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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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Characterization of XopD1-760 expression and function in N. benthamiana.(A) Confocal images of epidermal cells of N. benthamiana leaves expressing YFPv-tagged XopD216-760 and XopD1-760 36 hours after agroinfiltration. Bright field images are shown on the right. Bars  = 15 µm. (B) Western blot analysis using an anti-GFP antibody shows expression of YFPv-tagged XopD216-760 and XopD1-760 constructs, 36 hours after agroinfiltration. Ponceau S staining of the membrane illustrates equal loading. (C) Cell death development 4 (upper panel) and 7 (lower panel) days after agroinfiltration of YFPv-tagged XopD216-760 (left) and XopD1-760 (right) constructs. (D) Cell death was quantified by measuring electrolyte leakage in N. benthamiana leaves expressing YFPv-tagged XopD216-760 (open circles) and XopD1-760 (filled circles) at the indicated time points after agroinfiltration. Mean and SEM values are calculated from 3 independent experiments (8 replicates/experiment). The statistical significance in mean conductivity values obtained with leaves expressing XopD216-760 or XopD1-760 was assessed by using a Student's t test (P value <10−5). (E) SUMO-conjugates detected by Western blot analysis using an anti-HA antibody 36 hours after agroinfiltration of the indicated constructs. Expression of XopD proteins was revealed using an anti-GFP antibody. Ponceau S staining of the membrane illustrates equal loading.
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pone-0015773-g005: Characterization of XopD1-760 expression and function in N. benthamiana.(A) Confocal images of epidermal cells of N. benthamiana leaves expressing YFPv-tagged XopD216-760 and XopD1-760 36 hours after agroinfiltration. Bright field images are shown on the right. Bars  = 15 µm. (B) Western blot analysis using an anti-GFP antibody shows expression of YFPv-tagged XopD216-760 and XopD1-760 constructs, 36 hours after agroinfiltration. Ponceau S staining of the membrane illustrates equal loading. (C) Cell death development 4 (upper panel) and 7 (lower panel) days after agroinfiltration of YFPv-tagged XopD216-760 (left) and XopD1-760 (right) constructs. (D) Cell death was quantified by measuring electrolyte leakage in N. benthamiana leaves expressing YFPv-tagged XopD216-760 (open circles) and XopD1-760 (filled circles) at the indicated time points after agroinfiltration. Mean and SEM values are calculated from 3 independent experiments (8 replicates/experiment). The statistical significance in mean conductivity values obtained with leaves expressing XopD216-760 or XopD1-760 was assessed by using a Student's t test (P value <10−5). (E) SUMO-conjugates detected by Western blot analysis using an anti-HA antibody 36 hours after agroinfiltration of the indicated constructs. Expression of XopD proteins was revealed using an anti-GFP antibody. Ponceau S staining of the membrane illustrates equal loading.

Mentions: The N-terminal domain of XopD216-760 was previously shown to be required for targeting the effector to subnuclear foci [43]. To determine the effect of the newly identified N-terminal extension of XopD on its subcellular localization in planta, XopD1-760 was fused to the Yellow Fluorescent Protein venus (YFPv) and transiently expressed, under the control of the 35S promoter, in Nicotiana benthamiana leaf epidermal cells. As XopD216-760, XopD1-760 was also localized in subnuclear foci (Figure 5A). Both proteins were detected by Western blot using an anti-GFP antibody (Figure 5B).


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)

Characterization of XopD1-760 expression and function in N. benthamiana.(A) Confocal images of epidermal cells of N. benthamiana leaves expressing YFPv-tagged XopD216-760 and XopD1-760 36 hours after agroinfiltration. Bright field images are shown on the right. Bars  = 15 µm. (B) Western blot analysis using an anti-GFP antibody shows expression of YFPv-tagged XopD216-760 and XopD1-760 constructs, 36 hours after agroinfiltration. Ponceau S staining of the membrane illustrates equal loading. (C) Cell death development 4 (upper panel) and 7 (lower panel) days after agroinfiltration of YFPv-tagged XopD216-760 (left) and XopD1-760 (right) constructs. (D) Cell death was quantified by measuring electrolyte leakage in N. benthamiana leaves expressing YFPv-tagged XopD216-760 (open circles) and XopD1-760 (filled circles) at the indicated time points after agroinfiltration. Mean and SEM values are calculated from 3 independent experiments (8 replicates/experiment). The statistical significance in mean conductivity values obtained with leaves expressing XopD216-760 or XopD1-760 was assessed by using a Student's t test (P value <10−5). (E) SUMO-conjugates detected by Western blot analysis using an anti-HA antibody 36 hours after agroinfiltration of the indicated constructs. Expression of XopD proteins was revealed using an anti-GFP antibody. Ponceau S staining of the membrane illustrates equal loading.
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Related In: Results  -  Collection

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pone-0015773-g005: Characterization of XopD1-760 expression and function in N. benthamiana.(A) Confocal images of epidermal cells of N. benthamiana leaves expressing YFPv-tagged XopD216-760 and XopD1-760 36 hours after agroinfiltration. Bright field images are shown on the right. Bars  = 15 µm. (B) Western blot analysis using an anti-GFP antibody shows expression of YFPv-tagged XopD216-760 and XopD1-760 constructs, 36 hours after agroinfiltration. Ponceau S staining of the membrane illustrates equal loading. (C) Cell death development 4 (upper panel) and 7 (lower panel) days after agroinfiltration of YFPv-tagged XopD216-760 (left) and XopD1-760 (right) constructs. (D) Cell death was quantified by measuring electrolyte leakage in N. benthamiana leaves expressing YFPv-tagged XopD216-760 (open circles) and XopD1-760 (filled circles) at the indicated time points after agroinfiltration. Mean and SEM values are calculated from 3 independent experiments (8 replicates/experiment). The statistical significance in mean conductivity values obtained with leaves expressing XopD216-760 or XopD1-760 was assessed by using a Student's t test (P value <10−5). (E) SUMO-conjugates detected by Western blot analysis using an anti-HA antibody 36 hours after agroinfiltration of the indicated constructs. Expression of XopD proteins was revealed using an anti-GFP antibody. Ponceau S staining of the membrane illustrates equal loading.
Mentions: The N-terminal domain of XopD216-760 was previously shown to be required for targeting the effector to subnuclear foci [43]. To determine the effect of the newly identified N-terminal extension of XopD on its subcellular localization in planta, XopD1-760 was fused to the Yellow Fluorescent Protein venus (YFPv) and transiently expressed, under the control of the 35S promoter, in Nicotiana benthamiana leaf epidermal cells. As XopD216-760, XopD1-760 was also localized in subnuclear foci (Figure 5A). Both proteins were detected by Western blot using an anti-GFP antibody (Figure 5B).

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