2.1 Strain, media and plasmids
Cryptococcus neoformans B-3501A (serotype D) was used as WT strains. YPDA (a complete medium containing 1% yeast extract, 2% peptone 2% glucose and 0.003% adenine sulfate dihydrate, with or without 2% agar) was for the routine growth of C. neoformans.DOPA medium contained L-dopa (0.2 g/L), L-asparagine (1 g/L), glycine (1 g/L), glutamine (1 g/L), MgSO4 (2.5 g/L), KH2PO4 (4 g/L), vitamin B1 (10 mg/L), glucose (1 g/L), with or without agar (20 g/L). For strain selection, G418 (100μg/mL) or hygromycin B (100μg/mL) was added to the YPDA and the DOPA medium. For the preparation of RNA extraction, the DOPA medium without L-dopa and glucose was named laccase-inducing medium. All cultures and plates were incubated at 30℃, and the liquid cultures needed to continue shaking at 150 rpm/min. The pSilencer 4.1-CMV neo plasmid was bought from Miao Ling Plasmid Platform, China. Supplementary Fig. 1 shows the map of pSilencer 4.1-CMV neo plasmid. The PNK003 vector (Genbank: MW938321) was gifted by professors Xudong Zhu and Ping Zhang. Supplementary Fig. 3 shows the map of PNK003 vector, and Supplementary sequence 3 shows the sequencing result. E. coli DH5α grew on LB medium (containing 0.5% yeast extract, 1% tryptone, 1% NaCl and 2% agar) with ampicillin to amplify and extract target vectors in this study. All the reagents were brought from Sinopharm Chemical Reagent Co., Ltd and Solarbio Science & Technology Co., Ltd.
Enzymes and primers
BspQI, HindIII-HF and BamHI-HF restriction endonucleases were obtained from New England Biolabs (Beijing, China). T4 DNA ligase and Taq DNA polymerases were obtained from Takara (Dalian, China). Yeast Transformation Kit was obtained from Coolaber (Beijing, China). Ezup column fungal genomic DNA extraction kits, DNA gel purification kits, primer synthesis and sequencing were from Sangon Biotech (Shanghai, China). EndoFree Plasmid Maxi Kit and SYBR qPCR Master Mix were purchased from Vazyme (Nanjing, China). Evo M-MLV RT Mix Kit with gDNA Clean for qPCR were purchased from Accurate Biotechnology (Hunan, China). The primers used in this study are listed in Supplementary Table 1.