NIAA, THE STRUCTURAL NITRATE REDUCTASE GENE OF PHYTOPHTHORA-INFESTANS- ISOLATION, CHARACTERIZATION AND EXPRESSION ANALYSIS IN ASPERGILLUS-NIDULANS

The nitrate reductase (NR) gene niaA of the oomycete Phytophthora n in festans was selected from a gene library by heterologous hybridization . NiaA occurs as a single-copy gene and its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined a nd comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, partic ularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test w hether oomycete genes are functional in the ascomycete Aspergillus nid ulans, a fungus which is highly accessible for molecular genetic studi es. The complete niaA gene was stably integrated into the genome of a nia(-) deletion mutant of A. nidulans. However, transformants containi ng one or more copies of the niaA gene were not able to complement the nia(-) mutant. This suggests that there is no functional expression o f the introduced niaA gene in A. nidulans. In addition, the activity o f two other oomycete gene promoters was analyzed in a transient expres sion assay. Plasmids containing chimaeric genes with the promoter of t he P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gen e, fused to the coding sequence of the Escherichia coli beta-glucuroni dase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the reg ulatory sequences which are sufficient for gene activation in oomycete s are not functional in the ascomycete A. nidulans.