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.