An N-acetyltransferase influences AreA nuclear localization and nitrogen utilization in the basidiomycete Ganoderma...

Nitrogen metabolism is fundamental to fungal growth, development, and the biosynthesis of bioactive metabolites. The GATA transcription factor AreA plays a pivotal regulatory role in nitrogen metabolite repression. While the molecular mechanisms by which AreA responds to nitrogen sources have been relatively well elucidated in ascomycetes, they remain poorly understood in basidiomycetes. Here, using the macrofungus Ganoderma lucidum as a model, we elucidated a molecular mechanism by which an N-acetyltransferase (Nat) influences AreA nuclear entry under non-preferred nitrogen sources, thereby promoting nitrogen catabolic gene expression and the utilization of non-preferred nitrogen sources. Through a yeast two-hybrid library screen, we identified Nat as an AreA-interacting protein, and their physical interaction was subsequently validated by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. In vitro acetylation assays coupled with mass spectrometry analysis demonstrated that Nat specifically acetylates lysine residues 619 and 916 of AreA. In vivo experiments further showed that silencing of nat reduced the acetylation level of AreA. Nitrate conditions induced the expression of nat and increased NAT protein levels, compared with ammonium conditions. Silencing of nat did not affect areA transcription or total AreA protein abundance; however, it significantly reduced nuclear AreA accumulation with a concomitant increase in cytoplasmic retention. Compared with the wild-type strain, nuclear AreA accumulation in the nat-silenced strain was reduced by approximately 70% under nitrate conditions. Consequently, the expression and enzymatic activity of AreA-regulated nitrogen metabolic genes, including glutamine synthetase (gs) and nitrate reductase (nr), were markedly suppressed. This study illuminates a post-translational mechanism by which non-preferred nitrogen sources activate AreA in basidiomycetes. It further reveals a role for protein acetylation in the nitrogen metabolism of G. lucidum, thereby expanding our understanding of nitrogen metabolic networks in fungi.