The first single-cell sequencing of Plasmodiophora brassicae reveals genetic diversity and clonal dynamics

Clubroot, caused by the obligate Chromist pathogen <i>Plasmodiophora brassicae</i>, is an important disease of brassica crops but little is known about its reproductive biology. We enzymatically removed cell walls from dormant spores to generate protoplasts, enabling the first single-cell sequencing of <i>P. brassicae</i> with DNA free from host and soil microbial contamination. Analysis of 4,000 protoplasts from a single root showed moderate genetic diversity, with 2-5 distinct genotypes. A more detailed analysis of the 500 cells indicated the presence of seven distinct genotypes, accounting for rare haplotypes. This level of genetic diversity in a single root supports other indications that there is a high genetic diversity in field populations of <i>P. brassicae</i>. These results support the hypothesis that balancing selection maintains multiple genotypes within the pathogen population. This level of diversity complicates the use of single-gene resistance sources for clubroot management and explains the short durability of clubroot resistance. The predominance of distinct genotypes in a single root is a strong indication that reproduction of <i>P. brassicae</i> is predominantly clonal. This is the first whole genome DNA sequencing of a single-cell of a plant pathogen.