Fungal transformation

Transformation was performed as described by Ribot et al., (2013)[92]. Protoplasts of GY11 were prepared as described previously [93]. For the knock-out cks1 mutant, 1.2kb upstream and 1.2 kb downstream regions of the gene of interest were amplified by PCR using genomic GY11 DNA (100 ng) as template. The strategy used for constructing the gene replacement cassettes is derived from Kämper (2004) [94] and presented in the S2 Fig. Primers used are shown in S2 Table. Growing colonies were transferred to Tanaka-Hygromycin or Tanaka-Basta plates for assessing resistance. Resistant colonies were further grown on rice agar media for 7–10 days at 26°C, purified by single-spore isolation, tested for resistance, and stored at -20°C. At least 2 independent transgenic fungal lines were isolated for each plasmid. Resistant colonies were characterized by PCR using a Phire Plant Direct PCR kit (Thermo Scientific, Waltham, MA, USA).