Fig. 8: The P. syringae toxin syringolin-A (SylA) inhibits ex-proteasome activity and suppresses ROS bursts triggered by flagellin but not flg22.

a Chemical structure of SylA. b Arabidopsis ex-proteasome activity is inhibited by SylA. The crude lysate (CL), APF, and affinity-purified proteasomes were extracted from PAG1-FLAG pag1-1 leaves, pretreated for 20 min without or with 50 or 150 µM of SylA or an equivalent volume of DMSO (Mock), and assayed for proteasomes activity using the fluorogenic substrate Suc-LLVY-AMC. Bars reflect the mean (±SD) of four technical replicates with the values normalized to the reactions obtained without SylA. Individual data points are included. c SylA, like Epo, does not block the ROS burst elicited by flg22. Leaf discs were incubated with flg22 with or without a 10-min pretreatment with 62.5 μM of Epo or SylA, or an equivalent volume of DMSO, and assayed for ROS burst as in Fig. 6. The left panel show the time course for the mean RLUs measured with five biological replicates (±SD). Horizontal grey bar indicates the measurement timeframe for determining the total luminescence intensity (TLI). d Quantification of the ROS bursts measured by each treatment in panel (c) by TLI generated over minutes 2 to 40 (±SD). Individual data points are included. The letter a above the bars indicates no significant difference from others using a one-way ANOVA followed by Tukey’s test to determine significance. e Addition of SylA to the APF suppresses the ROS burst elicited by flagellin. APF was incubated with purified P. syringae flagellin for 4 hr with or without a 10-min pretreatment with increasing concentrations of SylA, or an equivalent volume of DMSO, and assayed for ROS burst as in Fig. 6. The left panel shows the time course for the mean RLUs measured as in panel (c) with four biological replicates (±SD). The right panel shows the inhibitory effect of SylA calculated by an IC50 value as in Fig. 6a-c. Each point represents the mean of four biological replicates (±SD).