Fig 2: SA Binds to the A Subunit of PP2A(A) DARTS assay suggests that PP2AA1 is potential target of SA. pPP2AA1::PP2AA1-GFP seedlings were used for the protein isolation. Samples were treated with DMSO (mock) and SA and digested by different concentrations of Pronase. Samples were further analyzed by western blot with an anti-GFP antibody.(B) DSC analysis suggesting the potential binding of SA to recombinant His-PP2AA1. 5 µM of purified His-PP2AA1 protein was analyzed by DSC with or without 50 µM SA. Tm = 48.01°C and 45.03°C for His-PP2AA1+DMSO and His-PP2AA1+SA, respectively.(C) SPR analysis of the His-PP2AA1 and SA interaction. An active synthetic SA analog (SA-f) was immobilized on a CM-5 sensor chip, and different concentrations of His-PP2AA1 were applied. The binding curve was plotted by values at the steady state, for which the sensorgram is shown is Figure S7D. A KD value of 3.623 µM was detected.(D) SPR assay reveals the binding of His-PP2AA3 to SA. The same sensor chip as above was used, and different concentrations of His-PP2AA3 were applied. The binding curve was plotted by values at the steady state, with the data points shown in the sensorgram in Figure S7E. A KD value of 1.916 µM was detected.See also Figures S5, S6, and S7.

Fig 3: PREP inhibition reduces PP2A phosphorylation and activates autophagy in p25a transfected OLN-AS7 cells. p25a transfection did not alter the levels of catalytic subunit of PP2A (A; PP2Ac) but Tyr307 phosphorylated PP2Ac (B; pPP2Ac) was significantly elevated after transfection and vehicle treatment (B). 10 µM KYP-2047 significantly reduced pPP2Ac levels compared to p25a+vehicle but did not change the levels of PP2Ac (A-B). The protein accumulation marker, p62, was significantly decreased in p25a transfected and KYP-2047 treated cells compared to vehicle treatment (C). Autophagosome marker LC3BII was elevated in p25a transfected cells and remained elevated after 10 µM KYP-2047 treatment (D). Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001, 1-way ANOVA with Tukey's post hoc test (compared to vehicle). #, p < 0.05; 1-way ANOVA with Tukey's post hoc test (p25a+veh vs. p25a+KYP-2047). See figure S5for uncut blots. Representative blots in Figure 4A and C are from the same membrane and therefore share the beta-actin loading control

Fig 4: SA Functions through PP2A in Regulating Root Development(A) SA treatment promoted the phosphorylation of PIN2 in Col-0 to a similar degree as that in pp2aa1-6. Roots of 7-day-old Col-0 and pp2aa1-6 seedlings were treated with DMSO or 40 µM SA for 60 min and were then sampled for protein isolation and western blot. The shifted bands indicate the phosphorylated PIN proteins (upper panel). Asterisk indicates a non-specific band that contributes partially to the signal. The same membrane was stripped and probed with a PIP2;1 antibody to indicate the loading (upper panel). Ponceau staining is shown in the bottom panel.(B) Phosphorylation with 32P-ATP revealed that SA treatment increased the phosphorylation of His-PIN2-HL in Col-0, whereas this increase was attenuated in pp2aa1-6. Upper panel: autoradiography is shown; lower panel: CBB is shown.(C) Representative images revealing the hypersensitivity of pp2aa1-6 to SA. Col-0 and pp2aa1-6 seedlings were grown on plates with SA. Scale bars, 2 cm.(D) pp2aa1-6 was hypersensitive to SA in root growth inhibition. Col-0 and pp2aa1-6 seedlings grew on plates with SA for 7 days and then the primary root length was measured. n = 11–28. p values were calculated by a two-tailed t test for indicated pairs of Col-0 and pp2aa1-6 at a certain concentration of SA.(E and F) pp2aa1-6 was hypersensitive to SA in terms of interfering with root gravitropism. Col-0 (E) and pp2aa1-6 (F) seedlings grew on plates containing different concentrations of SA for 7 days, and the root tip angles were measured by ImageJ and shown as polar bar charts. p values were calculated by a two-tailed t test in (E) and (F) and indicate differences of variances by a further F-test in (F).(G) The pp2aa1, a3 double mutant exhibited decreased sensitivity to SA. Col-0 and pp2aa1, a3 seedlings grew on plates with SA for 7 days and then the primary root length was measured. n = 11–25. p values were calculated by a two-tailed t test for indicated pairs of Col-0 and pp2aa1, a3 at the given concentration of SA.(H) The pp2ac3, c4 double mutant exhibited decreased sensitivity to SA. Col-0 and pp2ac3, c4 seedlings grew on plates with SA for 7 days and then the primary root length was measured. n = 10–21. p values were calculated by a two-tailed t test for indicated pairs of Col-0 and pp2ac3, c4 at the given concentration of SA.See also Figures S3 and S4.

Fig 5: Model for the Parallel SA Action in Immunity and Growth Regulation(A) SA plays a key role in the growth-immunity transition following pathogen attack: on one hand, SA activates the immune response, through stimulating NPR1 and repressing NPR3/4, all together increasing the expression of downstream defense genes; on the other hand, SA inhibits growth via suppressing PP2A activity and the subsequent dephosphorylation of substrates.(B) The auxin efflux carrier PIN2 is phosphorylated by different kinases, including PINOID/WAGs, D6PK/D6PKLs, and MAPKs, and dephosphorylated by PP2A. Following pathogen attack, the SA levels increase. SA binds to the A subunits of PP2A and thereafter represses its dephosphorylation activity toward PIN proteins, which leads to hyperphosphorylation of PIN, thereby a decrease in PIN activity ultimately resulting in a decrease in auxin export and attenuation of growth.(C and D) Induced stronger expression of pPR1::eYFP-NLS by SA was detected in pp2aa1-6.(C) pPR1::eYFP-NLS seedlings were constantly grown on plates with DMSO or 40 µM SA for 5 days from germination and were then imaged by CLSM. Scale bars, 10 µm.(D) For quantification, the average GFP florescence of 5–10 representative cells from 10 seedlings for each treatment was measured by Fiji. The data points were showed as dot plots, and lines indicate mean ± SD. Different letters represent significant difference; p < 0.05; by one-way ANOVA with a Tukey multiple comparison test.