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. 2013;9(6):e1003427.
doi: 10.1371/journal.ppat.1003427. Epub 2013 Jun 13.

The Xanthomonas campestris type III effector XopJ targets the host cell proteasome to suppress salicylic-acid mediated plant defence

Suayib Üstün  1 , Verena Bartetzko, Frederik Börnke
Affiliations

The Xanthomonas campestris type III effector XopJ targets the host cell proteasome to suppress salicylic-acid mediated plant defence

Suayib Üstün et al. PLoS Pathog. 2013.

Abstract

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) requires type III effector proteins (T3Es) for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA) and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Interaction of XopJ with RPT6 in Yeast Two-Hybrid Assays.
XopJ fused to the GAL4 DNA binding domain (BD) was expressed in combination with RPT6 fused to the GAL4 activation domain (AD) in yeast strain Y190. Cells were grown on selective media before a LacZ filter assay was performed. pSV40/p53 served as positive control while the empty AD vector served as negative control. NtRPT6, N. tabacum RPT6; AtRPT6a, A. thaliana RPT6 isoform a; AtRPT6b, A. thaliana RPT6 isoform b; ScRPT6, S. cerevisiae RPT6. – LT, yeast growth on medium without Leu and Trp. – HLT, yeast growth on medium lacking His, Leu, and Trp, indicating expression of the HIS3 reporter gene. LacZ, activity of the lacZ reporter gene.
Figure 2
Figure 2. Subcellular localization of NtRPT6 and XopJ – NtRPT6 interaction in planta.
(A) Subcellular localization of NtRPT6-GFP and XopJ-mCherry fusions in N. benthamiana leaves transiently transformed by Agro-infiltration. The green fluorescence (GFP), red fluorescence (mCherry) and chlorophyll autofluorescence (Chl) were monitored separately to prevent cross-talk of the fluorescence channels and the resulting fluorescence images were merged. Bars = 20 µm. Pictures show a representative result of at least three repetitions. (B) Visualization of protein interactions in planta by the BiFC assay. YFP confocal microscopy images show tobacco leaf epidermal cells transiently expressing constructs encoding the fusion proteins indicated. Merge indicates an overlay of the YFP and chlorophyll autofluorescence images. A close up of the same cells shows that the YFP fluorescence aligns with the PM. Bars = 20 µm. The experiment has been repeated three times with similar results. (C) Co-immunoprecipitation of NtRPT6-GFP with XopJ-myc and XopJ(C235A)-myc. NtRP6-GFP was transiently co-expressed in leaves of N. benthamiana using Agro-infiltration with either XopJ-myc or XopJ(C235A)-myc. After 48 h, total proteins (Input) were subjected to immunoprecipitation (Eluate) with GFP-Trap beads followed by immunoblot analysis using either anti-GFP or anti-myc antibodies. At least three repetitions with similar result have been conducted. (D) In vitro pull-down assay showing physical interaction of XopJ with RPT6. MBP-XopJ and GST-Rpt6 were expressed in E. coli. Pull down was performed using amylose resin. Proteins were detected in an immunoblot using antibodies as indicated.
Figure 3
Figure 3. Transient expression of XopJ in N. benthamiana leaves inhibits proteasome activity.
(A) Upper panel: Proteasome activity in N. benthamiana leaves transiently expressing XopJ-myc proteins. XopJ protein variants along with an empty vector (EV) control were transiently expressed in leaves of N. benthamiana using Agro-infiltration. After 48 h, relative proteasome activity in total protein extracts was determined by monitoring the breakdown of the fluorogenic peptide Suc-LLVY-AMC at 30°C in a fluorescence spectrophotometer. The empty vector (EV) control was set to 100%. Data represent the mean SD (n = 3). The experiment has been repeated more than three times with similar results. Lower panel: immunodetection of transiently expressed XopJ variants in the same leaves that were used for proteasome activity measurements. After immunodetection of proteins the membrane was stained with amido black to control for equal protein loading. (B) Upper panel: Proteasome activity in the presence of cysteine protease inhibitor E64 in N. benthamiana leaves transiently expressing XopJ-myc. XopJ protein along with an empty vector (EV) control was transiently expressed in leaves of N. benthamiana using Agro-infiltration. After 48 h, relative proteasome activity was determined by monitoring the breakdown of the fluorogenic peptide Suc-LLVY-NH-AMC at 30°C in a fluorescence spectrophotometer. Plant extracts were incubated with water or 100 µM E64 for 15 min at 30°C before measurements. The empty vector (EV) control was set to 100%. Data represent the mean SD (n = 4). Significant differences were calculated using Student's t-test and are indicated by: **, P<0.01; *** P<0.001. Lower panel: Proteasome inhibition by MG132. XopJ and empty vector (EV) control were transiently expressed in leaves of N. benthamiana using Agro-infiltration. After 48 h, relative proteasome activity was determined by monitoring the breakdown of the fluorogenic peptide Suc-LLVY-NH-AMC at 30°C in a fluorescence spectrophotometer. Plant extracts were incubated with 1% EtOH or 50 µM MG132 for 15 min at 30°C before measurements. The empty vector (EV) control was set to 100%. Data represent the mean SD (n = 3). (C) Distribution of ubiquitin conjugates in N. benthamiana leaves transiently expressing XopJ-myc proteins. Total proteins were extracted 48 h after infiltration with Agrobacteria harbouring the respective XopJ expression constructs. Ubiquinated proteins were detected using an anti-ubiquitin antibody. Expression of the XopJ variants was verified using an anti myc-antibody. After immunodetection of proteins the membrane was stained with amido black to control for equal protein loading.
Figure 4
Figure 4. XopJ contributes to bacterial growth in pepper at late stages of infection and dampens proteasome activity in infected pepper leaves.
(A) Growth of Xcv 85-10 (vector), Xcv ΔxopJ, Xcv ΔxopJ (XopJ), strains in pepper ECW leaves. Leaves were hand-infiltrated with a 105 cells/ml suspension of bacteria. The number of bacteria in each leaf was quantified at 0, 4, 6, 8 dpi. Data represent the mean SD (n = 3). Significant differences were calculated using Student's t-test and are indicated by: *, P<0.05; **, P<0.01. The experiment was repeated three times with almost identical results. A representative result is shown. (B) XopJ reduces proteasome activity during Xcv-pepper interaction. Leaves were infiltrated with strains indicated in the figure. After 3 dpi proteasome activity in total leaf extracts was determined by monitoring the breakdown of the fluorogenic peptide suc-LLVY-NH-AMC at 30°C in a fluorescence spectrophotometer. Data represent the mean SD (n = 4). Significant differences were calculated using Student's t-test and are indicated by: *, P<0.05; **, P<0.01. A representative result of more than three repetitions with independent sets of plants is shown.
Figure 5
Figure 5. XopJ suppresses cell death during the compatible Xcv-pepper interaction.
(A) Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA), XopJ (G2A-HA) and (C235A-HA) were inoculated at a bacterial density of 2×108 cfu ml−1 into leaves of pepper ECW plants. Leaf phenotype was photographed at 3 d post infection (dpi). (B) Protein extracts from pepper leaves infiltrated with 1 mM MgCl2, Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA), XopJ (G2A-HA) and (C235A-HA) at 3 dpi were prepared. Equal volumes representing approximately equal protein amounts of each extract were immunoblotted and proteins were detected using anti-HA antiserum. (C) Trypan blue staining of infected tissue of ECW plants reveals reduced cell death in the presence of functional XopJ. Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA), XopJ (G2A-HA) and (C235A-HA) were inoculated at a bacterial density of 2×108 cfu ml−1 into leaves of pepper ECW plants. Samples of infected and untreated leaves were taken 3 dpi and stained with trypan blue. Dead plant cells stain blue. Grey and black spots represent calcium oxalate crystals. (D) Summary of observed phenotypes. 20 pepper plants were infected with the different Xcv strains indicated in this figure and then scored for phenotype development at 3 dpi. (E) Delivery of XopJ by Xcv leads to reduced ion leakage in pepper. Ion leakage was measured in pepper plants infected with. Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA), XopJ (G2A-HA) and (C235A-HA). Conductivity was measured at the time points indicated. Data represent the mean SD (n = 3). Significant differences are indicated by asterisks (* P<0.05; ** P<0.01; *** P<0.001) and were calculated using Student's t-test.
Figure 6
Figure 6. The proteasome inhibitor MG132 prevents development of necrosis in Xcv ΔxopJ infected pepper leaves.
(A) Xcv ΔxopJ with 100 µM MG132 or 1% EtOH (control) were inoculated at a bacterial density of 2×108 cfu ml−1 into leaves of pepper ECW plants. Plant reactions were photographed at 3 d post infection (dpi). (B) MG132 treatment leads to a reduced ion leakage in pepper in the absence of XopJ. Ion leakage was measured in pepper plants infected with Xcv, Xcv ΔxopJ with MG132 or 1% EtOH. Conductivity was measured at the time point indicated. Data represent the mean SD (n = 3). Significant differences are indicated by asterisks (*** P<0.001) and were calculated using Student's t-test.
Figure 7
Figure 7. XopJ reduces levels of free and conjugated SA in leaves of Xcv infected pepper plants.
Susceptible pepper ECW leaves were hand-inoculated with MgCl2 or a 2×108 cfu/mL suspension of Xcv (vector), Xcv ΔxopJ (vector) and Xcv ΔxopJ (XopJ). Free SA and total SA (free SA+SAG) levels in infected tissue were measured 2 and 3 dpi. Data represent the mean SD (n = 3). Significant differences are indicated by asterisks (* P<0.05) and were calculated using Student's t-test. FW, Fresh weight.
Figure 8
Figure 8. XopJ alters the mRNA abundance of senescence- and SA-dependent genes.
Total RNA was isolated from pepper leaves infiltrated with 2×108 cfu/mL of Xcv or Xcv ΔxopJ, respectively. Quantitative real-time RT-PCR was performed to monitor mRNA levels. (A) SA-dependent-upregulated genes. (B) senescence-downregulated genes (CaSGR and CaCab-1B) and senescence-upregulated genes (CaSENU4). In both panels relative expression levels at 3 dpi are shown. Actin expression was used to normalize the expression value in each sample, and relative expression values were determined against the average value of the sample infected with wild- type Xcv. Leaf material from 4 independent pepper plants was pooled and analyzed in triplicates. Data represent the mean SD. Significant differences were calculated using Student's t-test and are indicated by: *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 9
Figure 9. SA treatment induces proteasome activity and RPT6 gene expression in N. benthamiana and pepper ECW plants.
(A) Pepper and N. benthamiana leaves were treated with 5 mM SA. Total RNA was isolated at 0 (untreated), 1, 3, 6 hour after SA application. Q-PCR was performed to monitor CaRPT6 and NbRPT6 mRNA levels. Relative expression levels at time points indicated are shown. Actin expression was used to normalize the expression value in each sample, and relative expression values were determined against the average value of the untreated sample. Data represent the mean SD (n = 4). Significant differences were calculated using Student's t-test and are indicated by: *, P<0.05; **, P<0.01; ***, P<0.001. (B) Pepper and N. benthamiana leaves were sprayed with 5 mM SA and proteasome activity in total leaf extracts was determined at time points indicated in the figure by monitoring the breakdown of the fluorogenic peptide sLLVY-NH-Mec at 30°C in a fluorescence spectrophotometer. Data represent the mean SD (n = 3) Significant differences are indicated by asterisks (* P<0.05; *** P<0.001) and were calculated using Student's t-test.
Figure 10
Figure 10. VIGS of NPR1 interferes with XopJ-dependent phenotypes in pepper.
(A) qRT-PCR analysis of NPR1 mRNA level in NPR1 silenced pepper plants. Relative expression levels at 21 dpi are shown. Actin expression was used to normalize the expression value in each sample, and relative expression values were determined against pTRV2-GFPsil plants. (B) Xcv (vector), Xcv ΔxopJ (vector), Xcv ΔxopJ (XopJ-HA) were infiltrated at a bacterial density of 2×108 cfu ml−1 into leaves of pTRV2-GFPsil and pTRV2-NPR1 plants. Plant reactions were photographed at 3 d post infection (dpi). The number of leaves showing necrosis is indicated below the appropriate construct. (C) Leaves were infiltrated with strains indicated in the figure. At 3 dpi proteasome activity in total leaf extracts was determined by monitoring the breakdown of the fluorogenic peptide suc-LLVY-NH-AMC at 30°C in a fluorescence spectrophotometer. Data represent the mean SD (n = 3). Significant differences were calculated using Student's t-test and are indicated by: *, P<0.05.
Figure 11
Figure 11. Inhibition of the proteasome by MG132 affects basal defence responses.
MG132 treatment blocks secretion and leads to the accumulation of secreted green fluorescent protein (secGFP) within a cytosolic reticulum. Confocal images of N. benthamiana leaf epidermis cells transiently expressing (A) secGFP alone (1% EtOH) and (B)/(C) together with Sp2/XopJ. (D) secGFP- expressing leaf infiltrated with MG132 (300 µM) (E) or 100 µM MG132. Pictures in (D) and (E) were taken 2 h after infiltration. Bars = 20 µm. (F) Plants were co-infiltrated with 1 µM flg22+1% EtOH or 100 µM MG132. Leaf tissue was collected 6 hours after treatment and stained for callose. (G) Quantification of callose depositions per field of view. Data represent the mean SD (n = 4). Significant differences are indicated by asterisks (** P<0.01) and were calculated using Student's t-test.
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