hyphae, which has the ability to penetrate the root. Here, we describe a genetic transformation of G. graminis var. graminis by using polyethylene glycol (PEG)-based protoplast transformation. Fungus cells were transformed with a plasmid, pHPG, containing the gusA reporter gene that codes for β-glucuronidase (GUS) and the hph gene for hygromycin resistance as the selectable marker. A de novo transformant selection assay was developed to identify the putative transformants that were expressing the hph gene. In addition, the transformed cells maintained the ability to infect the plant tissues. The GUS-expressing fungus can be used to study fungal infection processes including fungal penetration, colonization and the role(s) of melanin during pathogenesis. Thus, this study is the first report of G. graminis var. graminis transformed with a visibly detectable reporter gene that provides a useful tool to a better understanding of host-Gaeumannomyces interactions.