Effect of disruption of PsPCP gene on fruit body color of Pleurotus salmoneostramineus.

The pink color of Pleurotus salmoneostramineus is due to a complex of pink color protein (PsPCP) and 2-dehydro-3-deoxylaetiporic acid A, which is a polyene pigment with the yellow color of Laetiporus sulphureus. In this study, we used the Cas9 ribonucleoprotein as a plasmid-free genome-editing tool to perform gene destruction in P. salmoneostramineus. Gene disruption via split-marker recombination by targeting the PsPCP gene using a pre-assembled Cas9/single-guide RNA system was introduced into the protoplasts of the wild-type monokaryotic P. salmoneostramineus strain N13 (A1B1) or N83 (A2B2). Disruptions at the target site were identified using genomic PCR, followed by sequencing. Strains N13 ∆PsPCP and N83 ∆PsPCP were matured on PDA agar plates and then cultivated on sawdust media to form the fruit body of the dikaryotic mutant N13 ∆PsPCP × N83 ∆PsPCP. In addition, compatible mutants of N13 ∆PsPCP × N83 and N13 × N83 ∆PsPCP were cultivated, and the pink coloring of these fruiting bodies was compared. N13 ∆PsPCP × N83 ∆PsPCP showed completely white fruiting bodies. In vitro color-developing reaction using 2-dehydro-3-deoxylaetiporic acid A extracted from the mutant with recombinant PsPCP, which showed no color, suggested that the amount of the compound was trace or degraded during fruit body formation.