Generation of transformants

To generate gene knockout transformants, the deletion constructs prepared by a modified double-joint PCR were introduced to the wild-type protoplast via a previously reported (7, 9, 58). For complementation, the sequences of each gene containing the promoter, open reading frame (ORF), terminator, and a 1.5 kb Geneticin resistance cassette were co-transformed into protoplasts of desired deletion mutants (15). The deletion mutants and complemented strains were verified via Southern blotting and RT-PCR (59). To perform subcellular localization, the fragments containing the promoter and ORF of CsCZF1 minus stop codon were amplified with primers CAP_012989.1 5 F/CAP_012989.1 R (Table S5). Fragments of green fluorescent protein (GFP) were amplified with primers CAP_012989.1 _ GFP F/GFP NR (Table S5). The GFP sequence was fused to the C terminus of CsCZF1 and was further transformed into wild-type protoplasts. Localizations of CsCZF9 and CsSTE12 were performed according to the same method. To generate N-terminal fusion of GFP to CsATG8, the sequence of CsATG8 promoter, GFP, CsATG8 ORF, and CsATG8 ORF terminator were amplified with primers CsATG8 F/CsATG8_Pro_R, CsATG8_Pro_GFP F/GFP_CsATG8 R, and CsATG8_ORF F/CsATG8 R, respectively (Table S5). The amplified sequences were fused to generate recombinant construct Pro^CsATG8:GFP:CsATG8, which were further transformed into wild-type and ΔCscrz1 protoplasts. To generate constitutive expression construct of CsCZF1, promoter of Neurospora crassa isocitrate lyase gene was amplified with primers ICL F/ICL R (Table S5). The sequence of ORF and terminator of CsCZF1 was amplified with primers ICL_CAP_012989.1 F/CAP_012989.1 3R (Table S5). Two amplified fragments were fused and transformed into ΔCshox2 protoplasts. To generate C-terminal fusion of 6 × His to CsPMK1, a sequence containing promoter and ORF of CsPMK1 was amplified with primers CsPMK1 5 F/CsPMK1_His R (Table S5). The sequence containing terminator of CsPMK1 was amplified with primers His_CsPMK1_Ter F/CsPMK1_3R (Table S5). Amplified sequences were fused and transformed protoplasts of wild-type strain expressing CsCZF9:GFP. All transformants were screened using PCR or checked the fluorescence signals using fluorescence microscopy (Carl Zeiss Microscope Division, Germany).