Analysis of determinants of binding and transcriptional activation of the pisatin-responsive DNA binding factor of Nectria haematococca.
Pisatin is a fungistatic isoflavonoid produced by garden pea. Field isolates of the ascomycete Nectria haematococca mating population VI (anamorph: Fusarium solani) that are highly virulent on pea have been found to possess the PDA1 gene encoding a pisatin detoxifying activity. Expression of PDA1 is specifically and highly induced by exposure of mycelia to pisatin. A pisatin-responsive DNA-binding activity has previously been identified with properties suggestive of a transcriptional regulator of PDA1. In this study, the sequence determinants for binding this pisatin-responsive factor (PRF) were localized to a 14-bp region through analysis of sequence alterations that reduced PRF binding. Using a homologous in vitro transcription system, a transcriptional activator of PDA1 was shown to be present in mycelial extracts that shared the sequence specificity characteristic of the PRF, indicating function of the DNA-binding protein in transcriptional control. A 70-kDa protein was shown to be a DNA-binding component of PRF by three independent assays for DNA-binding proteins: Southwestern (DNA-protein) blotting, UV-crosslinking, and binding to immobilized DNA. These results characterize a transcriptional activator acting on the PDA1 promoter that is responsive to a host-specific compound and provide insight into the regulation of fungal genes in response to plant flavonoids.
He
J
J
Department of Plant Biology, Maryland Agriculture Experiment Station, University of Maryland, College Park 20742, USA.
Ruan
Y
Y
Straney
D
D
D001483:Base Sequence; D001578:Benzopyrans / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D006999:Hypocreales / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D008658:Inactivation, Metabolic; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000378:metabolism / Q000382:microbiology; D036343:Pterocarpans; D015533:Transcriptional Activation; D000094162:Phytoalexins
D001578:Benzopyrans; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D036343:Pterocarpans; C003161:pisatin; D000094162:Phytoalexins