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Title: GREEN FLUORESCENT PROTEIN AS A REPORTER TO MONITOR GENE EXPRESSION AND FOODCOLONIZATION BY ASPERGILLUS FLAVUS.

Author
item DU, WANGELI - NC STATE UNIVERSITY
item HUANG, ZHENGYU - DOW AGROS SCIENCES
item FLAHERTY, JOSEPH - NC STATE UNIVERSITY
item WELLS, KEVIN
item PAYNE, GARY - NC STATE UNIVERSITY

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/21/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: Aspergillus flavus is a filamentous fungi, or mold, that causes disease in plants. It is also responsible for aflatoxin contamination of food and feeds. A gene for a naturally fluorescent protein from a jellyfish (Aequorea victoria) was tested for its ability to differentiate genetically modified A. flavus from wild type fungus. This gene for green fluorescent protein (gfp) allowed detection of genetically modified fungus. When fuse to the regulatory region of an aflatoxin control gene (aflR), gfp also allowed the production of aflatoxin to be estimated by its correlation with green fluorescence. When kernels of corn from strains of various sensitivity to infection were inoculated with A. flavus that contained a gfp gene, it was possible to correlate the degree of sensitivity of the corn with the amount of gfp visualized on the kernel. This advancement will allow faster and easier detection of plants resistance in to A. flavus. Detection of infection with the modified A. flavus could be performed without expensive equipment. No microscope or light filters were required. Any black light was sufficient for detection of the gfp gene.

Technical Abstract: The purpose of this work was to test the utility of a reporter transgene in the detection of transgenic Aspergillus flavus. This filimentous fungi is a plant pathogen that is responsible for aflatoxin contamination. The ability to easily detect transgenic fungi is a prerequisite to any field trials with pathogens modified for biocontrol. Transformants of A. flavus containing the Aequorea victoria gfp gene fused to a viral promoter region and 483 bp of the coding region of A. flavus aflR expressed green fluorescence detectable without a microscope or fluorescence filters. Expression of green fluorescence was correlated with resistance to aflatoxin accumulation in five corn genotypes inoculated with these transformants. This study demonstrates for the first time that a simple, inexpensive, non-invasive method can be used to follow transgenic filimentous fungi. In addition, this study demonstrates that an aflR/gfp transgene can be used to estimate A. flavus resistance in different strains of corn. It is also noteworthy that a mammalin viral promoter (cmv) and nuclear localization domain (SV40) can function in Aspergillus.