Targeted Gene Replacement in Fon

The double-joint PCR method was used to prepare the targeted replacement constructs for the genes of interest (44 (link)). The upstream and downstream fragments of the target genes were amplified with gene-specific primers (Table S2), and the PCR products were transformed into protoplasts of Fon strains (46 (link)). Putative positive transformants were selected on PDA plates supplemented with 100 mg/mL hygromycin B and identified by PCR with gene-specific primer pairs (Table S2), followed by further confirmation using Southern blotting. For construction of complementation vectors, fragments containing an ~1.5-kb native promoter region and ORF (without stop codon) of the genes were cotransformed with XhoI-digested vector pYF11 into yeast strain XK-125 using the alkali-cation yeast transformation kit (MP Biomedicals, Solon, OH, USA), yielding the GFP-tag fused vectors. The recombined vectors were transformed into protoplasts of the corresponding deletion mutants, and neomycin-resistant transformants were characterized by PCR and examined for GFP signal. Site-specific point mutations in FonPAT1, FonPAT2, FonPAT4, and FonAP-2 complex subunits FonAP-2α, FonAP-2β, and FonAP-2μ were created using the Mut Express MultiS fast mutagenesis kit (Vazyme Biotech, Nanjing, China) and cloned into pYF11 for construction of complementation strains (46 (link)).