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 extreme ly useful vital marker in a large number of organisms, but good expres sion in filamentous ascomycetes has not been reported. To facilitate t he research of fungal development and fungal-plant interaction, we con structed two plasmid vectors for the expression of the synthetic SGFP- TYG gene in ascomycete species, and used these vectors for transformat ion of the maize pathogen Cochliobolus heterostrophus. High level expr ession 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 change s that occur in the fungus, and to follow phytopathogenic development of the fungus on and inside maize leaves. The hyphae within the leaf f orm a unique parallel growth pattern, closely associated with, and app arently determined by, the anatomy of the leaf. Fluorescence intensity was quantified by digital analysis of the green fluorescence image an d was highly correlated with the amount of mycelium and levels of dise ase. Expression of GFP was obtained in additional ascomycetes that wer e transformed with the new constructs, indicating that SGFP-TYG can be used as a highly effective vital marker in ascomycetes.