Extended Data Figure 2. Characterization of uORFs_TBF1 and uORFs_bZIP11 in translational control, related to Fig. 1.

a, Subcellular localization of the LUC-YFP fusion (a) and GFP_ER (b). SP, signal peptide from Arabidopsis basic chitinase; HDEL, ER retention signal. Representative of 8 images. Scale bar, 10 μm. c–e, mRNA levels of LUC in (Fig. 1b; n = 3), GFP_ER in (Fig. 1c; n = 4), and TBF1-YFP in (Fig. 1d; n = 3) 2 dpi before cell death was observed in plants expressing TBF1. f, Schematics of the 5′ leader sequences used in studying the translational activities of WT uORFs_bZIP11, mutant uorf2a_bZIP11 (ATG to CTG) or uorf2b_bZIP11 (ATG to TAG). g–i, uORFs_bZIP11-mediated translational control of cytosol-synthesized LUC (g; chemiluminescence with pseudo colour); ER-synthesized GFP_ER (h; fluorescence under UV); and cell death induced by overexpression of TBF1-YFP fusion (i; cleared using ethanol) after transient expression in N. benthamiana for 2 d (g, h) and 3 d (i), respectively. Representative of 4 images. j–l, mRNA levels of LUC in (g; n = 2 experiments with 3 technical replicates), GFP_ER in (h; n = 3 experiments with 3 technical replicates), and TBF1-YFP in (i; n = 3 experiments with 3 technical replicates). m, TE changes in LUC controlled by the 5′ leader sequence containing WT uORFs_bZIP11, mutant uorf2a_bZIP11 or uorf2b_bZIP11 in response to elf18 in N. benthamiana. Mean of the LUC/RLUC activity ratios (n = 12). n, LUC/RLUC mRNA changes in (m). Mean of LUC/RLUC mRNA normalized to Mock from 2 experiments with 3 technical replicates. Bar with solid circles, mean with individual biological replicates.