Significance Mycoviruses are viruses that infect fungi and replicate in fungi. Previously, no mycoviruses had been discovered with negative-stranded (−)ssRNA genomes. Here, we characterize a (−)ssRNA mycovirus that infects a fungal plant pathogen. Although its genome and organization are significantly different from those of current mononegaviruses, this virus is closely related to viruses in families Nyamiviridae and Bornaviridae that infect animals. This discovery may provide insights into the global ecology and evolution of (−)ssRNA viruses. Furthermore, since many (−)ssRNA viruses are serious human pathogens, this system is likely to provide a less hazardous way to study replication of a (−)ssRNA virus and could be useful in establishing a system to screen antiviral compounds against (−)ssRNA viruses.

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As a soilborne protist pathogen, Plasmodiophora brassicae causes the devastating clubroot disease on Brassicaeae crops worldwide. Due to its intracellular obligate biotrophic nature, the life cycle of P. brassicae is still not fully understood. Here, we used fluorescent probe-based confocal microscopy and transmission electron microscopy (TEM) to investigate the infection process of P. brassicae on the susceptible host Arabidopsis under controlled conditions. We found that P. brassicae 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 P. brassicae 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 P. brassicae may represent the diploid sexual life stage. From these findings, we propose a refined life cycle of P. brassicae which will contribute to understanding of the complicated infection biology of P. brassicae.