NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans

Abstract

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 niadeletion 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 P-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.C. M. J. Pietersel J. van't Klooster G. C. M. van den Berg-Velthuis F. Govers (2i)

Metabolic Pathway Gene Clusters in Filamentous Fungi

“…Although homologs of A. nidulans nitrate assimilation and metabolism genes have been identified in several fungi (Diolez et al, 1993;Unkles et al, 1992) and the oomycete Phytophthora infestans (Pieterse et al, 1995), niiA and niaD linkage is not always conserved. Still, the fact that niiA and niaD clusters have been reported in the yeast Hansenula polymorpha (Brito et al, 1996) is evidence that a nutrient utilization pathway cluster can be widespread in the fungal kingdom.…”

Are elicitins cryptograms in plant-Oomycete communications?

“…Another domain found in elicitin sequences is a myristoylation site, also located in the carboxy-terminal part of the sequence (residues 79-84). Finally, a helixloop-helix, dimerization domain signature was observed in all elicitin sequences, although it corresponds to a small region (residues [76][77][78][79][80][81][82][83][84][85][86][87][88][89][90][91]. This domain may be constituted by two amphipathic helices linked by a region, which would form a loop.…”

Molecular Genetics of Pathogenic Oomycetes

“…2). A motif, CTAAC, believed to be important for lariat formation during splicing, has been reported in several intron sequences (48,75). P. infestans and P. sojae transcribed sequences are characterized by a GC content of approximately 58% (3,78).…”