Use of green fluorescent protein (GFP) for studying development and fungal-plant interaction in Cochliobolus heterostrophus

The green fluorescent protein (GFP) has been widely used as an extremely useful vital marker in a large number of organisms, but good expression in filamentous ascomycetes has not been reported. To facilitate the research of fungal development and fungal-plant interaction, we constructed two plasmid vectors for the expression of the synthetic SGFP-TYG gene in ascomycete species, and used these vectors for transformation of the maize pathogen Cochliobolus heterostrophus. High level expression of GFP was obtained, as detected by anti-GFP antibodies and by fluorescence microscopy. The intense fluorescence was used as a highly efficient vital marker to detect cytoplasmic and developmental changes that occur in the fungus, and to follow phytopathogenic development of the fungus on and inside maize leaves. The hyphae within the leaf form a unique parallel growth pattern, closely associated with, and apparently determined by, the anatomy of the leaf. Fluorescence intensity was quantified by digital analysis of the green fluorescence image and was highly correlated with the amount of mycelium and levels of disease. Expression of GFP was obtained in additional ascomycetes that were transformed with the new constructs, indicating that SGFP-TYG can be used as a highly effective vital marker in ascomycetes.