Breeding strains without basidiospores is important for the mushroom industry. However, target genes for sporeless breeding remain limited. To identify a new gene essential for basidiospore production in Pleurotus ostreatus, the RNA sequencing data obtained in our previous study were re-analyzed. Among the 36 P. ostreatus genes that are exclusively expressed in the gills where basidiospores are formed and produced, candidate genes were narrowed down using transcriptome data during the fruiting stage of Coprinopsis cinerea, in which the meiotic steps progress synchronously. Three C. cinerea genes homologous to the gill-specific P. ostreatus genes were upregulated during meiosis. One of these three genes encodes a protein homologous to Saccharomyces cerevisiae Mek1p, a protein kinase important in the meiotic recombination checkpoint. Plasmids containing expression cassettes for hygromycin B-resistance screening, Cas9, and single-guide RNA targeting mek1 were introduced into the protoplasts of the dikaryotic P. ostreatus strain PC9×#64, which showed that the obtained dikaryotic gene disruptant produced no basidiospores. Microscopic analysis suggests that meiosis is suspended during telophase I. These results suggested that P. ostreatus Mek1 is essential for meiosis II progression and basidiospore production. In addition, disturbed orientation and loss of negative gravitropism during fruiting were observed.

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Nakazawa T, Kobukata T, Yamasaki F, Sugano J, Oh M, Kawauchi M, Honda Y

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