Episomal maintenance of introduced plasmid with or without AMA1 sequence in the basidiomycete fungus Pleurotus ostreatus.

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Citation abstract

White-rot basidiomycete fungi have been the focus of research as edible mushrooms and for industrial utilization, especially due to their unique wood-degrading enzymes. Nevertheless, establishing highly efficient transformation systems for these organisms remains a substantial challenge. In filamentous ascomycetes, plasmids carrying the specific sequence, AMA1, have been widely used to enhance transformation efficiency. However, their functionality in basidiomycetes has not been systematically assessed. In this study, a pAMAHyg plasmid was constructed that harbored AMA1, in addition to a hygromycin B resistance marker gene derived from the pPHT1 plasmid. The pAMAHyg was introduced into the white-rot fungus Pleurotus ostreatus and yielded more hygromycin-resistant colonies than those with the pPHT1, despite its larger size, suggesting a positive impact on transformation efficiency. However, unlike previous reports in ascomycetes, frequent loss of drug resistance was not observed upon subculturing. Plasmid rescue experiments in Escherichia coli suggested that the circular form and original sequence of pPHT1 or pAMAHyg could be maintained in P. ostreatus, with few exceptions. Methylation-sensitive restriction enzyme analysis of the rescued plasmids confirmed the loss of bacterial Dam methylation and the acquisition of CpG methylation, regardless of AMA1 presence, suggesting the replication of these plasmids in P. ostreatus. Southern blot analysis revealed the genomic integration of the introduced plasmid as well. Collectively, these results suggest that the integrated and episomal plasmid sequences may coexist in P. ostreatus transformants. This study provides experimental evidence supporting the extrachromosomal existence of circular plasmids in the fungus, even in the absence of AMA1.