Supplementary Figure 10: CRISPR-Cas9 editing of SWEET13 and SWEET14 for knock-out lines and predicted truncated form of transporters.
Mutagenesis of SWEET13 and SWEET14 using CRISPR/Cas9 genome editing. The guide RNA-targeting site is marked with an underline, and the PAM is marked in green. a, Mutagenesis scheme of SWEET13 and SWEET14. The dashed line denotes a deleted nucleotide in sweet13-1 (10 nt), sweet13-2 (4 nt) and sweet14-1 (1 nt), respectively. 1nt insertion in sweet14-2 is marked in blue. Both deletion and frameshift of amino acids occurred in the 1st exon and caused early termination. b, Predicted amino acid sequence of sweet13-1, sweet13-2, sweet14-1 and sweet14-2, respectively. In sweet13-1 and sweet13-2, frameshifts occured at the position of codons 8 and 7 of the original open reading frame, respectively, leading to polypeptides with altered sequence and length due to premature stop codons. c, If we assume that the second ATG (codon 58 in wild-type SWEET13) was used for protein production, only truncated proteins could be formed. In both mutants, the mutations will lead to loss of the first two transmembrane spanning domains, most likely leading to non-functional transporters. d, Predicted topology of the truncated SWEET14 protein in the sweet14-1 and sweet14-2 mutants in case codon 23 would serve a start codon. In both mutants, the mutations will lead to loss of the first transmembrane-spanning domain, most likely leading to non-functional transporters. Typically, premature stop codons affect RNA stability. Moreover, typically only the first ATG is used; thus, it is likely that all four lines completely lost the transport functions for the respective SWEETs.
