Abstract
Bidirectional 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.
Highlights
A bidirectional promoter is an intergenic region of ~1–1.5 kb that regulates the flanking protein coding gene pair[1,2]
Bidirectional promoters (BDPs) are abundant in plants, and divergent gene pairs were identified in most plant species ranging from Arabidopsis to rice and Populus wherein these account ~30% of their genomes[12]
Most of the bidirectional gene pairs identified are functionally associated with their intergenic regions enriched of regulatory elements that respond to a particular inducer
Summary
A bidirectional promoter is an intergenic region of ~1–1.5 kb that regulates the flanking protein coding gene pair[1,2]. Most of the bidirectional gene pairs identified are functionally associated with their intergenic regions enriched of regulatory elements that respond to a particular inducer These inducible BDPs could be tissue-specific thereby rendering appropriate for expressing proteins in the host at a particular stage of their growth cycle. This intergenic region behaves as a switch; ‘on’ state in presence of nitrate and ‘off ’ in presence of ammonium and glutamine This nitrate-inducible promoter was used to express the transcription factor AflR and its cofactor AflS that were responsible for activating eight sterigmatocystin (precursor for aflatoxin B1) promoters for sterigmatocystin production in Aspergillus nidulans[21]. This genetic circuit was used to produce β-carotene in Aspergillus nidulans from the carotenoid gene cluster of Fusarium fujikuroi[21] These BDPs are regulated by activators, transcription factors and enhancers that facilitate binding of the protein complexes driving expression of the gene pair. This is the first study that reports the isolation of a BDP from an economically important phytopathogen Fusarium oxysporum
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