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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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Analysis of the XopD protein sequence by mass spectrometry.XopD216-760 protein sequence is shadowed. All possible translation starts situated in frame and upstream the annotated M216 are underlined. Peptides identified by Nano LC/ESI MS/MS analysis following trypsin (A) or V8 protease (B) digestion of the purified XopD protein are shown in bold. The 30 amino acid low complexity KAE-rich region is indicated in red.
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pone-0015773-g003: Analysis of the XopD protein sequence by mass spectrometry.XopD216-760 protein sequence is shadowed. All possible translation starts situated in frame and upstream the annotated M216 are underlined. Peptides identified by Nano LC/ESI MS/MS analysis following trypsin (A) or V8 protease (B) digestion of the purified XopD protein are shown in bold. The 30 amino acid low complexity KAE-rich region is indicated in red.

Mentions: A mass spectrometry analysis was thus conducted to determine the starting amino acid of the XopD protein produced by Xcv. HA-tagged XopD was immunopurified from Xcv 85* cultures and subjected to electrophoresis. After Coomassie staining of the gel, a ∼85 kDa band corresponding to XopD was excised, digested with trypsin and analyzed by mass spectrometry (Nano LC/ESI MS/MS). Trypsin cleaves at the C-terminal side of arginine (R) and lysine (K) residues, which are well distributed throughout the XopD sequence. In total, 28 peptides corresponding to XopD were detected (Figure 3A; Table 1), which represents a total XopD sequence coverage of 44%. The N-terminal region of the protein was particularly well covered (58% coverage for the newly identified N-terminal extension). MS/MS fragmentations for all peptides are provided in Figure S1.


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)

Analysis of the XopD protein sequence by mass spectrometry.XopD216-760 protein sequence is shadowed. All possible translation starts situated in frame and upstream the annotated M216 are underlined. Peptides identified by Nano LC/ESI MS/MS analysis following trypsin (A) or V8 protease (B) digestion of the purified XopD protein are shown in bold. The 30 amino acid low complexity KAE-rich region is indicated in red.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015773-g003: Analysis of the XopD protein sequence by mass spectrometry.XopD216-760 protein sequence is shadowed. All possible translation starts situated in frame and upstream the annotated M216 are underlined. Peptides identified by Nano LC/ESI MS/MS analysis following trypsin (A) or V8 protease (B) digestion of the purified XopD protein are shown in bold. The 30 amino acid low complexity KAE-rich region is indicated in red.
Mentions: A mass spectrometry analysis was thus conducted to determine the starting amino acid of the XopD protein produced by Xcv. HA-tagged XopD was immunopurified from Xcv 85* cultures and subjected to electrophoresis. After Coomassie staining of the gel, a ∼85 kDa band corresponding to XopD was excised, digested with trypsin and analyzed by mass spectrometry (Nano LC/ESI MS/MS). Trypsin cleaves at the C-terminal side of arginine (R) and lysine (K) residues, which are well distributed throughout the XopD sequence. In total, 28 peptides corresponding to XopD were detected (Figure 3A; Table 1), which represents a total XopD sequence coverage of 44%. The N-terminal region of the protein was particularly well covered (58% coverage for the newly identified N-terminal extension). MS/MS fragmentations for all peptides are provided in Figure S1.

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