Figure 4.

Disruption and complementation of FST1. (A) A hygromycin‐resistance cassette (HYG^R) was inserted into the FST1 gene by homologous recombination to create a Δfst1 strain. For complementation, protoplasts of Δfst1 were co‐transformed with pHT‐10 (constructed by cloning the FST1 locus into the pGEM‐T‐Easy vector; dashed lines represent vector sequence) and pKS‐GEN, a vector harbouring a geneticin resistance cassette. For Southern analysis, genomic DNA was digested with ClaI (C) and probed with a 473‐bp region of FST1 (P). (B) The presence or absence of FST1 was verified by Southern analysis in the wild‐type (lane 1), Δfst1 (lane 2), FST‐COMP1 (lane 3) and FST‐COMP2 (lane 4) strains. Integration of pHT‐10 was ectopic in the transformants, and the FST‐COMP1 strain appeared to contain two copies of the complementation construct and thus was not used in subsequent experiments.