Extended Data Fig. 7: Several mechanisms are involved in the upregulation of PTI signalling components during ETIAvrRps4.
From: Mutual potentiation of plant immunity by cell-surface and intracellular receptors
a, Relative gene expression of ICS1, BIK1, RBOHD, MPK3 and MPK6 in seedlings with pre-activation of ETIAvrRps4 for 3 h before treatment with cycloheximide and MG132. Data are mean ±s.e.m. (from three independent experiments). A two-sided Welch’s t-test was used to analyse significant differences of 3-h data points from 0 h (*P ≤ 0.05; ****, P ≤ 0.001). Exact P values are provided in Supplementary Table 5. b, c, Protein accumulation of MPK3, RBOHD, BIK1, MPK6 and actin in seedlings pre-treated with mock (DMSO) for 3 h (b), and RPS4–HA, FLS2 and BAK1 in seedlings pre-treated with mock or oestradiol and subsequently treated with cycloheximide (50 μM), MG132 (10 μM) or both for indicated times (2 h, 4 h and 8 h) (c). Actin was used as the loading control. Ponceau staining of corresponding blots is shown below. For FLS2 and actin, as well as BAK1 and BIK1, immunoblotting was performed with membranes cut in half (above 70 kDa for FLS2 and BAK1; below 70 kDa for actin and BIK1 immunoblot). Therefore, the Ponceau staining for FLS2 and actin, BAK1 and BIK1, respectively, is identical. d, Schematic of ribosome enrichment. e, f, Ribosomes were enriched, and total extract (T), supernatant (S) and ribosomal pellet (P) samples were blotted with RPS6 and RPL10 antibody (e). For b, c, e, Ponceau staining was used as loading control. Molecular weight markers (in kDa) are indicated on the left. f, RNA extracted from total extract (total RNA), and ribosomal pellet (ribosome RNA) from mock and oestradiol-treated Est:AvrRps4 samples were loaded on an agarose gel; 28S and 18S ribosomal RNA (rRNA) are indicated. g, Expression of ICS1, SOBIR1, BAK1, BIK1, RBOHD and MPK3 to relative to EF1A from total RNA (total) and ribosomal pellet (ribosomal). Data are mean ±s.e.m. (from three independent experiments). A two-sided Welch’s t-test was used to analyse significant differences of 6-h data points from 0 h (*P ≤ 0.05; ***, P ≤ 0.005; ****, P ≤ 0.001). Exact P values are provided in Supplementary Table 5. h, Ratio of ribosomal RNA to total RNA (relative to EF1A) of ICS1, SOBIR1, BAK1, BIK1, RBOHD and MPK3 in mock and ETI-activated samples. Values are calculated from the transcripts retained in the ribosomal samples over total samples. Data are mean ±s.e.m. (from three independent experiments). A two-sided Welch’s t-test was used to analyse significant differences in the translation efficiency (T.E.) between mock and ETI-activated samples. Exact P values are provided in Supplementary Table 5. All experiments were repeated at least three times with similar results.
