The perennial ryegrass endophyte Neotyphodium lolii genetically transformed with the green fluorescent protein gene (gfp) and visualization in the host plant

The green fluorescent protein gene was inserted into isolates of the endophyte Neotyphodium lolii from perennial ryegrass Lolium perenne using the method with polyethylene glycol treatment of protoplasts. The transformation rate was 3.2 x 10(-6) of the original number of protoplasts 5 weeks after the transformation. Freshly isolated mycelium from the host plant was used in the transformation experiments, since difficulties of infecting the host plant with older isolates were observed. Isolates were identified using the universal primed (UP) PCR fingerprinting method. The transformed mycelium readily expressed the green fluorescent protein gene. Fluorescence was observed in the cytoplasm of the mycelium cells in pure culture, both before infection and after re-isolation from the host plant. The fluorescent hyphae were easily detected in the host plant. No disturbing autofluorescence from the host plant leaf sheaths or from the wild type isolate was observed. The importance of mitotic stability is discussed, based on the transformed phenotype and southern hybridization pattern, since the Neotyphodium lolii transformants lost 1-2 copies of the integrated gene after re-isolation from the host plant without losing their GFP phenotype. This is the first report on transformation of a Neotyphodium sp. with a gfp gene. In the future, the green fluorescent protein may serve as a tool for studying gene expression and protein localization in the endophyte/host plant interaction using heterologous fusion proteins.