Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes.
Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.
Marui
Junichiro
J
ABE-project, New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
Yoshimi
Akira
A
Hagiwara
Daisuke
D
Fujii-Watanabe
Yoshimi
Y
Oda
Ken
K
Koike
Hideaki
H
Tamano
Koichi
K
Ishii
Tomoko
T
Sano
Motoaki
M
Machida
Masayuki
M
Abe
Keietsu
K
D000199:Actins / Q000235:genetics*; D000935:Antifungal Agents / Q000494:pharmacology*; D019677:Artificial Gene Fusion; D001236:Aspergillus oryzae / Q000187:drug effects* / Q000235:genetics*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D005656:Fungal Proteins / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic
D000199:Actins; D000935:Antifungal Agents; D005656:Fungal Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins