A set of heterologous promoters useful for investigating gene functions in Fusarium graminearum

Abstract

  The activities of four constitutive promoters from Aspergillus nidulans were compared in Fusarium graminearum by using β-glucuronidase (GUS) as a reporter gene. The promoter-GUS constructs were integrated into the Tri14 locus at the terminus of the trichothecene gene cluster and crude cell extracts were used for the reporter assay. The translation elongation factor 1-alpha (TEF1α) promoter yielded by far the strongest induction of GUS with little or no effect seen with the glyceraldehyde 3-phosphate dehydrogenase (GPD), polyubiquitin (UBI), and β-tubulin (TUB) gene promoters. The promoters of TUB and TEF1α, with or without an original trichothecene regulator gene (Tri6) opal codon, were connected to a transcriptional fusion of Tri6 and enhanced green fluorescent protein (EGFP) gene, and targeted to a locus downstream of the trichothecene 3-O-acetyltransferase gene (Tri101). Northern blot analysis revealed expression levels of these fusion genes to be proportional to the activities of the promoters as demonstrated by the GUS assay. In addition, analysis of trichothecene levels demonstrated drastically decreased activity of a translational fusion of TRI6 with EGFP (TRI6::EGFP) as a trichothecene transcription factor. These results indicate that the set of promoters reported in this study could be used to investigate biological functions of master genes by modulating their expression levels in F. graminearum.