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.

Ustilago maydis可以利用硝酸盐作为唯一的氮源。该过程是通过硝酸盐转运蛋白将硝酸盐从细胞外环境运输到细胞中开始的，然后分别通过硝酸盐还原酶和亚硝酸盐还原酶将硝酸盐两步还原为铵盐。在这里，我们根据它们在交配和毒力中的作用表征了U. maydis 中编码硝酸盐转运蛋白um03849和亚硝酸盐还原酶um03848的基因。um03848、um03849或这两种基因的缺失突变体在交配的相容单倍体菌株1/2和2/9中构建。此外，um03849采用了CRISPR-Cas9基因编辑技术基因在U. maydis交配菌株中产生 INDEL 突变。对于所有突变体，检查了生长能力、交配效率和发病机制等表型。当在以硝酸盐为氮源的培养基中生长时，所有突变体的生长均减弱。尽管对于单突变体、双 Δ um03848 Δ um03849均未观察到对单倍体成丝或交配的明显影响突变体表现出交配减少，但在低铵时增加了成丝，特别是在 1/2 背景下。关于宿主的发病机制，所有突变体都表现出疾病症状程度的降低。此外，当缺失突变体与相反交配型的野生型配对时，观察到毒力降低，以特定于突变体祖细胞的遗传背景的方式。植物致病性的这种背景特异性降低与美国玉米中的交配程序基因的差异表达相关。