Refining the Life Cycle of .

As a soilborne protist pathogen, causes the devastating clubroot disease on Brassicaeae crops worldwide. Due to its intracellular obligate biotrophic nature, the life cycle of is still not fully understood. Here, we used fluorescent probe-based confocal microscopy and transmission electron microscopy (TEM) to investigate the infection process of on the susceptible host under controlled conditions. We found that can initiate the primary infection in both root hairs and epidermal cells, producing the uninucleate primary plasmodium at 1 day postinoculation (dpi). After that, the developed multinucleate primary plasmodium underwent condensing and cytoplasm cleavage into uninucleate zoosporangia from 1 to 4 dpi. This was subsequently followed by the formation of multinucleate zoosporangia and the production of secondary zoospores within zoosporangium. Importantly, the secondary zoospores performed a conjugation in the root epidermal cells after their release. TEM revealed extensive uninucleate secondary plasmodium in cortical cells at 8 dpi, indicating the establishment of the secondary infection. The subsequently developed into binucleate, quadrinucleate, and multinucleate secondary plasmodia from 10 to 15 dpi, during which the clubroot symptoms appeared. The uninucleate resting spores were first observed in the cortical cells at 24 dpi, marking the completion of a life cycle. We also provided evidence that the secondary infection of may represent the diploid sexual life stage. From these findings, we propose a refined life cycle of which will contribute to understanding of the complicated infection biology of .