Constructing CcPtc1 Gene Deletion Mutants

The split marker method was used to construct the CcPtc1 gene deletion (ΔCcPtc1) mutants as previously described (Catlett et al., 2003 (link); Goswami, 2012 (link)). According to this method, the upstream (∼1.3 kb) and downstream (∼1.2 kb) flanking sequences of CcPtc1 were amplified by primer pairs CcPtc1-5Ffor/CcPtc1-5Frev and CcPtc1-3Ffor/CcPtc1-3Frev, respectively (Supplementary Table 1). The hygromycin B resistance cassette (HPH) was amplified by specific primer pairs hygromycinfor and hygromycinrev, which included approximately 20 bp overlapped 5′ and 3′flanking sequences, respectively. Then, the resulting upstream and downstream fragments were fused with two-thirds of the hygromycin B resistance cassette by overlap PCR using primer pairs CcPtc1-5Ffor/HY-R and YG-F/CcPtc1-3Frev, respectively. The two overlapping fragments were directly transformed into protoplasts of the WT strain by using the PEG-mediated transformation, and the transformants were selected on TB₃ agar medium supplemented with 20 μg/ml hygromycin B. All transformants were identified by PCR assays with the primer pairs External-CcPtc1for/External-CcPtc1rev and Internal-CcPtc1for/Internal-CcPtc1rev to screening the successful replacement transformants. In addition, to analyze homologous recombination events in the transformants, southern blotting analysis was performed with the DIG High Prime DNA Labeling and Detection Starter Kit I, following the manufacturer’s protocol (Roche, Germany). BglI was used to digest the genomic DNA extracted from the WT strain and the transformants. The probes were amplified by the primers ProbeHPHfor and ProbeHPHrev from HPH and the primers ProbeCcPtc1for and ProbeCcPtc1rev for CcPtc1.
To generation the CcPtc1 gene complementation construct, a fragment containing the entire length of the CcPtc1 coding region along with native promoter sequence and terminator sequence was cloned from gDNA using the primer pair CcPtc1-Compfor/CcPtc1-Comprev. The resulting PCR products were co-transformed into protoplasts of the ΔCcPtc1-11 strains with a geneticin-resistant cassette. After that, we selected the transformants in TB₃ medium supplemented with 40 μg/ml geneticin. Successful complementation was confirmed by PCR with the primer pair Internal-CcPtc1for/Internal-CcPtc1rev. The complementation strain was named ΔCcPtc1/PTC1 in this study. All primers used in gene deletion and complementation were listed in Supplementary Table 1.