Functional analysis of the C 6 zinc finger gene pro1 involved in fungal sexual development

Abstract

The pro1 gene, controlling fruiting body development in the homothallic ascomycete Sordaria macrospora, encodes a C 6 zinc finger protein with a typical DNA binding domain of GAL4-like C 6 zinc finger proteins as well as a putative nuclear targeting signal. In the corresponding mutant pro1, the pro1 gene is deleted, and the transition of primordia into mature fruiting bodies is prevented. To further characterize the PRO1 polypeptide, the yeast system was used for identifying a transactivation domain in the N-terminal half of PRO1, which probably also functions in S. macrospora. The functional analysis was extended by using truncated versions of the pro1 gene in complementation transformations of a Δ pro1 mutant. Interestingly, the 5 ′ part of the pro1 gene encoding the DNA binding and transactivation domain as well as putative nuclear targeting signals was sufficient to restore fertility in the sterile pro1 mutant. In vitro mutagenesis verified that the DNA binding domain is essential for normal fruiting body development. This was concluded from transformation experiments with eight pro1 derivatives containing triplet substitutions in conserved codons of the DNA binding domain; some, but not all, failed in restoring the wild-type phenotype in mutant pro1. Using a PCR-based cloning strategy, pro1 homologs from the two related heterothallic species Neurospora crassa and Sordaria brevicollis were isolated, showing similarities in the predicted amino acid sequences of 91 and 90%, respectively. When a N. crassa pro1 cDNA clone was used in complementation transformations, we succeeded in restoring the wild-type phenotype to the S. macrospora pro1 mutant. These data suggest that pro1 homologs from heterothallic species can provide the pro1 function in homothallic ascomycetes. Based on the published sequence of the N. crassa genome, we identified hpro1A, another transcriptionally expressed gene, with a similarity of 40% to the pro1 genes, which is present as a single copy gene in N. crassa as well as in S. macrospora.