Construction of GFP-Pcf1 Fusion Protein

A GFP fusion protein vector, pKD8B-GFP-PCF1, was constructed using a high-throughput gene knockout system as described previously [30 (link)]. Briefly, the fragment of PCF1 coding sequence was amplified from the M. oryzae genome was co-transformed into Saccharomyces cerevisiae strain FY834 with a linearized pKD8B-GFP that was digested by XbaI and SalI [30 (link)]. The plasmids of yeast transformants were extracted using TIANprep yeast plasmid DNA kit (Tiangen Biotech, Beijing, China) and transformed into Escherichia coli strain DH5α. After confirming the correctness of the vector, pKD8B-GFP-PCF1 and pKD9-H₂B-mCherry [41 (link)] was co-transformed into the wild-type strain through Agrobacterium tumefaciens-mediated transformation (ATMT) [30 (link)]. Transformants that were expressing both GFP-Pcf1 and H₂B-mCherry were screened on CM plates that were supplemented with 200 μg/mL hygromycin and 50 mg/mL G418 sulfate. The colocalization of GFP-Pcf1 and H₂B-mCherry and the fluorescence intensity of GFP-Pcf1 in M. oryzae transformants were observed using fluorescence microscopy (Nikon Eclipse Ni) (Nikon, Tokyo, Japan) under the same exposure conditions. The primers for PCF1 amplification are listed in Supplemental Table S1.