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Ustilago maydis, the basidiomycete smut-fungus, can infect and cause tumors in corn plants. For this, mating between compatible haploid cells is important. The mating and subsequent dimorphic transition in U. maydis require starvation for nutrients such as nitrogen, in addition to pheromone-receptor interactions between compatible partners. In this research, the CRISPR-Cas9 gene-editing technique was used to create INDEL mutations (sequence insertion or deletion) in the nitrate transporter gene, um03849, in U. maydis. The gene was edited in mating compatible haploid strains 1/2 and 2/9. The phenotypes were characterized for the um03849 mutants as to growth ability, mating efficiency and pathogenesis. 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 matting assay results showed that any forms of mutation in um03849 in U. maydis didn’t affect 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 2 bp substitution crossed with 2/9 WT strain showed dramatically reduced infection. Base substitution in the 1/2 strain resulted in arginine being substituted for lysine. Thus, this study suggests that the nitrate transporter affects the growth and pathogenesis of U. maydis on its host plant in a manner dependent on the 1/2 background.
CRISPR-Cas9, Nitrate Transporter, Ustilago Maydis, Maize
Biology | Microbiology | Pathogenic Microbiology
Schroeder, Luke A.; Khanal, Sunita; and Perlin, Michael H., "CRISPR-Cas9 Editing of Nitrate Transporter Gene, um03849, in Ustilago maydis" (2020). Undergraduate Arts and Research Showcase. 6.