Role for nitrate assimilatory genes in virulence of Ustilago maydis

Ustilago maydis can utilize nitrate as a sole source of nitrogen. This process is initiated by transporting nitrate from the extracellular environment into the cell by a nitrate transporter and followed by a two-step reduction of nitrate to ammonium via nitrate reductase and nitrite reductase enzymes, respectively. Here, we characterize the genes encoding nitrate transporter, um03849 and nitrite reductase, um03848 in U. maydis based on their roles in mating and virulence. The deletion mutants for um03848, um03849 or both genes were constructed in mating compatible haploid strains 1/2 and 2/9. In addition, CRISPR-Cas9 gene editing technique was used for um03849 gene to create INDEL mutations in U. maydis mating strains. For all the mutants, phenotypes such as growth ability, mating efficiency and pathogenesis were examined. The growth of all the mutants was diminished when grown in a medium with nitrate as the source of nitrogen. Although no clear effects on haploid filamentation or mating were observed for either single mutant, double Δum03848 Δum03849 mutants showed reduction in mating, but increased filamentation on low ammonium, particularly in the 1/2 background. With respect to pathogenesis on the host, all the mutants showed reduced degrees of disease symptoms. Further, when the deletion mutants were paired with wild type of opposite mating-type, reduced virulence was observed, in a manner specific to the genetic background of the mutant's progenitor. This background specific reduction of plant pathogenicity was correlated with differential expression of genes for the mating program in U. maydis.