Functional characterization of an inducible bidirectional promoter from Fusarium oxysporum f. sp. cubense

Abstract

AbstractBidirectional promoters (BDPs) are regulatory DNA sequences (~1000 bp long) intervening two genes arranged on opposite strands with their 5′ ends in close proximity. These genes are mostly co-expressed; but, instances of anti-correlation and independent transcription have been observed. In fungal systems, BDPs have shown to provide an improved genetic circuit by assembling and regulating transcription of different genes of a common metabolic pathway. We have identified an intergenic region (1063 bp) from the genome of Fusarium oxysporum f. sp. cubense (Foc), a banana root pathogen. This intergenic region regulates the expression of a gene pair required for the breakdown of hemicellulose. For characterization, it was cloned into pCSN44 vector backbone between two reporter genes, namely β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP). The newly formed vector was transformed into Foc and tested for its bidirectional expression activity. Using histochemical staining and fluorescence microscopy, the kinetics for both, GUS and EGFP expression were tested under different growth conditions respectively. The activity was differentially regulated by inducers such as xylan, arabinogalactan and pectin. This is the first report on the isolation of the intergenic region with inducible bidirectional promoter activity from Fusarium. Characterization of such BDPs will find applications in genetic engineering, metabolic engineering and synthetic biology using fungal systems.