G.C.M. van den Berg-Velthuis

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 infestans 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 and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli β-glucuronidase (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 regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.

tef1, a Phytophthora infestans gene encoding translation elongation factor 1α ☆

From a set of Phytophthora infestans cDNA clones randomly selected from a potato-P. infestans interaction cDNA library, three out of 22 appeared to correspond to a gene encoding translation elongation factor 1alpha. The gene, called tef1, is a single copy gene in P. infestans. During the life cycle of P. infestans, tef1 is expressed in all developmental stages. Alignment and phylogeny analysis based on EF-1alpha proteins from several taxonomic groups, including fungi, slime molds, algae, higher plants and archeabacteria, support the view that oomycetes evolved completely independently from the true fungi. In the phylogenetic tree, P. infestans EF-1alpha forms one branch with EF-1alpha from the unicellular alga Cyanophora paradoxa, an organism belonging to a taxonomic group that occupies a key position in the evolution of plastids.