Computational Methods

Binding site motifs for N. benthamiana transcription factors were obtained from the PlantTFDB (Tian et al., 2020), and Find Individual Motif Occurrences (FIMO; Grant et al., 2011) was used to predict their occurrence in the 35S core enhancer. Fragments of pZS*11_4enh were aligned to the reference sequence using Bowtie2 (Langmead and Salzberg, 2012). For analysis of the STARR-seq experiments, the reads for each barcode were counted in the input and cDNA samples. Barcode counts below 5 and barcodes present in only one of three replicates were discarded. Barcode enrichment was calculated by dividing the barcode frequency (barcode counts divided by all counts) in the cDNA sample by that in the input sample. For the pZS*11_4enh fragment library (Figure 5; Supplemental Figure 1) and the mutagenesis (Figures 6A to 6D) experiments, the enrichment of the fragments or variants was calculated as the median enrichment of all barcodes linked to them. Boxplots were created using all corresponding barcodes from all replicates performed and were normalized to the median enrichment of constructs without an enhancer. The enrichment coverage of pZS*11_4enh was calculated by summing up the enrichment of all fragments containing a given nucleotide and dividing this sum by the number of fragments. Nucleotides covered by fewer than five fragments were excluded from analysis. Light dependency of enhancers or enhancer fragments was calculated as log₂ of the enrichment in the light condition divided by the enrichment from the dark condition. Spearman correlations were calculated using the base R function. The code used for analyses is available at https://github.com/tobjores/tobacco-STARR-seq.