Figure 3.

The URA3B expression cassette driven by the Tef (TRANSLATION ELONGATION FACTOR) promoter works as a uridine auxotrophy selection marker in ura3a/b. (a) Schematic diagrams of PKS1 (Cob_09513) knock out in ura3a/b. The pNK059 plasmid has 2 kb of upstream (Up) and downstream (Down) sequences of the PKS1 locus. The Down sequence includes the partial PKS1 coding sequence (CDS). The URA3B expression cassette driven by the Tef constitutive promoter (Tef::URA3B) is located between the Up and Down sequences as a uridine auxotrophy marker. Green arrows represent a 500‐bp sequence located upstream of the Up sequence on the Colletotrichum orbiculare genome. The 500‐bp sequence is designed to allow homologous recombination induced by 5‐fluoroorotic acid (5‐FOA) treatment for excision of Tef::URA3B. (b) Genomic DNA polymerase chain reaction (PCR) showed that PKS1 was knocked out. The primer set Po13/Po14 generates the 1200‐bp amplicon if the Tef::URA3B cassette is present. The primer set Po15/Po16, generates the 1000‐bp band if the PKS1 CDS is present. The primers used are listed in Table S3 (see Supporting Information). (c) Wild‐type (WT), ura3a/b#1 and two independent pks1/ura3a/b‐Tef::URA3B strains were cultured on potato dextrose agar (PDA) for 6 days at 25 °C in the dark. The details of each strain are listed in Table S1 (see Supporting Information). [Colour figure can be viewed at wileyonlinelibrary.com]