Functional analyses of Pleurotus ostreatus pcc1 and clp1 using CRISPR/Cas9

• Pleurotus ostreatus pcc1 or clp1 mutants were obtained using CRISPR/Cas9. • Pseudoclamp was not observed in monokaryotic Popcc1 mutants. • Fruiting body formation was induced in some monokaryotic Popcc1 mutants. • Dikaryosis was impaired in Poclp1 mutants. 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 ).