Molecular evolution of nitrate assimilation genes in fungi

Nitrogen is a necessary element for a wide range of biological molecules and can be acquired from the environment in many forms by living organisms. One inorganic form of nitrogen, nitrate, can be acquired by organisms from terrestrial and aquatic habitats with the high affinity nitrate transporter NRT2. NRT2 is a protein composed of 12 transmembrane alpha-helices and is a member of the Nitrate/Nitrite Porter (NNP) family of the Major Facilitator Superfamily. To date, NRT2 is the only characterized means of nitrate acquisition in fungi. The NNP gene family is supported to have originated in eukaryotic cells via a single gene transfer from an unknown proteobacterium. There is evidence for a secondary origin of nrt2 in a derived subkingdom of fungi, Dikarya, possibly facilitated by horizontal gene transfer of a gene cluster encoding the nitrate assimilation pathway (HANT-AC) from heterokonts. The HANT-AC is strongly supported by phylogenetic analyses to have been horizontally transferred at one point between the Dikarya phyla Ascomycota and Basidiomycota, a transfer that may have facilitated niche shift within the class Sordariomycetes. Surveys of fungal genome projects reveal modest diversification of the nrt2 gene family through sporadic duplications and selective loss leading to lineages in Dikarya with zero, one or two paralogous forms of the gene. Duplications in nrt2 have led to its functional diversification in Ascomycota (Aspergillus) and Basidiomycota (Hebeloma ). Functional diversification in nrt2 is supported by previous physiological studies of affinity for nitrate in Aspergillus and by the current studies of structural and regulatory divergence between nrt2 paralogs in Hebeloma. Structural differences in NRT2 between paralogs and between groups of mushroom forming fungi ( Hebeloma, Laccaria and Psathyrellaceae) from different niches include the size of a central large intracellular loop, the number of putative Protein Kinase C binding motifs and the hydrophobicity of amino acids in close spatial proximity to a conserved arginine residue in the second trans-membrane helix. Positive selection on fungal nrt2 is supported by six amino acid sites in Laccaria.