The LaeB homology protein FpLaeB was identified via querying the F. pseudograminearum genomic sequence (GenBank accession NC_031951.1). The conserved domains of FpLaeB were predicted via the Conserved Domain Search Service (CD Search) in the National Center for Biotechnology Information (NCBI). The phylogenetic tree of FpLaeB and its homology proteins were constructed via the neighbour-joining method with the MEGA version 7.02 software package (Xia et al., 2021 (link)).
Open reading frame (ORF) was replaced by the hygromycin phosphotransferase gene to construct the deletion mutant of FpLaeB. Two primer pairs FpLaeB-1F/2R and FpLaeB-3F/4R were used to amplify the upstream and downstream flanking fragment of the FpLaeB gene. The hygromycin phosphotransferase (hph) gene was amplified via the primer pair HYG-F/R. The replacement fragment was constructed by joining the three fragments via double-joint polymerase chain reaction (PCR) (Yu et al., 2004 (link)). The FpLaeB replacement fragment was transformed into protoplasts of wild-type 2035 by the polyethylene glycol (PEG) approach (Liu and Friesen, 2012 ). Following screening by hygromycin, the transformants were screened and confirmed using PCR and Southern blot analyses, respectively (Tang et al., 2018 (link)). For complementation assays, XhoI-digested pFL2 and the FpLaeB fragments with promoters cotransformed into yeast strain XK1-25. FpLaeB–pFL2 plasmid was constructed by the yeast gap repair method (Zhang et al., 2017 (link)). Then, FpLaeB–pFL2 was transformed into the protoplasts of the FpLaeB deletion mutant by the PEG approach as well. After geneticin screening, the primer pair FpLaeB-5F/6R was used to confirm the complementation strain from geneticin-resistant transformants. Primers used for deletion, complementarity, and gene expression are listed inSupplementary Table S1
Open reading frame (ORF) was replaced by the hygromycin phosphotransferase gene to construct the deletion mutant of FpLaeB. Two primer pairs FpLaeB-1F/2R and FpLaeB-3F/4R were used to amplify the upstream and downstream flanking fragment of the FpLaeB gene. The hygromycin phosphotransferase (hph) gene was amplified via the primer pair HYG-F/R. The replacement fragment was constructed by joining the three fragments via double-joint polymerase chain reaction (PCR) (Yu et al., 2004 (link)). The FpLaeB replacement fragment was transformed into protoplasts of wild-type 2035 by the polyethylene glycol (PEG) approach (Liu and Friesen, 2012 ). Following screening by hygromycin, the transformants were screened and confirmed using PCR and Southern blot analyses, respectively (Tang et al., 2018 (link)). For complementation assays, XhoI-digested pFL2 and the FpLaeB fragments with promoters cotransformed into yeast strain XK1-25. FpLaeB–pFL2 plasmid was constructed by the yeast gap repair method (Zhang et al., 2017 (link)). Then, FpLaeB–pFL2 was transformed into the protoplasts of the FpLaeB deletion mutant by the PEG approach as well. After geneticin screening, the primer pair FpLaeB-5F/6R was used to confirm the complementation strain from geneticin-resistant transformants. Primers used for deletion, complementarity, and gene expression are listed in
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