Deletion and Complementation of Fungal Genes

For the generation of deletion mutant of each gene POX04853, POX06496, POX07588, and POX07948, the knockout cassette, containing 5′ and 3′ DNA fragments of the target gene and the G418-resistance gene fragment, was constructed by fusion PCR with specific primer pairs (Supplementary Table S1) according to previously published protocols [25 (link)]. Subsequently, the knockout cassette was directly transformed into fresh protoplasts of strain ΔPoxKu70, the transformants were selected on G418-containing PDA plates, and then knockout candidates were verified by PCR amplification using the special primer pairs (Supplementary Table S1). Similarly, the deletion mutant of PoxMK1 could also be obtained by the above method [23 (link)].
For the creation of a complementary strain of mutant ΔPOXO7948 (ΔPoxMKK1)-, the complementary cassette was used to replace the protease gene PoxPepA, composed of the complete coding region of PoxMKK1, its native promoter and terminator, the bleomycin resistance gene fragment, and the upstream- and downstream-flanking DNA sequence of PoxPepA. The transformants were screened on PDA plate containing 80 μg/mL bleomycin (Sigma-Aldrich, Darmstadt, Germany) and was validated by the special primer pairs (Supplementary Table S1), as previously described by Yan et al. [26 (link)].