Ultraviolet Mutagenesis Coupled with Next-Generation Sequencing as a Method for Functional Interrogation of Powdery Mildew Genomes.

Abstract

Powdery mildews are obligate biotrophic fungal pathogens causing important diseases of plants worldwide. Very little is known about the requirements for their pathogenicity at the molecular level. This is largely due to the inability to culture these organisms in vitro or to modify them genetically. Here, we describe a mutagenesis procedure based on ultraviolet (UV) irradiation to accumulate mutations in the haploid genome of the barley powdery mildew pathogen Blumeria graminis f. sp. hordei. Exposure of B. graminis f. sp. hordei conidia to different durations of UV-C radiation (10 s to 12 min) resulted in a reduced number of macroscopically visible fungal colonies. B. graminis f. sp. hordei colony number was negatively correlated with exposure time and the total number of consecutive cycles of UV irradiation. Dark incubation following UV exposure further reduced fungal viability, implying that photoreactivation is an important component of DNA repair in B. graminis f. sp. hordei. After several rounds of UV mutagenesis, we selected two mutant isolates in addition to the parental B. graminis f. sp. hordei K1 isolate for whole-genome resequencing. By combining automated prediction of sequence variants and their manual validation, we identified unique UV-induced mutations in the genomes of the two isolates. Most of these mutations were in the up- or downstream regions of genes or in the intergenic space. Some of the variants detected in genes led to predicted missense mutations. As an additional insight, our bioinformatic analyses revealed a complex population structure within supposedly clonal B. graminis f. sp. hordei isolates.