Sun, Yue, Shao, Xiaolong, Zhang, Yingchao, Han, Liangliang, Huang, Jiadai, Xie, Yingpeng, Liu, Jingui, and Deng, Xin
Additional file 1: Figure S1. Induction curves of PopNPA14 and non-T3SS proteins (AdhBPS, AlpAPA, MexAPA14, GumBXO and SucCRS) induced in 1× T3SS-inducing conditions and 1× T3SS-repressing conditions. (A) The red curve showed the delayed expression levels of adhBPS-pro-CDS-lux reporter in 1× MM compared with that in 1× KB. (B) The red curve showed the slightly fast expression levels of alpAPA-pro-CDS-lux reporter in LB + 5 mM EGTA compared with that in LB. (C) The red curve showed the delayed expression levels of gumBXO-pro-CDS-lux reporter in 1× XOM2 compared with that in 1× M210. (D) The red curve showed the delayed expression levels of sucCRS-pro-CDS-lux reporter in 1× MMRS compared with that in 1× B. (E) The red curve showed the slightly fast expression levels of popNPA14-pro-CDS-lux reporter in LB + 5 mM EGTA compared with that in LB. (F) The red curve showed the slightly fast expression levels of mexAPA14-pro-CDS-lux reporter in LB + 5 mM EGTA compared with that in LB. (G) The expression of HrpGRS in MMRS at 50 and 60 min were induced rapidly so that the synthesis time of HrpGRS was not calculated. Figure S2. Expression levels of T3SS regulators, effectors and structural protein of P. syringae, X. oryzae and R. solanacearum in 0.5× T3SS-inducing conditions were less stable than that in 1× T3SS-inducing conditions. The red curve and green curve denoted the expression levels in 1× and 0.5× T3SS-inducing conditions, respectively. (A) The expression levels of hrpLPS-pro-CDS-lux reporter were extremely low in 0.5× MM compared with those in 1× MM. (B-D, F) The expression levels of hopG1PS, hrpRPS, hopX1PS and hrpGRS-pro-CDS-lux reporters in 0.5× T3SS-inducing conditions rapidly increased, but declined to a low level immediately compared with those in 1× T3SS-inducing conditions. (E) The expression levels of hpa1XO-pro-CDS-lux reporter in 0.5× XOM2 were slightly lower than those in 1× XOM2. Figure S3. Translational elongation rate of PopNPA14 and non-T3SS proteins (AdhBPS, AlpAPA, MexAPA14, GumBXO and SucCRS) in multiple nutrition conditions and at various time. (A-B) Translational ERs of PopNPA14 under different EGTA concentrations and at various culture time (0–60 min). (C) The translational ERs of MexAPA14 in LB with EGTA (~ 1.0- ~ 2.2 aa/s) were much lower than that in LB (~ 5.2 aa/s) with no significant difference. (D-E) The translational ERs of MexAPA14 in both LB and LB with 5 mM EGTA during various culture times kept at a low level (~ 0.5- ~ 2.0 aa/s and ~ 0.8- ~ 1.8 aa/s, respectively). (F) The translational ER of AdhBPS in 1× MM (~ 8 aa/s) was more than fourfold than those in richer or poorer nutrition conditions (less than 2 aa/s). (G) In 1× KB, the translational ER of AdhBPS at the beginning of culture (~ 8 aa/s) had significant difference than those at various culture time (less than 2 aa/s). (H) In 1× MM, the translational ERs of AdhBPS at 0–4 h of culture were much lower than that in 1× KB, which had no difference. (I) The translational ER of AlpAPA in LB (~ 13 aa/s) was more than threefold than those in LB + EGTA (less than 5 aa/s). (J) In LB, the translational ERs of AlpAPA at 0–40 min of culture (10–15 aa/s) had no difference. The ER decreased rapidly at 50–60 min (less than 5 aa/s). (K) In LB + 5 mM EGTA, the translational ERs of AlpAPA were much lower than those in LB. (L) The translational ER of GumBXO in 1× M210 (~ 0.5 aa/s) had no significant differences with those in 0.5× M210 and 0.125× M210 (~ 0.8 aa/s). (M) The translational ERs of GumBXO at 10, 30, 40 and 50 min (~ 0.7–0.8 aa/s) showed no differences with that at 0 min (~ 0.5 aa/s) apart from those at 20 and 60 min (~ 0.9–1.0 aa/s). (N) The ERs of SucCRS in 0.5× , 0.25× and 0.125× B (~ 0.9–1.4 aa/s) were much lower than that in 1× B (~ 13.4 aa/s). (O) The translational ERs of SucCRS for 10–60 min (~ 1.3–2.7 aa/s) were much lower than that at 0 min (~ 13.4 aa/s). Figure S4. Induction assay of full-length PopNPA14 and non-T3SS proteins (AdhBPS, AlpAPA and MexAPA14) mRNAs in 1× T3SS-inducing conditions. (A) The mRNA synthesis level of popNPA14 was rapidly induced in LB with 5 mM EGTA. $$\sqrt{E\left(t\right)-E(0)}$$ E t - E ( 0 ) plot showed that Ttest of PopNPA14 was ~ 2191 s in LB with 5 mM EGTA. (B) The mRNA synthesis level of adhBPS rapidly decreased in 1× MM and maintained a low level within 26–420 min. $$\sqrt{E\left(t\right)-E(0)}$$ E t - E ( 0 ) plot showed that Ttest of AdhBPS was ~ 343.7 min in 1× MM. (C) The mRNA synthesis level of alpAPA increased after adding EGTA and dropped to the starting level. $$\sqrt{E\left(t\right)-E(0)}$$ E t - E ( 0 ) plot showed that Ttest of AlpAPA was ~ 47 s in LB + 5 mM EGTA. (D) The mRNA synthesis level of mexAPA14 increased after adding EGTA and dropped to the starting level. $$\sqrt{E\left(t\right)-E(0)}$$ E t - E ( 0 ) plot showed that Ttest of MexAPA14 was ~ 5 min in LB + 5 mM EGTA. Figure S5. Relative expression levels of tRNAs and EFs of PA14 in LB with 5 mM EGTA. (A-C) tRNAs of PA14 were detected at 10, 20, 30, 40, 50, 60 min. The data at 0 min for each individual tRNA was set as 1. (D) The relative EFs levels of PA14 were detected by qRT-PCR. The EFs levels were shown together with the translational ER of PopNPA14 in LB with 5 mM EGTA. The translational ERs at specific times were shown as green hollow symbols. The standard deviations were shown but were very small in the plot. *p < 0.05; **p < 0.01; and ***p < 0.001. Results were indicated in mean ± SD. All experiments were repeated at least three times. Figure S6. The expression levels of T3SS genes during 0–6-h inducing culture. (A–E) The expression levels of hrpLPS, popNPA, hpa1XO, hrpGRS and popNPA14 in 1× T3SS-inducing conditions for 0–6 h. Figure S7. The Schleif plot of T3SS regulators, effectors and structural protein of P. syringae, P. aeruginosa, X. oryzae and R. solanacearum in 1× T3SS-inducing conditions. The Schleif plot was used to detect the translational time of the first newly synthesized protein in 1× T3SS-inducing conditions. The square root of newly synthesized protein ( $$\sqrt{E\left(t\right)-E(0)}$$ E t - E ( 0 ) ) was linear correlated with time during the initial dozens of minutes. E(t) denoted the expression levels of the tested proteins at specific times in 1× T3SS-inducing conditions, and E(0) denoted the basal expression levels of the culture. The green line and red line described the linear line of transcriptional fusion reporters and reporters containing both transcription and translation, respectively. The x-intercepts of the green line and red line denoted the time for the initial cost (Tinitiation) and the transcription and translation of the tested proteins (Ttest), respectively. Therefore, the difference of the two intercepts corresponded to the time to translate the full-length protein molecule (Tfirst). The initial cost included the sense of cells to nutrient conditions, RNA polymerase transcriptional initiation and ribosome translational initiation (Ref. 4 of the main text). The initiation time of various proteins were different (~ 1702s for HrpLPS, ~ 1791s for HopG1PS, ~ 1699 s for HrpRPS, ~ 2454 s for HopX1PS, ~ 2485 s for HrpA2PS, ~ 759.8 s for PopNPA, ~ 17.03 s for Hpa1XO and ~ 2553 s for HrpGRS). Figure S8. Ttests of Hpa1XO and HrpGRS in 1× T3SS-repressing conditions were much longer than those in 1× T3SS-inducing conditions. The red line and blue line denoted the time containing both transcription and translation (Ttest) in 1× T3SS-inducing conditions and 1× T3SS-repressing conditions, respectively. (A) Ttests of hpa1XO-pro-CDS-lux reporter in 1× XOM2 and 1× M210 were ~ 3.352 min and ~ 300.4 min. (B) Ttests of hrpGRS-pro-CDS-lux reporter in 1× MMRS and 1× B medium were ~ 42.35 s and ~ 181.2 s. Table S1. Bacterial strains, plasmid, and primers used in this study.