Understanding the molecular mechanisms controlling dikaryon formation in Agaricomycetes, which is basically controlled by A and B mating-type loci, contributes to improving mushroom cultivation and breeding. In Coprinopsis cinerea, various mutations in the SRY-type high mobility group protein-encoding gene, pcc1, were shown to activate the A-regulated pathway to induce pseudoclamp (clamp cells without clamp connection) and fruiting body formation in monokaryons. The formation of clamp cells was blocked in AmutBmut strain 326 with clp1-1 mutation in C. cinerea. However, considering the diverse mechanisms of sexual development among Agaricomycetes, it remains unclear whether similar phenotypes are also observed in clp1 or pcc1 mutants in cultivated mushrooms. Therefore, phenotypic analyses of Pleurotus ostreatus pcc1 or clp1 (Popcc1 or Poclp1) mutants generated using CRISPR/Cas9 were performed in this study. Plasmids with Cas9 expression cassette and different single guide RNAs targeting Popcc1 or Poclp1 were individually introduced into a monokaryotic P. ostreatus strain PC9 to obtain the mutants. Unlike in C. cinerea, the pseudoclamp cell was not observed in monokaryotic Popcc1 mutants, but it was observed after crossing two compatible strains with Popcc1 mutations. In Poclp1 mutants, dikaryosis was impaired as clamp cells were not observed after crossing, suggesting that Poclp1 functions may be essential for clamp cell formation, like in C. cinerea. These results provided a clue with respect to conserved and diverse mechanisms underlying sexual development in Agaricomycetes (at least between C. cinerea and P. ostreatus).

了解基本由A和B交配型位点控制的蘑菇纲双核形成的分子机制，有助于改善蘑菇的栽培和育种。在Coprinopsis cinerea 中，SRY 型高迁移率组蛋白编码基因pcc1中的各种突变显示激活A调节途径以诱导单核中的假钳（没有钳连接的钳细胞）和子实体形成。在C. cinerea 中具有clp1-1突变的AmutBmut菌株 326中钳细胞的形成被阻断. 然而，考虑到伞菌纲中性发育的不同机制，尚不清楚在栽培蘑菇的clp1或pcc1突变体中是否也观察到类似的表型。因此，本研究对使用 CRISPR/Cas9 产生的平菇 pcc1或clp1（Popcc1或Poclp1）突变体进行了表型分析。将带有 Cas9 表达盒的质粒和针对Popcc1或Poclp1 的不同单向导 RNA单独引入单核P. ostreatus菌株 PC9 中以获得突变体。与C. cinerea不同，在单核Popcc1突变体中未观察到假钳细胞，但在与具有Popcc1突变的两个相容菌株杂交后观察到。在Poclp1突变体中，双核功能受损，因为在杂交后未观察到钳细胞，这表明Poclp1功能可能对钳细胞形成至关重要，如在C. cinerea 中。这些结果提供了关于蘑菇菌（至少在C. cinerea和P. ostreatus 之间）性发育的保守和多样化机制的线索。