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Detection and functional characterization of a 215 amino acid N-terminal extension in the Xanthomonas type III effector XopD.

Canonne J, Marino D, Noël LD, Arechaga I, Pichereaux C, Rossignol M, Roby D, Rivas S - PLoS ONE (2010)

Bottom Line: XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins.The full length XopD protein identified in this study (XopD(1-760)) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD(216-760)).The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire des Interactions Plantes Micro-organismes, UMR CNRS-INRA 2594/441, Castanet Tolosan, France.

ABSTRACT
During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD(1-760)) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD(216-760)). Furthermore, the N-terminal extension of XopD, which is absent in XopD(216-760), is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.

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In planta analysis of XopD1-760-mediated virulence functions.(A) Transactivation of the PR1 promoter after SA treatment in transient assays in N. benthamiana. Leaves were inoculated with A. tumefaciens carrying a 35S:PR1p-GUS fusion either alone (lanes 1, 2) or together with HA-tagged XopD216-760 (lane 3) or XopD1-760 (lane 4). 18 hours after agroinfiltration, leaves were mock-treated (white bar) or treated with 2 mM SA (grey bars). Fluorimetric GUS assays in leaf discs were performed 12 hours later. Mean values and SEM values were calculated from the results of four independent experiments, with four replicates per experiment. Statistical differences according to a Student's t test P value <0.05 are indicated by letters. MU, methylumbelliferone. (B) Western blot analysis showing expression of HA-tagged XopD216-760 and XopD1-760. Ponceau S staining illustrates equal loading. (C) Susceptible Pearson tomato leaves were inoculated with Xcv 85* or Xcv 85* ΔxopD, expressing an HA-tagged GUS control, Xcv 85* ΔxopD expressing HA-tagged XopD216-760 or Xcv 85* ΔxopD expressing HA-tagged XopD1-760. Inoculation was performed with bacterial suspensions of 1×105 cfu/ml. Representative symptoms observed 10 dpi are shown. Similar phenotypes were observed in four independent experiments. (D) Strains Xcv 85* expressing a GUS control (1) and 85* ΔxopD expressing either a GUS control (2), XopD216-760 (3) or XopD1-760 (4) were incubated in MOKA rich medium (total extract, left) or secretion medium (supernatant, right). Total protein extracts (10-fold concentrated) and TCA-precipitated filtered supernatants concentrated (200-fold concentrated) were analysed by immunoblotting using anti-HA antibodies (upper panel) to detect the presence of GUS, XopD216-760 and XopD1-760, or anti-GroEL antibodies (lower panel) to show that bacterial lysis had not occurred.
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pone-0015773-g006: In planta analysis of XopD1-760-mediated virulence functions.(A) Transactivation of the PR1 promoter after SA treatment in transient assays in N. benthamiana. Leaves were inoculated with A. tumefaciens carrying a 35S:PR1p-GUS fusion either alone (lanes 1, 2) or together with HA-tagged XopD216-760 (lane 3) or XopD1-760 (lane 4). 18 hours after agroinfiltration, leaves were mock-treated (white bar) or treated with 2 mM SA (grey bars). Fluorimetric GUS assays in leaf discs were performed 12 hours later. Mean values and SEM values were calculated from the results of four independent experiments, with four replicates per experiment. Statistical differences according to a Student's t test P value <0.05 are indicated by letters. MU, methylumbelliferone. (B) Western blot analysis showing expression of HA-tagged XopD216-760 and XopD1-760. Ponceau S staining illustrates equal loading. (C) Susceptible Pearson tomato leaves were inoculated with Xcv 85* or Xcv 85* ΔxopD, expressing an HA-tagged GUS control, Xcv 85* ΔxopD expressing HA-tagged XopD216-760 or Xcv 85* ΔxopD expressing HA-tagged XopD1-760. Inoculation was performed with bacterial suspensions of 1×105 cfu/ml. Representative symptoms observed 10 dpi are shown. Similar phenotypes were observed in four independent experiments. (D) Strains Xcv 85* expressing a GUS control (1) and 85* ΔxopD expressing either a GUS control (2), XopD216-760 (3) or XopD1-760 (4) were incubated in MOKA rich medium (total extract, left) or secretion medium (supernatant, right). Total protein extracts (10-fold concentrated) and TCA-precipitated filtered supernatants concentrated (200-fold concentrated) were analysed by immunoblotting using anti-HA antibodies (upper panel) to detect the presence of GUS, XopD216-760 and XopD1-760, or anti-GroEL antibodies (lower panel) to show that bacterial lysis had not occurred.

Mentions: We next investigated whether the newly identified N-terminal protein extension in XopD may have an effect on its function in planta. First, a previous report showed that Agrobacterium-mediated transient expression of XopD216-760 in N. benthamiana prevents the induction of the expression of the PR1 promoter (PR1p) fused to the GUS reporter gene after salicylic acid (SA) treatment [46]. Consistent with previous results, SA treatment induced PR1p transcriptional activation whereas, in the presence of XopD216-760, PR1p activation was significantly reduced (Figure 6A). Interestingly, co-expression of XopD1-760 in these assays led to a stronger repression of PR1p transcriptional activation, suggesting that the N-terminal extension of XopD is necessary to modulate XopD function in the host (Figure 6A).


Detection and functional characterization of a 215 amino acid N-terminal extension in the Xanthomonas type III effector XopD.

Canonne J, Marino D, Noël LD, Arechaga I, Pichereaux C, Rossignol M, Roby D, Rivas S - PLoS ONE (2010)

In planta analysis of XopD1-760-mediated virulence functions.(A) Transactivation of the PR1 promoter after SA treatment in transient assays in N. benthamiana. Leaves were inoculated with A. tumefaciens carrying a 35S:PR1p-GUS fusion either alone (lanes 1, 2) or together with HA-tagged XopD216-760 (lane 3) or XopD1-760 (lane 4). 18 hours after agroinfiltration, leaves were mock-treated (white bar) or treated with 2 mM SA (grey bars). Fluorimetric GUS assays in leaf discs were performed 12 hours later. Mean values and SEM values were calculated from the results of four independent experiments, with four replicates per experiment. Statistical differences according to a Student's t test P value <0.05 are indicated by letters. MU, methylumbelliferone. (B) Western blot analysis showing expression of HA-tagged XopD216-760 and XopD1-760. Ponceau S staining illustrates equal loading. (C) Susceptible Pearson tomato leaves were inoculated with Xcv 85* or Xcv 85* ΔxopD, expressing an HA-tagged GUS control, Xcv 85* ΔxopD expressing HA-tagged XopD216-760 or Xcv 85* ΔxopD expressing HA-tagged XopD1-760. Inoculation was performed with bacterial suspensions of 1×105 cfu/ml. Representative symptoms observed 10 dpi are shown. Similar phenotypes were observed in four independent experiments. (D) Strains Xcv 85* expressing a GUS control (1) and 85* ΔxopD expressing either a GUS control (2), XopD216-760 (3) or XopD1-760 (4) were incubated in MOKA rich medium (total extract, left) or secretion medium (supernatant, right). Total protein extracts (10-fold concentrated) and TCA-precipitated filtered supernatants concentrated (200-fold concentrated) were analysed by immunoblotting using anti-HA antibodies (upper panel) to detect the presence of GUS, XopD216-760 and XopD1-760, or anti-GroEL antibodies (lower panel) to show that bacterial lysis had not occurred.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008746&req=5

pone-0015773-g006: In planta analysis of XopD1-760-mediated virulence functions.(A) Transactivation of the PR1 promoter after SA treatment in transient assays in N. benthamiana. Leaves were inoculated with A. tumefaciens carrying a 35S:PR1p-GUS fusion either alone (lanes 1, 2) or together with HA-tagged XopD216-760 (lane 3) or XopD1-760 (lane 4). 18 hours after agroinfiltration, leaves were mock-treated (white bar) or treated with 2 mM SA (grey bars). Fluorimetric GUS assays in leaf discs were performed 12 hours later. Mean values and SEM values were calculated from the results of four independent experiments, with four replicates per experiment. Statistical differences according to a Student's t test P value <0.05 are indicated by letters. MU, methylumbelliferone. (B) Western blot analysis showing expression of HA-tagged XopD216-760 and XopD1-760. Ponceau S staining illustrates equal loading. (C) Susceptible Pearson tomato leaves were inoculated with Xcv 85* or Xcv 85* ΔxopD, expressing an HA-tagged GUS control, Xcv 85* ΔxopD expressing HA-tagged XopD216-760 or Xcv 85* ΔxopD expressing HA-tagged XopD1-760. Inoculation was performed with bacterial suspensions of 1×105 cfu/ml. Representative symptoms observed 10 dpi are shown. Similar phenotypes were observed in four independent experiments. (D) Strains Xcv 85* expressing a GUS control (1) and 85* ΔxopD expressing either a GUS control (2), XopD216-760 (3) or XopD1-760 (4) were incubated in MOKA rich medium (total extract, left) or secretion medium (supernatant, right). Total protein extracts (10-fold concentrated) and TCA-precipitated filtered supernatants concentrated (200-fold concentrated) were analysed by immunoblotting using anti-HA antibodies (upper panel) to detect the presence of GUS, XopD216-760 and XopD1-760, or anti-GroEL antibodies (lower panel) to show that bacterial lysis had not occurred.
Mentions: We next investigated whether the newly identified N-terminal protein extension in XopD may have an effect on its function in planta. First, a previous report showed that Agrobacterium-mediated transient expression of XopD216-760 in N. benthamiana prevents the induction of the expression of the PR1 promoter (PR1p) fused to the GUS reporter gene after salicylic acid (SA) treatment [46]. Consistent with previous results, SA treatment induced PR1p transcriptional activation whereas, in the presence of XopD216-760, PR1p activation was significantly reduced (Figure 6A). Interestingly, co-expression of XopD1-760 in these assays led to a stronger repression of PR1p transcriptional activation, suggesting that the N-terminal extension of XopD is necessary to modulate XopD function in the host (Figure 6A).

Bottom Line: XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins.The full length XopD protein identified in this study (XopD(1-760)) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD(216-760)).The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire des Interactions Plantes Micro-organismes, UMR CNRS-INRA 2594/441, Castanet Tolosan, France.

ABSTRACT
During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD(1-760)) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD(216-760)). Furthermore, the N-terminal extension of XopD, which is absent in XopD(216-760), is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.

Show MeSH
Related in: MedlinePlus