Fig 1.
RipAC contributes to Ralstonia solanacearum virulence in Arabidopsis and tomato.
(A, B) Soil-drenching inoculation assays in Arabidopsis were performed with GMI1000 WT, ΔripAC mutant, and RipAC complementation (ripAC+) strains. In (A) the results are represented as disease progression, showing the average wilting symptoms in a scale from 0 to 4. Values from 3 independent biological repeats were pooled together (mean ± SEM; n = 45). Curves for each replicate are shown in S2A Fig. (B) Survival analysis of the data in (A); the disease scoring was transformed into binary data with the following criteria: a disease index lower than 2 was defined as ‘0’, while a disease index equal or higher than 2 was defined as ‘1’ for each specific time point. Statistical analysis was performed using a Log-rank (Mantel-Cox) test (n = 45 for each strain), and the corresponding p value is shown in the graph with the same colour as each curve. (C, D) Soil-drenching inoculation assays in tomato were performed with GMI1000 WT, ΔripAC mutant, and RipAC complementation (ripAC+) strains. In (C) and (D) the analyses were performed the same as in (A) and (B) (n = 36 for each strain), and the corresponding p value is shown in the graph in (D) with the same colour as each curve. Curves for each replicate are shown in S2B Fig. (E) Growth of R. solanacearum GMI1000 WT, ΔripAC mutant, and RipAC complementation (ripAC+) strains in tomato plants. 5 μL of bacterial inoculum (106 cfu mL-1) were injected into the stem of 4-week-old tomato plants and xylem sap was taken from each infected plants for bacterial titer calculation 3 days post-inoculation (dpi). Different colours represent values obtained in 3 independent biological repeats, and horizontal bars represent average values (n = 6 plants per strain in each repeat). Asterisks indicate significant differences (*** p<0.001, **** p<0.0001, t-test). (F, G) Soil-drenching inoculation assays in Col-0 WT and RipAC-expressing Arabidopsis lines (AC #3, AC #31, two independent lines) with GMI1000 WT strain. The analyses were performed the same as in (A) and (B) (n = 45 for each genotype), and the corresponding p value is shown in the graph in (G) with the same colour as each curve. Curves for each replicate are shown in S3C Fig.
Fig 2.
RipAC associates with SGT1 in plant cells.
(A) CoIP to determine interactions between RipAC and SGT1s transiently expressed in Nicotiana benthamiana. The signal in the interaction between RipAC and AtSGT1a was weak and is shown with longer exposure. (B) Split-LUC assay to determine direct interaction between RipAC and SGT1 transiently expressed in N. benthamiana. (C) Pull-down assay to determine direct interaction between RipAC and SGT1a/b. His-tagged RipAC was incubated with immobilized GST-AtSGT1a/AtSGT1b/GUS. After 4 rounds of wash, bound proteins were eluted and subjected to western blot analysis using anti-RipAC antibody. Coomassie blue (CBB) staining showed the visualization of both input and GST pull-down proteins. (D) CoIP to determine interactions between RipAC and different truncated versions of NbSGT1 in N. benthamiana. The diagram summarizes the different domains of SGT1: TPR, N-terminal tetratricopeptide repeat (TPR) domain; CS, CHORD-SGT1 domain; SGS, C-terminal SGT1-specific domain; VR, variable region. The experiments in (A-C) were repeated at least 3 times with similar results. The experiment in (D) was repeated 2 times with similar results. In western blot assays, protein marker sizes are provided for reference. In (A) and (D), blots were stained with Coomassie Brilliant Blue (CBB) to verify equal loading.
Fig 3.
RipAC inhibits SGT1-dependent immune responses.
(A) RipAC suppresses RPS2-, Avr3a/R3a-, or RipE1-associated cell death in Nicotiana benthamiana. (B) RipAC does not suppress BAX- or INF1-induced cell death in N. benthamiana. In (A) and (B) Photographs were taken 5 days after the expression of the cell death inducer. The numbers beside the photographs indicate the ratios of infiltration showing the presented result among the total number of infiltrations. (C-F) RipAC suppresses MAPK activation triggered by the overexpression of RPS2 (C), Avr3a/R3a (D), or RipE1 (E) in N. benthamiana. Plant tissues were taken one day after cell death inducer expression. (F) Quantification of relative pMAPK in (C-E). The quantification of the pMAPK band in RipAC expression spots is represented relative to the intensity in the GFP control in each assay (mean ± SEM of 4 independent biological replicates). (G-J) RipAC suppresses effector-triggered immunity in Arabidopsis. Leaves of Col-0 WT and RipAC-transgenic lines (AC #3 and AC #31) were hand-infiltrated with Pto empty vector (EV, G), Pto AvrRpm1 (H), Pto AvrRpt2 (I), or Pto AvrRps4 (J). Samples were taken 3dpi (mean ± SEM, n = 6, ** p<0.01, t-test). These experiments were repeated at least 3 times with similar results. In western blot assays, protein marker sizes are provided for reference. Blots were stained with Coomassie Brilliant Blue (CBB) to verify equal loading.
Fig 4.
MAPKs associate with SGTs in plant cells.
(A) AtSGT1a and AtSGT1b associate with MAPK4 and MAPK6 in Nicotiana benthamiana. CoIPs were performed as in Fig 2A. (B, C) AtSGT1a/b associate with AtMAPK3/4, but not with MAPK6, in Split-LUC assay in N. benthamiana. Agrobacterium combinations with different constructs were infiltrated in N. benthamiana leaves and luciferase activities were examined with microplate luminescence reader. The graphs show the mean value ± SEM (n = 8, ** p<0.01, t-test). The MAPK-PIP2A combination was used as negative control. (D) Phosphorylation of recombinant AtSGT1a/AtSGT1b by activated MPK3 and MPK6 in vitro. Recombinant MPK3/MPK6 were first activated by MKK5DD and subsequently incubated with AtSGT1a/AtSGT1b. Phosphorylated proteins (upper panel) were visualized by western blots using anti-SGT1 phosphorylation antibody, and the presence of SGT1 was visualized by western blots using anti-SGT1 antibody (middle panel). The equal input of MKK5DD and MPK3/MPK6 was confirmed by Coomassie blue (CBB) (lower panel). In all the blots, asterisks indicate non-specific bands. (E) MAPK activation triggered by MKK5DD expression correlates with increased AtSGT1 phosphorylation at T346 in Arabidopsis. Twelve-day-old Dex:MKK5DD plants were treated with 30 mM Dex, and samples were taken at the indicated time points. In (E), a custom antibody was used to detect the phosphorylated peptide containing AtSGT1 T346. The induction of MKK5DD was determined by western blot using anti-FLAG antibody. (F) Quantification of pSGT1b signal relative to Coomassie blue staining control in (E) (mean ± SEM of 6 independent biological repeats, p-values are shown, t-test). (G) MAPK activation triggered by AvrRpt2 expression correlates with increased AtSGT1 phosphorylation at T346 in Arabidopsis. Twelve-day-old Dex:AvrRpt2/Col-0 crossing F1 plants were treated with 30 mM Dex, and samples were taken at the indicated time points. In (G), a custom antibody was used to detect the phosphorylated peptide containing AtSGT1 T346. The induction of AvrRpt2 was determined by RT-PCR. (H) Quantification of pSGT1b signal relative to Coomassie blue staining control in (G) (mean ± SEM of 6 independent biological repeats, p-values are shown, t-test). (I) MPK3 and MPK6 are required for Pto AvrRpt2-induced SGT1 phosphorylation. Twelve-d-old Col-0, MPK3SR, and MPK6SR plants grown in liquid medium were first treated with 2 μM NA-PP1 or DMSO (mock) for 1 h. Then the plants were immersed in Pto AvrRpt2 (OD600 = 0.02) for the indicated periods of time. In (I), a custom antibody was used to detect the phosphorylated peptide containing AtSGT1 T346. The induction of MAPK activation was determined by western blot using anti-pMAPK antibody. (J) Quantification of pSGT1b signal relative to Coomassie blue staining control in (I) (mean ± SEM of 3 independent biological repeats, p-values, compared to their control at 0 hpi, are shown, t-test). These experiments were repeated at least 3 times with similar results. In western blot assays, protein marker sizes are provided for reference. Blots were stained with Coomassie Brilliant Blue (CBB) to verify equal loading.
Fig 5.
MAPK-mediated phosphorylation is important for SGT1 function in the activation of ETI.
(A) A phospho-mimic mutation in AtSGT1b T346 (T346D) promotes cell death triggered by RPS2 overexpression in N. benthamiana. Agrobacterium expressing AtSGT1b variants or the GUS-FLAG control (OD600 = 0.5) were infiltrated into N. benthamiana leaves 1 day before infiltration with Agrobacterium expressing RPS2 (OD600 = 0.15). Leaf discs were taken 21 hpi for conductivity measurements at the indicated time points. The time points in the x-axis are indicated as hpi with Agrobacterium expressing RPS2 (mean ± SEM, n = 4, ** p<0.01, t-test, 3 replicates). (B, C) Observation of RPS2-triggered cell death by visible light (B) and UV light (C) in N. benthamiana. The Agrobacterium combinations were infiltrated as described in (A), and the cell death phenotype was recorded 4 dpi with Agrobacterium expressing RPS2. (D, E) A phospho-mimic mutation in AtSGT1b T346 (T346D) disrupts RPS2-SGT1b association in N. benthamiana. Agrobacterium combinations with different constructs were infiltrated in N. benthamiana leaves and luciferase activities were examined in both qualitative (CCD imaging machine, D) and quantitative assays (microplate luminescence reader, E). Quantification of the luciferase signal was performed with microplate luminescence reader (mean ± SEM, n = 8, ** p<0.01, t-test, 3 replicates). The nomenclature of the AtSGT1b mutants used is: 1A = S271A, 1D = S271D, 2A = T346A, 2D = T346D, AA = S271AT346A, DD = S271DT346D. The experiments were performed at least 3 times with similar results.
Fig 6.
RipAC interferes with MAPK-SGT1 interaction to suppress SGT1 phosphorylation.
(A) RipAC reduces SGT1 phosphorylation in Arabidopsis. SGT1 phosphorylation was determined in twelve-day-old Col-0 WT and RipAC transgenic Arabidopsis lines (AC #3 and AC #31) by western blot. Custom antibodies were used to detect the accumulation of SGT1 and the presence of a phosphorylated peptide containing AtSGT1 T346. (B) Quantification of SGT1 and pSGT1b signal normalized to actin and relative to Col-0 WT samples in (A) (mean ± SEM, n = 9, p-values are shown, t-test). (C) RipAC suppresses ETI-triggered SGT1 phosphorylation in Arabidopsis. ETI triggered MAPK activation and SGT1 phosphorylation were determined in twelve-day-old Dex::AvrRpt2/Col-0 or Dex::AvrRpt2/RipAC crossing F1 plants by western blot. Custom antibodies were used to detect the accumulation of SGT1 and the presence of a phosphorylated peptide containing AtSGT1 T346. The induction of AvrRpt2 was determined by RT-PCR. (D) Quantification of pSGT1b signal relative to coomassie blue staining control in (C) (mean ± SEM of 6 independent biological replicates, p-values are shown, t-test). (E) Inoculation of ΔripAC mutant on Col-0 wild-type plants showing increased SGT1 phosphorylation. Four to five-week-old Col-0 WT plant leaves were infiltrated with GMI1000 WT or ΔripAC mutant strain (OD600 = 0.02) and the samples were taken at the indicated time points. Custom antibodies were used to detect the phosphorylated peptide containing AtSGT1 T346 and endogenous SGT1. (F) Quantification of pSGT1b signal relative to Coomassie blue staining control in (E) (mean ± SEM of 3 independent biological repeats, p-values, compared to their control at 2 hpi, are shown, t-test). (G, H) Competitive Split-LUC assays showing that RipAC interferes with the interaction between MAPK3 (G) / MAPK4 (H) and AtSGT1b in Nicotiana benthamiana. In quantitative assays, the graphs show the mean value ± SEM (n = 8, ** p<0.01, t-test). (I) Competitive CoIP showing that RipAC interferes with the interaction between MAPK6 and AtSGT1b in N. benthamiana. Anti-FLAG beads were used to IP MAPK6. In all the competitive interaction assays, in addition to the interaction pair, RipAC or CBL-GFP (as negative control) were expressed to determine interference. In all the blots, asterisks indicate non-specific bands. These experiments were repeated at least 3 times with similar results. In western blot assays, protein marker sizes are provided for reference. Blots were stained with Coomassie Brilliant Blue (CBB) to verify equal loading.
Fig 7.
SGT1 phosphorylation contributes to resistance against R. solanacearum in Arabidopsis.
(A-D) Mutation of AtSGT1 increases the susceptibility to R. solanacearum in Arabidopsis. Soil-drenching inoculation assays in different Arabidopsis genotypes (AtSGT1a-1 and its wild-type control Ws-0, AtSGT1b-3 and its wild-type control La-er) were performed with GMI1000 WT strain. In (A, C) the results are represented as disease progression, showing the average wilting symptoms in a scale from 0 to 4. Values from 3 independent biological repeats were pooled together (mean ± SEM; n = 36 in (A) and n = 48 in (C)). Curves for each replicate are shown in S12A and S12B Fig. (B, D) Survival analysis of the data in (A, C); the disease scoring was transformed into binary data with the following criteria: a disease index lower than 2 was defined as ‘0’, while a disease index equal or higher than 2 was defined as ‘1’ for each specific time point. Statistical analysis was performed using a Log-rank (Mantel-Cox) test (n = 36 in (A) and n = 48 in (C)), and the corresponding p value is shown in the graph with the same colour as each curve. (E, F) Soil-drenching inoculation assays in Arabidopsis transgenic lines overexpressing AtSGT1b variants were performed with GMI1000 ΔripAC mutant. In (E) and (F) the analyses were performed the same as in (A) and (B) (n = 45 for each genotype), and the corresponding p value is shown in the graph in (F) with the same colour as each curve. Curves for each replicate are shown in S12C Fig.