Extended Data Figure 3: Ectopic expression of T474D-D downregulates translational-machinery-related genes and suppresses de novo protein synthesis.

a, Representation of the translational-machinery-related genes differentially expressed in the T474D lines. The ‘MA’ plots show the log of the ratio of expression levels versus the log concentration, and each dot represents a gene. This plot represents the contrast between the T474D mock-inoculated lines and the Col-0 mock-inoculated lines. The red points denote ribosomal and protein synthesis-related genes (as shown in Supplementary Table 3). b, Downregulation of global translation by ectopic expression of T474D in Arabidopsis. The in vivo labelling of leaf proteins with [³⁵S]Met was performed in 20-day-old Col-0 plants and T474D transgenic lines. The total protein extracts were fractionated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), and the radioactive bands were quantified by densitometric analysis of the images obtained by autoradiography. The labelling percentage was normalized to the leaf chlorophyll content, and the protein loading on the gel was adjusted to the Coomassie-stained band of the large subunit of rubisco. c, Induction of T474D expression by dexamethasone (DEX) in Arabidopsis transgenic seedlings. The constitutive expression of T474D was associated with stunted growth in the transgenic lines (Extended Data Fig. 1a) and repression of global protein synthesis (Fig. 1b). These phenotypes precluded the use of an appropriate normalization method for protein loading in comparative gels of contrasting genotypes to estimate precisely the T474D-mediated protein synthesis inhibition in our assay. To overcome this limitation, we used a dexamethasone-inducible promoter to control the T474D expression in the transgenic lines. Arabidopsis seedlings independently transformed with a T474D–GFP fusion under the control of a dexamethasone-inducible promoter (TR1 and TR2) were treated with 30 μM dexamethasone for 8 h, and the induction of T474D–GFP expression was monitored by semi-quantitative RT–PCR. The dexamethasone-induced expression of T474D for 8 h led to a higher inhibition of de novo protein synthesis in the transgenic lines, as measured by TCA-precipitable radioactivity, which could be normalized to total protein (TR1 and TR2; Fig. 1c). d, Inhibition of de novo protein synthesis by protein synthesis inhibitors in Col-0, untransformed lines. We also compared the T474D-mediated suppression of translation with known global translation inhibitors, such as the cytosolic protein synthesis inhibitors cycloheximide and puromycin and the chloroplast translation suppressor chloramphenicol. Arabidopsis seedlings (10 days old) were treated with 10 μM cycloheximide (Cyclo), 10 μM puromycin (Pur), 25 μM chloramphenicol (Chlor) or 30 μM dexamethasone (Dex) for the indicated periods of time, and then they were pulse labelled with l-[³⁵S]Met for 60 min. Lysates of treated cells were measured by liquid scintillation counting and normalized to total protein. The relative [³⁵S]Met incorporation was normalized to wild-type (WT = 1) control without treatment. Means ± 95% confidence intervals (n = 3) based on bootstrap resampling replicates of three independent experiments are shown. e, Inhibition of de novo protein synthesis by inducible expression of T474D. Seedlings (10 days old) from the TR1 transgenic line were treated with dexamethasone and the protein synthesis inhibitors for the times as indicated in the figure, and then they were pulse labelled with l-[³⁵S]Met for 60 min. Lysates were processed as described in d. Means ± 95% confidence interval (n = 3) based on bootstrap resampling replicates of three independent experiments are shown. Cycloheximide was the most effective inhibitor of translation in both the wild-type and T474D-expressing lines. T474D expression inhibited global translation to the same extent as puromycin and caused a further inhibition in the level of chloramphenicol translational inhibition in a combined treatment. The increase in translational inhibition by combining T474D expression and chloramphenicol treatment may indicate that T474D inhibits cytosolic protein synthesis, which is consistent with the T474D-mediated downregulation of components of the cytosolic translational machinery (Supplementary Table 3).

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