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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 silico analysis of the xopD locus.(A) The xopD locus in Xcv 85-10 is shown (the genome interval for the Xcv 85-10 shown sequence and the xopD gene are respectively: 486209–489200 and 486544–488826). Open arrows indicate ORFs and filled arrows show promoter elements, such as the PIP box and the −10 sequence. The position of XopD translation start annotated in the public databases is indicated by an asterisk. (B) Schematic representation of XopD functional domains in Xcv 85-10, in comparison to its known protein homologues. The N-terminal extension, essential V and L residues in the DBD, tandemly repeated EAR motifs, conserved catalytic residues in the cysteine protease domain, and NLS motif are shown. (C) Sequence alignment of XopD from Xcv 85-10, hypothetical protein from Xcc B100 (YP_001902662), virulence protein from Xcc 8004 (AAY48282) or Xcc ATCC 33913 (AAM42168), peptidase C48 SUMO from Acidovorax avenae (EFA39722) and XopD from Xcc 147. The figure shows the longest ORF possible for all proteins. For XopD from Xcv, the first shown M residue corresponds to a UUG codon, not conserved among Xcc B100 and A. avenae coding sequences. The M residue previously annotated as the starting amino acid of the XopD protein is indicated by a red arrowhead. Putative translation starts situated in frame and upstream the previously annotated starting M are also indicated as follows: translation starts conserved among Xcv 85-10, Xcc B100 and Aea are indicated with a full red dot, otherwise, they appear indicated by an empty red dot. Yellow box: putative N-terminal extension; green box: DNA-binding domain (V and L residues in the helix-loop-helix domain essential for maximal DNA-binding [46] are indicated by an empty green dot); red boxes: tandemly-repeated EAR motifs [L/FDNLL/F(X)P] [44]; black box: SUMO protease domain (H, D and C catalytic core residues are indicated by a black empty dot); blue box: NLS. Gray and black highlighting of amino acids indicates, respectively, 70–80% and 90–100% of similarity.
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pone-0015773-g001: In silico analysis of the xopD locus.(A) The xopD locus in Xcv 85-10 is shown (the genome interval for the Xcv 85-10 shown sequence and the xopD gene are respectively: 486209–489200 and 486544–488826). Open arrows indicate ORFs and filled arrows show promoter elements, such as the PIP box and the −10 sequence. The position of XopD translation start annotated in the public databases is indicated by an asterisk. (B) Schematic representation of XopD functional domains in Xcv 85-10, in comparison to its known protein homologues. The N-terminal extension, essential V and L residues in the DBD, tandemly repeated EAR motifs, conserved catalytic residues in the cysteine protease domain, and NLS motif are shown. (C) Sequence alignment of XopD from Xcv 85-10, hypothetical protein from Xcc B100 (YP_001902662), virulence protein from Xcc 8004 (AAY48282) or Xcc ATCC 33913 (AAM42168), peptidase C48 SUMO from Acidovorax avenae (EFA39722) and XopD from Xcc 147. The figure shows the longest ORF possible for all proteins. For XopD from Xcv, the first shown M residue corresponds to a UUG codon, not conserved among Xcc B100 and A. avenae coding sequences. The M residue previously annotated as the starting amino acid of the XopD protein is indicated by a red arrowhead. Putative translation starts situated in frame and upstream the previously annotated starting M are also indicated as follows: translation starts conserved among Xcv 85-10, Xcc B100 and Aea are indicated with a full red dot, otherwise, they appear indicated by an empty red dot. Yellow box: putative N-terminal extension; green box: DNA-binding domain (V and L residues in the helix-loop-helix domain essential for maximal DNA-binding [46] are indicated by an empty green dot); red boxes: tandemly-repeated EAR motifs [L/FDNLL/F(X)P] [44]; black box: SUMO protease domain (H, D and C catalytic core residues are indicated by a black empty dot); blue box: NLS. Gray and black highlighting of amino acids indicates, respectively, 70–80% and 90–100% of similarity.

Mentions: XopD was previously predicted to code for a protein of 612 amino acids [47]. However, secretion assays and Western blot analysis, following expression of c-myc-tagged XopD within its native Xcv chromosomic environment, allowed the detection of a unique band of a molecular mass close to 100 kDa [47]. Importantly, this protein was undetectable in culture supernatants of T3SS mutant bacteria cultured in secretion medium, demonstrating that this protein is secreted in a T3SS-dependent manner. In contrast, the XopD protein sequence annotated in the public databases predicts a protein of 545 amino acids with an expected molecular mass of 61 kDa. Noël and co-workers could not detect a (consensus) PIP box in the xopD promoter, but rather a putative hrp box, which is found in all hrpL-dependent promoters in Pseudomonas syringae and Erwinia spp [47]–[49]. Later inspection of the xopD promoter region identified the presence of a PIP box (ATCGC-N15-TTCGT), bound by HrpX, and a −10 putative sequence (TAAATT), which are situated 747 and 690 bp upstream the annotated start, respectively (Figure 1A) [35]. In agreement with these observations, the HrpX-dependent expression of XopD has been confirmed experimentally [35], [47].


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 silico analysis of the xopD locus.(A) The xopD locus in Xcv 85-10 is shown (the genome interval for the Xcv 85-10 shown sequence and the xopD gene are respectively: 486209–489200 and 486544–488826). Open arrows indicate ORFs and filled arrows show promoter elements, such as the PIP box and the −10 sequence. The position of XopD translation start annotated in the public databases is indicated by an asterisk. (B) Schematic representation of XopD functional domains in Xcv 85-10, in comparison to its known protein homologues. The N-terminal extension, essential V and L residues in the DBD, tandemly repeated EAR motifs, conserved catalytic residues in the cysteine protease domain, and NLS motif are shown. (C) Sequence alignment of XopD from Xcv 85-10, hypothetical protein from Xcc B100 (YP_001902662), virulence protein from Xcc 8004 (AAY48282) or Xcc ATCC 33913 (AAM42168), peptidase C48 SUMO from Acidovorax avenae (EFA39722) and XopD from Xcc 147. The figure shows the longest ORF possible for all proteins. For XopD from Xcv, the first shown M residue corresponds to a UUG codon, not conserved among Xcc B100 and A. avenae coding sequences. The M residue previously annotated as the starting amino acid of the XopD protein is indicated by a red arrowhead. Putative translation starts situated in frame and upstream the previously annotated starting M are also indicated as follows: translation starts conserved among Xcv 85-10, Xcc B100 and Aea are indicated with a full red dot, otherwise, they appear indicated by an empty red dot. Yellow box: putative N-terminal extension; green box: DNA-binding domain (V and L residues in the helix-loop-helix domain essential for maximal DNA-binding [46] are indicated by an empty green dot); red boxes: tandemly-repeated EAR motifs [L/FDNLL/F(X)P] [44]; black box: SUMO protease domain (H, D and C catalytic core residues are indicated by a black empty dot); blue box: NLS. Gray and black highlighting of amino acids indicates, respectively, 70–80% and 90–100% of similarity.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015773-g001: In silico analysis of the xopD locus.(A) The xopD locus in Xcv 85-10 is shown (the genome interval for the Xcv 85-10 shown sequence and the xopD gene are respectively: 486209–489200 and 486544–488826). Open arrows indicate ORFs and filled arrows show promoter elements, such as the PIP box and the −10 sequence. The position of XopD translation start annotated in the public databases is indicated by an asterisk. (B) Schematic representation of XopD functional domains in Xcv 85-10, in comparison to its known protein homologues. The N-terminal extension, essential V and L residues in the DBD, tandemly repeated EAR motifs, conserved catalytic residues in the cysteine protease domain, and NLS motif are shown. (C) Sequence alignment of XopD from Xcv 85-10, hypothetical protein from Xcc B100 (YP_001902662), virulence protein from Xcc 8004 (AAY48282) or Xcc ATCC 33913 (AAM42168), peptidase C48 SUMO from Acidovorax avenae (EFA39722) and XopD from Xcc 147. The figure shows the longest ORF possible for all proteins. For XopD from Xcv, the first shown M residue corresponds to a UUG codon, not conserved among Xcc B100 and A. avenae coding sequences. The M residue previously annotated as the starting amino acid of the XopD protein is indicated by a red arrowhead. Putative translation starts situated in frame and upstream the previously annotated starting M are also indicated as follows: translation starts conserved among Xcv 85-10, Xcc B100 and Aea are indicated with a full red dot, otherwise, they appear indicated by an empty red dot. Yellow box: putative N-terminal extension; green box: DNA-binding domain (V and L residues in the helix-loop-helix domain essential for maximal DNA-binding [46] are indicated by an empty green dot); red boxes: tandemly-repeated EAR motifs [L/FDNLL/F(X)P] [44]; black box: SUMO protease domain (H, D and C catalytic core residues are indicated by a black empty dot); blue box: NLS. Gray and black highlighting of amino acids indicates, respectively, 70–80% and 90–100% of similarity.
Mentions: XopD was previously predicted to code for a protein of 612 amino acids [47]. However, secretion assays and Western blot analysis, following expression of c-myc-tagged XopD within its native Xcv chromosomic environment, allowed the detection of a unique band of a molecular mass close to 100 kDa [47]. Importantly, this protein was undetectable in culture supernatants of T3SS mutant bacteria cultured in secretion medium, demonstrating that this protein is secreted in a T3SS-dependent manner. In contrast, the XopD protein sequence annotated in the public databases predicts a protein of 545 amino acids with an expected molecular mass of 61 kDa. Noël and co-workers could not detect a (consensus) PIP box in the xopD promoter, but rather a putative hrp box, which is found in all hrpL-dependent promoters in Pseudomonas syringae and Erwinia spp [47]–[49]. Later inspection of the xopD promoter region identified the presence of a PIP box (ATCGC-N15-TTCGT), bound by HrpX, and a −10 putative sequence (TAAATT), which are situated 747 and 690 bp upstream the annotated start, respectively (Figure 1A) [35]. In agreement with these observations, the HrpX-dependent expression of XopD has been confirmed experimentally [35], [47].

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