Functional Characterization of SUCs by Heterologous Expression in Baker’s Yeast

The coding sequences of AtSUC6_Col-0 and AtSUC7_Col-0 were amplified from pCF12 and pTR73 with primer pairs that introduced a NotI site on both sides of the PCR products as well as the sequence 5′-AAGCTTGTAAAAGAA-3′ (part of the STP1 5′UTR) (Stadler et al., 1995) upstream of the start codon (Supplementary Table 5). The fragments were ligated into the NotI site of the vector NEV-N (Sauer and Stolz, 1994) in both sense and antisense orientation, yielding plasmids pFC14 (AtSUC6c sense), pFC19 (AtSUC6c antisense), pTR10 (AtSUC7c sense), and pTR11 (AtSUC7c antisense). The AtSUC6 constructs were then used for lithium acetate-mediated transformation (Soni et al., 1993) of S. cerevisiae CSY4000 (Rottmann et al., 2016), yielding strains TRY1039 (AtSUC6c sense) and TRY1040 (AtSUC6c antisense). AtSUC7 constructs were transformed into S. cerevisiae strain SEY2102 (Emr et al., 1983), yielding TRY1001 (AtSUC7c sense), and TRY1002 (AtSUC7c antisense). Transport tests with ¹⁴C-labeled sucrose were performed as described (Sauer and Stadler, 1993). AtSUC7_Col-0 was additionally amplified with primers attaching BspHI sites to both ends of the fragment (Supplementary Table 5) and ligated into NEV-CGFP (Rottmann et al., 2018) and NEV-NGFP (see below) linearized with NcoI for analyses of the subcellular localization in yeast. The transformation of SEY2102 led to strains TRY1006 (AtSUC7c-NEV-NGFP) and TRY1008 (AtSUC7c-NEV-CGFP).