Genes involved in nitrogen assimilation in Ustilago maydis.

Abstract

U. maydis can utilize nitrate as a source of nitrogen from its environment, a process requiring a nitrate transporter to bring the nitrate inside the cell. This is followed by a two-step reduction of nitrate to ammonium. At first, nitrate reductase reduces nitrate into nitrite; in the second reaction, nitrite is reduced to ammonium by nitrite reductase. The goal of the study was to identify the nitrate transporter and nitrite reductase genes in U. maydis and characterize them based on their role in mating and virulence. The genes um03848 and um03849 were identified bioinformatically as encoding, respectively, the nitrite reductase and nitrate transporter in U. maydis. In this study, the deletion mutants for um03848, um03849 or both genes were constructed. In addition, the CRISPR-Cas9 gene-editing technique was used to create INDEL mutations in the U. maydis um03849 gene. The gene deletion or editing was done in mating compatible haploid strains 1/2 and 2/9. For all the mutants, the phenotypes such as the growth ability, mating efficiency and pathogenesis were examined. For the CRISPR-Cas9 edited um03849 gene, DNA sequence analysis confirmed isolates with 3 bp-deletion, 19 bp-deletion and 2 bp-substitution in the 1/2 mating strain, while a 3 bp-deletion and a 66 bp-insertion were found in independent isolates of the 2/9 strain. The mating assay in these mutants showed that none of these mutations in um03849 in U. maydis affected mating with its compatible partner, as assessed by “fuzz” on charcoal media. However, the growth of mutated 1/2 strains was affected when grown in a medium with nitrate or nitrite as a source of nitrogen. With respect to host plant pathogenesis, the 1/2 strain with a 2 bp substitution (resulting in a Arg-> Lys missense mutant) and frameshift mutation showed dramatically reduced infection. In addition to growth and virulence of the fungus, other phenotypes were characterized in the mutants. Though no effect on mating was seen with deletion of either gene um03848 or um03849 alone, deletion of both genes affected the mating. The growth of all deleted mutants was impaired when grown in a medium with nitrate as a source of nitrogen. For the double deletion mutant, ∆um03848 ∆um03849, the growth on different stress media was also affected, depicting the interaction of the genes. Deletion of either gene alone, or both genes, led to reduced virulence. Further, when the deleted mutant strain was paired with wild type opposite mating-type partner, reduced