Fig 1.
RipE1 undergoes phosphorylation in N. benthamiana, which contributes to protein stability.
(A) Schematic representation of RipE1 protein, indicating the position of phosphorylation sites, domain A, and catalytic sites. (B) Western blot showing protein accumulation of GFP, RipE1-GFP, and RipE1 phosphodeficient mutants. Agrobacterium expressing GFP (as control), RipE1-GFP, 3A-GFP, or 5A-GFP (OD600 = 0.5) were infiltrated into the same leaf of N. benthamiana. Samples were taken at 30 hours post-infiltration (hpi), before the appearance of cell death. Blots were incubated with anti-Actin antibody to verify equal loading. This experiment was repeated 4 times, and the quantitation of the different repeats is shown in (C). (C) Quantification of the relative protein accumulation of the different repeats of the assay shown in (B), measured using Image J. RipE1 values were normalized using the respective actin values and represented as relative to RipE1 (WT) for each repeat. Values indicate mean ± SE (n = 4 biological replicates). Different letters indicate significant differences (one-way ANOVA, Tukey’s test, p < 0.05). (D) Quantitative RT–PCR (qRT–PCR) to determine the expression of RipE1 in (B). Expression values are relative to the expression of the housekeeping gene NbEF1a. Values indicate mean ± SE (n = 9 biological replicates). Different letters indicate significant differences (one-way ANOVA, Tukey’s test, p < 0.05). Composite data from 3 independent biological replicates. Nd: not detected. (E) Western blot showing protein accumulation of GFP, RipE1-GFP, 5D-GFP and 5A-GFP. This experiment was performed as in (B), repeated 4 times, and the quantitation of the different repeats is shown in (F). (F) Quantification of the relative protein accumulation of the different repeats of the assay shown in (E), measured and represented as in (C). (G) Quantitative RT–PCR (qRT–PCR) to determine the expression of RipE1 in (E), performed and represented as in (D). (H) Immunoprecipitation assays to determine the ubiquitination status of RipE1 and phosphodeficient mutants. Samples were collected 30 hpi, before the appearance of cell death. Anti-GFP beads were used for immunoprecipitation. An anti-ubiquitin (P4D1) antibody was used to detect ubiquitinated proteins. Protein marker sizes are shown for reference. This experiment was repeated 3 times, and the quantitation of the different repeats is shown in (I). A similar assay where the sample loading was adjusted to show similar accumulation of all RipE1 variants for comparison of their ubiquitination is shown in S2A Fig. (I) Quantification of the relative protein ubiquitination of the different repeats of the assay shown in (H), measured using Image J. Ubiquitination values were normalized using the respective protein accumulation and represented as relative to the GFP control for each repeat. Values indicate mean ± SE (n = 3 biological replicates). Different letters indicate significant differences (one-way ANOVA, Tukey’s test, p < 0.05).
Fig 2.
RipE1 interacts with NbUCH12 and NbUCH15.
(A) Selected RipE1-GFP interactors identified by immunoprecipitation followed by LC-MS/MS. The table includes protein ID, peptide counts in GFP and RipE1-GFP samples, closest Arabidopsis orthologs, and the protein names used in this work. (B) Co-immunoprecipitation assays to determine interactions between RipE1 (WT or C172A mutant) and NbUCH12/15. Agrobacterium containing the indicated constructs were infiltrated in N. benthamiana leaves and samples were taken 2 days post-infiltration (dpi). Immunoblots were analyzed using anti-GFP and anti-Flag antibodies, and protein marker sizes are provided for reference. These experiments were repeated 3 times with similar results. (C) Interaction between RipE1-RFP and NbUCH15-GFP determined using FRET-FLIM upon transient co-expression in N. benthamiana leaves. Free RFP was used as a negative control. Fluorescence was visualized 30-36 hpi. Lines represent average values (n = 33), and error bars represent standard error. Asterisks indicate significant differences with the RFP control according to a Student’s t-test (**p < 0.01). Composite data from 3 independent biological replicates.
Fig 3.
Silencing NbUCHs triggers SGT1-dependent immunity in N. benthamiana.
NbUCH genes were silenced using RNAi in N. benthamiana leaves. An RNAi construct carrying the appropriate gene fragments (to silence each NbUCH gene) or an empty vector (as control) was expressed in the same leaf side-by-side using Agrobacterium (OD 600 = 0.5). (A) Tissue collapse, indicative of cell death, triggered by silencing NbUCH genes. Photographs were taken at the indicated days post-infiltration (dpi) using a CCD camera. The infiltrated areas are delimited using dotted lines. (B) Graph showing sample conductivity, indicating ion leakage from plant tissues caused by cell death. Leaf discs were collected 8 dpi , and ion leakage was measured at the indicated times after sampling. Values indicate mean ± SE (n = 3 biological replicates). (C) Quantitative RT-PCR to determine the expression of the defense-related gene NbPR1. Samples were taken 8 dpi . Expression values are relative to the expression of the housekeeping gene NbEF1a. Values indicate mean ± SE (n = 3 biological replicates). (D) Growth of Ralstonia solanacearum Y45 in N. benthamiana. R. solanancearum was inoculated into N. benthamiana leaves after silencing NbUCH15 for 8 days, before the appearance of cell death. Leaf discs were collected 2 dpi for bacterial quantification. Values indicate mean ± SEM (n = 18 biological replicates from 3 independent repeats; each color corresponds to values from an independent replicate. Asterisks indicate significant differences compared to control according to a Student’s t test (**** p < 0.0001). (E) RNAi constructs for silencing NbUCHs or an empty vector (as control) were expressed in N. benthamiana undergoing VIGS of NbSGT1 or VIGS using an empty vector ( EV ) construct (as control). Tissue collapse, indicative of cell death, was recorded 16 dpi using a CCD camera. The infiltrated areas are delimited using dotted lines. Each experiment was repeat at least three times with similar results.
Fig 4.
Immunity triggered by silencing NbUCH does not require NbPtr1.
Agrobacterium expressing an empty vector or a construct to induce virus-induced gene silencing (VIGS) of NbPtr1 were infiltrated in N. benthamiana leaves. (A) Nine days after infiltration to induce VIGS of NbPtr1, Agrobacterium expressing RipE1 or a GFP control were infiltrated into N. benthamiana leaves. Tissue collapse, indicative of cell death, triggered by RipE1 expression was recorded 2 dpi. The infiltrated areas are delimited using dotted lines. Upper photographs were taken using a CCD camera from the adaxial side of the leaves, and lower pictures were captured using a UV camera from the abaxial side of the leaves and were flipped horizontally for representation. (B and C) Nine days after infiltration to induce VIGS of NbPtr1, RNAi constructs for silencing NbUCHs or an empty vector (as control) were expressed in N. benthamiana leaves. (B) Tissue collapse, indicative of cell death, was recorded 12 dpi using a CCD camera. The infiltrated areas are delimited using dotted lines. Tissue collapse triggered by RipE1 (4 dpi) was used as control. (C) Quantitative RT-PCR to determine the expression of the defense-related gene NbPR1. Samples were taken 8 dpi. Expression values are relative to the expression of the housekeeping gene NbEF1a. Values indicate mean ±SE (n = 3 biological replicates).
Fig 5.
NbUCH positive regulates RipE1 accumulation in N. benthamiana.
(A and B) NbUCH genes were silenced using RNAi in N. benthamiana leaves. An RNAi construct carrying the appropriate gene fragments (to silence each NbUCH gene) or an empty vector (as control) was expressed in the same leaf side-by-side using Agrobacterium (OD600=0.5). One day later, RipE1-GFP or RipAA-FLAG (as control) were expressed in same leaf using Agrobacterium (OD600=0.1). (A) Western blot showing the accumulation of RipE1-GFP and RipAA-FLAG protein accumulation. Blots were incubated with anti-Actin antibody to verify equal loading. Protein marker sizes are shown for reference. This experiment was repeated 3 times, and the quantitation of the different repeats is shown in (B). (B) Quantification of the relative protein accumulation of the different repeats of the assay shown in (A), measured using Image J. RipE1 or RipAA values were normalized using the respective actin values and represented as relative to their respective empty vector control for each repeat. Values indicate mean ± SE (n = 3 biological replicates). Asterisks indicate significant differences compared to each control according to a Student’s t test (* p < 0.05, ** p < 0.01). (C) Western blot to determine RipE1 protein accumulation after expression of NbUCH15 or Flag-RFP (as control). Agrobacterium expressing RipE1 was infiltrated 24 hours after expression of Flag-RFP (as control) or NbUCH15-Flag side-by-side in the same N. benthamiana leaf. Blots were incubated with anti-Actin antibody to verify equal loading. Protein marker sizes are shown for reference. This experiment was repeated 12 times, and the quantitation of the different repeats is shown in (D). (D) Quantification of the relative protein accumulation of the different repeats of the assay shown in (C), measured using Image J. RipE1 values were normalized using the respective actin values and represented as relative to the control expressing Flag-RFP for each repeat. Values indicate mean ± SE (n = 12 biological replicates). Asterisks indicate significant differences compared to each control according to a Student’s t test (** p < 0.01).
Fig 6.
NbUCH15 promotes RipE1 stability by deubiquitination.
(A and C) Immunoprecipitation assays to determine the ubiquitination status of RipE1. (A) Agrobacterium expressing RipE1-GFP was infiltrated 1 day after RNAi-mediated silencing of NbUCH15. An empty vector was used as RNAi negative control. Samples were collected 30 hpi, before the appearance of cell death. Given that NbUCH15 silencing compromises RipE1 protein accumulation, different volumes of the protein samples were loaded to show a comparable RipE1-GFP protein accumulation between different lanes after immunoprecipitation, allowing the detection of ubiquitination in the same amount of RipE1 protein. The relative loading volumes and protein abundance are indicated above lanes. A similar assay showing the loading of the same sample volumes (with different RipE1 accumulation) is shown in S6A Fig. Anti-GFP beads were used for immunoprecipitation. An anti-ubiquitin (P4D1) antibody was used to detect ubiquitinated proteins. The accumulation of native NbUCH proteins was detected using a custom anti-NbUCH antibody and the same volume of each sample. Protein marker sizes are shown for reference. This experiment was repeated 3 times, and the quantitation of the different repeats is shown in (B). (B) Quantification of the relative RipE1 ubiquitination of the different repeats of the assay shown in (A), measured using Image J. Ubiquitination values were normalized using the respective protein accumulation and represented as relative to the empty vector control for each repeat. Values indicate mean ± SE (n = 3 biological replicates). The p value compared to the control value using a Student’s t test is shown. (C) Agrobacterium expressing RipE1-GFP was infiltrated 1 day after expression of NbUCH15-Flag or Flag-RFP (as control). Samples were collected 30 hpi, before the appearance of cell death. Given that NbUCH15 overexpression enhances RipE1 protein accumulation, different volumes of the protein samples were loaded to show a comparable RipE1-GFP protein accumulation between different lanes after immunoprecipitation, allowing the detection of ubiquitination in the same amount of RipE1 protein. The relative loading volumes and protein abundance are indicated above lanes. Anti-GFP beads were used for immunoprecipitation. An anti-ubiquitin (P4D1) antibody was used to detect ubiquitinated proteins. Protein marker sizes are shown for reference. This experiment was repeated 5 times, and the quantitation of the different repeats is shown in (D). (D) Quantification of the relative RipE1 ubiquitination of the different repeats of the assay shown in (C), measured using Image J. Ubiquitination values were normalized using the respective protein accumulation and represented as relative to the control expressing Flag-RFP for each repeat. Values indicate mean ± SE (n = 5 biological replicates). Asterisks indicate significant differences compared to the control according to a Student’s t test (* p < 0.05).
Fig 7.
RipE1 phosphorylation and NbUCH-mediated deubiquitination are independent mechanisms leading to RipE1 stability.
(A) Co-immunoprecipitation assay to analyse the interaction between RipE1-5A and NbUCH15. Agrobacterium containing the indicated constructs were infiltrated in N. benthamiana leaves. In order to avoid stability issues with the RipE1-5A mutant, an estradiol (EST)-inducible vector was used, and samples were treated with 100 μM EST for 2.5 hours before being harvested at 2.5 dpi. Immunoblots were analyzed using anti-GFP and anti-Flag antibodies, and protein marker sizes are provided for reference. These experiments were repeated 3 times with similar results. (B) Western blot to determine the accumulation of the RipE1-5A mutant after expression of NbUCH15 or Flag-RFP (as control). Agrobacterium expressing the RipE1 versions were infiltrated 24 hours after expression of Flag-RFP (as control) or NbUCH15-Flag side-by-side in the same N. benthamiana leaf. Blots were incubated with anti-Actin antibody to verify equal loading. Protein marker sizes are shown for reference. This experiment was repeated 9 times, and the quantitation of the different repeats is shown in (C). (C) Quantification of the relative protein accumulation of the different repeats of the assay shown in (B), measured using Image J. RipE1 values were normalized using the respective actin values and represented as relative to the control expressing Flag-RFP for each repeat. Values indicate mean ± SE (n = 9 biological replicates). Asterisks indicate significant differences compared to the control according to a Student’s t test (**** p < 0.0001). (D) Agrobacterium expressing RipE1-5A-GFP was infiltrated 1 day after expression of NbUCH15-Flag or Flag-RFP (as control). Samples were collected 30 hpi, before the appearance of cell death. Given that NbUCH15 overexpression enhances RipE1 protein accumulation, different volumes of the protein samples were loaded to show a comparable RipE1-GFP protein accumulation between different lanes after immunoprecipitation, allowing the detection of ubiquitination in the same amount of RipE1 protein. The relative loading volumes and protein abundance are indicated above lanes. Anti-GFP beads were used for immunoprecipitation. An anti-ubiquitin (P4D1) antibody was used to detect ubiquitinated proteins. Protein marker sizes are shown for reference. This experiment was repeated 4 times, and the quantitation of the different repeats is shown in (E). (E) Quantification of the relative RipE1 ubiquitination of the different repeats of the assay shown in (D), measured using Image J. Ubiquitination values were normalized using the respective protein accumulation and represented as relative to the control expressing Flag-RFP for each repeat. Values indicate mean ± SE (n = 4 biological replicates). Asterisks indicate significant differences compared to the control according to a Student’s t test (**** p < 0.0001).
Fig 8.
Simplified diagram showing a schematical model of RipE1 protein stability and activity in plant cells.
Upon injection inside plant cells, RipE1 is subjected to ubiquitination and subsequent degradation. RipE1 phosphorylation and UCH-mediated deubiquitination contribute to RipE1 stability. In susceptible hosts, phosphorylated stable RipE1 exerts its virulence activity through the association with virulence target(s). In resistant hosts, RipE1 activity is perceived by the presence of NbPtr1, leading to the activation of immune responses and disease resistance.