Ustilago maydis Nit2 Regulates Nitrate Utilisation During Biotrophy and Affects Amino Acid Metabolism of Galls Under Nitrogen Depletion

In previous work, we have shown that the transcription factor Nit2 plays a major role in the utilisation of non-favoured nitrogen sources like nitrate, minor amino acids or nucleobases in saprotrophic sporidia of the basidiomycete corn smut fungus Ustilago maydis. Addressing the knowledge gap regarding how filamentous phytopathogens adapt to nitrogen limitation in the host plant, we employed Δnit2 mutants in the natural FB1 × FB2 background to identify Nit2-regulated genes during biotrophy. We further investigated the impact of Nit2 on the physiology of leaf galls in nitrogen-replete versus nitrogen-limited host plants by comparative RNA-Seq and metabolic steady state analysis. About one third of the fungal genes affected by Nit2 during biotrophy were involved in nitrogen metabolism and transport, only showing minor overlap to saprotrophic sporidia. Induction of the nitrate assimilation cluster was completely dependent on Nit2 during biotrophy. In nitrogen-limited host plants, Δnit2 leaf galls accumulated nitrate and showed reduced accumulation of the nitrogen-rich phloem transport amino acids asparagine and glutamine compared to wild-type galls. However, total protein content in galls and pathogenicity were comparable between fungal genotypes in both nitrogen regimes. The findings of our physiological and transcriptomic analysis demonstrate that nitrate utilisation is dispensable for U. maydis during biotrophy and can likely be actively compensated by increased utilisation of abundant organic nitrogen sources, like asparagine, GABA and glutamine in a partially Nit2-dependent fashion.