Cloning and characterization of a general amino acid control transcriptional activator from the chestnut blight fungus Cryphonectria parasitica

Abstract

We have cloned and characterized a homologue of the Neurospora crassa general amino acid control gene cpc-1 from the chestnut blight fungus Cryphonectria parasitica. The deduced amino acid sequence of C. parasitica CPC1 (cpCPC1) contains regions with significant homology to the transcriptional activation, DNA binding, and dimerization domains previously defined for N. crassa CPC1 (ncCPC1) and the equivalent "b-ZIP" transcription factor from Saccharomyces cerevisiae, GCN4 (scGCN4). Treatment of C. parasitica with low levels of the protein synthesis inhibitor cycloheximide caused cpc-1 transcript levels to undergo a rapid, transient increase similar to that reported for the mammalian b-ZIP transactivators, c-Jun and c-Fos. Northern analysis also revealed that amino acid starvation of C. parasitica elicits an increase in cpc-1 transcript levels. Hypovirus infection did not affect this increase, although transcript accumulation for several amino acid biosynthetic genes was slightly diminished in the hypovirus-containing strain. Recombinant cpCPC1 specifically bound to the consensus DNA binding element (AP-1), 5'-A/GTGACTCAT-3', also located upstream of the C. parasitica cpc-1 coding region. Constitutive transgenic expression of a DNA binding defective cpCPC1 mutant impaired the ability of C. parasitica to adjust to amino acid starvation. Moreover, these transformants showed reduced ability to grow on host chestnut tissue. Our results define a general amino acid control transactivator in a plant pathogenic fungus and suggest that functional modulation of this factor can influence fungal virulence.