Construction of gene replacement, GFP-, Flag-, CYFP-, NYFP- and mCherry- fusion cassettes

The double-joint PCR approach [80] was used to generate the gene replacement construct for each target gene. Briefly, for each gene, 5’ and 3’ flanking regions were amplified with the primer pairs listed in S1 Table and the resulting amplified sequences were then fused with the hygromycin resistance gene cassette (HPH) driven by the constitutive trpC promoter which was amplified from the pBS-HPH1 vector [81]. Protoplast transformation of F. graminearum was carried out using the protocol described previously [82]. Putative gene deletion mutants were identified by PCR assays with relevant primers (S1 Table) and the FgATM1 deletion mutation was further confirmed by Southern hybridization assays (S3 Fig).

To construct the FgAtm1-GFP cassette, FgATM1 containing the promoter region and open-reading frame (without the stop codon) was amplified with the relevant primers (S1 Table). The resulting PCR products were co-transformed with XhoI-digested pYF11 containing a geneticin resistance gene (NEO) [83] into the yeast strain XK1-25 [84] using the Alkali-Cation Yeast Transformation Kit (MP Biomedicals, Solon, USA) to generate the recombined FgAtm1-GFP fusion vector. Subsequently, the FgAtm1-GFP fusion vector was recovered from the yeast transformant using the Yeast Plasmid Kit (Solarbio, Beijing, China) and then transferred into Escherichia coli strain DH5α for amplification. Using the similar strategy, FgYak1 (FGSG_05418)—and FgTri1 (FGSG_00071) -GFP fusion cassettes were constructed. Using the similar strategy, FgGrx4 (FGSG_01317)—and FgYak1-Flag fusion cassettes were constructed by co-transformation with XhoI-digested PHZ126 vector. Similarly, FgHapX-CYFP and FgGrx4-NYFP fusion cassettes were constructed by co-transformation with XhoI-digested PHZ68 and PHZ65 vectors, respectively.

The double-joint PCR approach [80] was also used to construct FgHapX-, FgHapX^S245A/S338A- and FgLeu1 (FGSG_09589)-mCherry cassettes. Briefly, the target gene containing the promoter region and open-reading fragment and the geneticin resistance gene (NEO) were amplified, and then fused with mCherry fragment. Before protoplast transformation, each fusion construct was verified by DNA sequencing. The transformation of F. graminearum was carried out using the previously described protocol [82]. All the mutants generated in this study were preserved in 15% glycerol at −80°C.