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. 2011 Oct;12(8):715-30.
doi: 10.1111/j.1364-3703.2011.00706.x. Epub 2011 Feb 21.

Comparative analysis of the XopD type III secretion (T3S) effector family in plant pathogenic bacteria

Jung-Gun Kim  1 , Kyle W Taylor, Mary Beth Mudgett
Affiliations

Comparative analysis of the XopD type III secretion (T3S) effector family in plant pathogenic bacteria

Jung-Gun Kim et al. Mol Plant Pathol. 2011 Oct.

Abstract

XopD is a type III effector protein that is required for Xanthomonas campestris pathovar vesicatoria (Xcv) growth in tomato. It is a modular protein consisting of an N-terminal DNA-binding domain, two ethylene-responsive element binding factor-associated amphiphilic repression (EAR) transcriptional repressor motifs and a C-terminal small ubiquitin-related modifier (SUMO) protease. In tomato, XopD functions as a transcriptional repressor, resulting in the suppression of defence responses at late stages of infection. A survey of available genome sequences for phytopathogenic bacteria revealed that XopD homologues are limited to species within three genera of Proteobacteria--Xanthomonas, Acidovorax and Pseudomonas. Although the EAR motif(s) and SUMO protease domain are conserved in all XopD-like proteins, variation exists in the length and sequence identity of the N-terminal domains. Comparative analysis of the DNA sequences surrounding xopD and xopD-like genes led to revised annotation of the xopD gene. Edman degradation sequence analysis and functional complementation studies confirmed that the xopD gene from Xcv encodes a 760-amino-acid protein with a longer N-terminal domain than previously predicted. None of the XopD-like proteins studied complemented Xcv ΔxopD mutant phenotypes in tomato leaves, suggesting that the N-terminus of XopD defines functional specificity. Xcv ΔxopD strains expressing chimeric fusion proteins containing the N-terminus of XopD fused to the EAR motif(s) and SUMO protease domain of the XopD-like protein from X. campestris pathovar campestris strain B100 were fully virulent in tomato, demonstrating that the N-terminus of XopD controls specificity in tomato.

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Figures

Figure 1
Figure 1
Revised genome annotation and characterization of the xopD locus in Xanthomonas campestris pv. vesicatoria (Xcv) 85‐10. (A) Original xopD annotation compared with the revised xopD annotation. Analysis of the Xcv 85‐10 genome predicted the xopD open reading frame (ORF) to start 3′ of the putative hrp box (Thieme et al., 2005). The original xopD gene annotation predicted an ORF that encodes a protein of 545 amino acid residues. A 3.2‐kb genomic fragment was required to complement Xcv ΔxopD mutant phenotypes (Kim et al., 2008), suggesting that this annotation might be incorrect, and an alternative xopD start site occurs near the putative pathogen‐inducible promoter (PIP)‐box. The revised xopD annotation shows that the ORF starts 3′ of the putative PIP‐box. The xopD gene encodes a much larger polypeptide with a total of 760 amino acid residues. Schematic diagrams of the original and revised XopD protein coding regions are shown below the respective gene annotation arrows to compare the amino acid residues and functional domains for each predicted protein. The grey bar represents the C‐terminal small ubiquitin‐related modifier (SUMO) protease domain containing the catalytic core residues: histidine (H), aspartic acid (D) and cysteine (C). Black bars represent the putative ethylene‐responsive element binding factor‐associated amphiphilic repression (EAR) motifs. The white bar defines the N‐terminal region including a DNA‐binding domain. Based on the original xopD annotation, valine 118 (V118) and leucine 128 (L128) were shown to be required for DNA binding, and cysteine 470 (C470) for SUMO peptidase/isopeptidase activity (Hotson et al., 2003; Kim et al., 2008). Based on the revised xopD annotation, amino acids 1–545 in the old XopD protein sequence are equal to amino acids 216–760 in the new XopD protein sequence. Similarly, V118, L128 and C470 in the original coding sequence are equal to V333, L343 and C685, respectively, in the revised coding sequence. (B) Growth of the Xcv ΔxopD null mutant in tomato leaves is complemented by XopD (1–760 amino acids), but not XopD216–760. Leaves were hand‐inoculated with a 1 × 105 colony‐forming units (cfu)/mL suspension of Xcv (vector) containing pVSP61 (white bar), Xcv ΔxopD (vector) containing pVSP61 (grey bar), Xcv ΔxopD (XopD) containing pVSP61(lacZ promoter‐xopD‐His) (dark grey bar) and Xcv ΔxopD (XopD216–760) containing pVSP61(lacZ promoter‐xopD216–760‐His) (black bar). Bacterial growth was quantified from 0 to 12 days post‐inoculation (dpi). Data points represent mean log10 cfu/cm2± standard deviation (SD) of three tomato plants. Error bars indicate SD. The asterisks above the bars indicate statistically significant (t‐test, P < 0.05) differences between the bacterial numbers for Xcv (vector) and Xcv ΔxopD (vector) or Xcv ΔxopD (XopD216–760). (C) Symptom development in the infected tomato leaves sampled in (B). Hole punches were used for the quantification of bacterial numbers depicted in (B). Leaves were photographed at 12 dpi. Similar phenotypes were observed in three independent experiments.
Figure 3
Figure 3
XopD and XopD‐like proteins of plant pathogenic bacteria. (A) Phylogenetic tree of the XopD protein family. Bootstrap values are indicated on each branch and the scale bar represents branch lengths equivalent to 0.1 amino acid changes per amino acid residue. The proteins were grouped according to genus: Xanthomonas, Acidovorax and Pseudomonas. XopD, XopDXccB100, PsvAXcc8004, PsvAATCC33913, XopDAac, XopDAaa, PSA3335‐4544, PsvAPse, PsvAPsm, PSA3335‐0157 and PsvAPsd proteins are from X. campestris pv. vesicatoria (Xcv) 85‐10, X. campestris pv. campestris (Xcc) B100, Xcc 8004, Xcc ATCC 33913, A. avenae ssp. citrulli (Aac) AAC00‐1, A. avenae ssp. avenae (Aaa) ATCC 19860, P. savastanoi pv. savastanoi NCPPB 3335, P. syringae pv. eriobotryae (Pse), P. syringae pv. myricae, P. savastanoi pv. savastanoi NCPPB 3335 and P. syringae pv. dendropanacis, respectively. (B) Domain structure of XopD and XopD‐like proteins. Black bars represent putative ethylene‐responsive element binding factor‐associated amphiphilic repression (EAR) motifs. The grey rectangles represent C‐terminal small ubiquitin‐related modifier (SUMO) protease domains. For the PsvAPse protein, the dotted bar represents the putative type III secretion (T3S) signal (1–97 amino acids) sharing 41% identity with AvrA1PstT1. The hatched bar encodes a putative DNA‐binding domain (40–409 amino acids) sharing 31% identity with HsvBPab. The amino acid length of each protein is denoted at the C‐terminal end. XopD, PsvAXcc8004, XopDXccB100, XopDAac, XopDAaa and PsvAPse are from Xcv 85‐10, Xcc 8004, Xcc B100, Aac AAC00‐1, Aaa ATCC 19860 and Pse strains, respectively. All six proteins have the conserved catalytic core residues (His, Asp and Cys) in the SUMO protease domain. GenBank accession numbers: BK007963 (XopD), YP_242302(PsvAXcc8004), YP_001902662(XopDXccB100), YP_972673(XopDAac), ZP_06211344(XopDAaa) and BAA87062(PsvAPse).
Figure 2
Figure 2
Comparison of the chromosomal regions spanning the xopD and xopD‐like loci in different Xanthomonas strains. (A) The promoter and open reading frame (ORF) for xopD from Xanthomonas campestris pv. vesicatoria strain 85‐10 (Xcv 85‐10), xopDXccB100 from X. campestris pv. campestris strain B100 (Xcc B100), psvAXcc8004 from X. campestris pv. campestris strain 8004 (Xcc 8004) and psvAXccATCC33913 from X. campestris pv. campestris strain ATCC 33913 (Xcc ATCC 33913). Black boxes, putative pathogen‐inducible promoter (PIP)‐boxes. Red asterisk, confirmed ATG start codon for the XopD protein. The proposed start site for xopDXccB100 is shown at the corresponding ATG denoted in red. The original start sites annotated for xopD (Noël et al., 2002; Thieme et al., 2005) and xopDXccB100 (Vorhölter et al., 2008) are noted by thin black arrows. The grey shading indicates identical DNA sequence shared between xopD and xopD‐like genes. The percentage sequence identity is noted for the adjacent highlighted regions. The xopD‐like loci in Xcc 8004 and Xcc ATCC 33913 are disrupted by an insertion sequence (ISXac3) containing two genes (i.e. XC_1211 and XC_1212 in Xcc 8004 and XCC2898 and XCC2897 in Xcc ATCC) resulting in a natural 5′ deletion, creating psvAXcc8004 and psvAXccATCC33913, respectively. (B) Sequence alignment of the xopD and xopD‐like promoter regions. Boxes indicate the putative PIP‐box sequences sharing identity with the consensus motif (TTCGC—N15—TTCGC). Red asterisk indicates the confirmed ATG region in the xopD gene.
Figure 4
Figure 4
Small ubiquitin‐related modifier (SUMO) protease activity for XopD and XopD‐like proteins. Nicotiana benthamiana leaves were co‐infiltrated with a suspension of A. tumefaciens expressing tomato HA‐SUMO1 with vector, XopD(wt)‐His, XopD(C685A)‐His, XopD216–760‐His, PsvAXcc8004‐His, XopDXccB100‐His or XopDAac‐His. For co‐inoculations, strains were mixed equally and infiltrated into the leaf at a final density of 8 × 108 cells/mL. Sixty hours after inoculation, total protein was extracted from infected leaves and analysed by protein gel blot analysis using anti‐haemagglutinin (HA) (top panel) or anti‐His (bottom panel) sera, as described previously (Hotson et al., 2003). Ponceau S‐stained ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) large subunit is shown as a loading control. These experiments were repeated three times with similar results.
Figure 5
Figure 5
Phenotypes of Agrobacterium‐mediated transient expression of XopD and XopD‐like proteins in Nicotiana benthamiana. (A) Necrosis phenotype in N. benthamiana leaves. Leaves were infiltrated with a 6 × 108 cells/mL suspension of Agrobacterium tumefaciens expressing the following: 1, vector control; 2, XopD; 3, XopD216–760; 4, PsvAXcc8004; 5, XopDXccB100; 6, XopDAac. All proteins were tagged with the 6 × His epitope at the C‐terminus. The leaves were photographed at 6 and 8 days post‐inoculation (dpi). Protein expression was confirmed by protein gel blot analysis (Fig. S2). (B) Subcellular localization of XopD and XopD‐like proteins in N. benthamiana. Leaves were infiltrated with a 6 × 108 cells/mL suspension of Agrobacterium tumefaciens expressing yellow fluorescent protein (YFP), YFP‐XopD216–760, YFP‐XopD, YFP‐PsvAXcc8004, YFP‐XopDXccB100 or YFP‐XopDAac. At 60 h post‐inoculation, leaf epidermal cells were visualized by confocal microscopy at × 63. Scale bar, 20 µm. Protein expression was confirmed by protein gel blot analysis (Fig. S4). The experiments were repeated three times with similar results.
Figure 6
Figure 6
XopDXccB100, PsvAXcc8004 and XopDAac cannot complement Xanthomonas campestris pv. vesicatoria (Xcv) ΔxopD mutant phenotypes in tomato leaves. (A) Growth of Xcv strains in tomato leaves. Leaves were hand‐inoculated with a 1 × 105 cells/mL suspension of Xcv (vector) containing pVSP61 (white bar), Xcv ΔxopD (vector) containing pVSP61 (light grey bar), Xcv ΔxopD (XopDXccB100) containing pVSP61(lacZ promoter‐XopDXccB100‐His) (grey bar), Xcv ΔxopD (PsvAXcc8004) containing pVSP61(lacZ promoter‐PsvAXcc8004‐His) (dark grey bar) or Xcv ΔxopD (XopDAac) containing pVSP61(lacZ promoter‐xopDAac‐His) (black bar). Bacterial growth was quantified from 0 to 12 days post‐inoculation (dpi). Data points represent mean log10 colony‐forming units (cfu)/cm2± standard deviation (SD) of three tomato plants. Error bars indicate SD. The asterisks above the bars indicate statistically significant (t‐test, P < 0.05) differences between the bacterial numbers for Xcv (vector) and Xcv ΔxopD (vector) or Xcv ΔxopD expressing XopDXccB100, PsvAXcc8004 or XopDAac. (B) Phenotype of Xcv‐infected tomato leaves sampled in (A). Hole punches were used for the quantification of the bacterial numbers depicted in (A). Leaves were photographed at 12 dpi. Similar phenotypes were observed in three independent experiments.
Figure 7
Figure 7
Chimeric XopD‐XopDXccB100 fusion proteins complemented Xanthomonas campestris pv. vesicatoria (Xcv) ΔxopD mutant phenotypes in infected tomato leaves. (A) Schematic diagram of wild‐type XopD (1–760 amino acids) and XopDXccB100 (1–801 amino acids) and two chimeric fusion proteins, XopDvc1 and XopDvc2. XopDvc1 contains amino acids 1–457 of XopD fused to amino acids 533–801 of XopDXccB100. XopDvc2 contains amino acids 1–379 of XopD fused to amino aids 399–801 of XopDXccB100. The yellow rectangles represent XopD's N‐terminal domain before its small ubiquitin‐related modifier (SUMO) protease domain (grey rectangle), and the black bars represent the putative ethylene‐responsive element binding factor‐associated amphiphilic repression (EAR) motifs. The white rectangles represent XopDXccB100's N‐terminal domain before its SUMO protease domain (green rectangle) and the red bars represent putative EAR motifs. (B) Growth of Xcv strains in tomato leaves. Leaves were hand‐inoculated with a 1 × 105 cells/mL suspension of Xcv ΔxopD (vector) containing pVSP61 (white bar), Xcv ΔxopD (XopD) containing pVSP61(lacZ promoter‐XopD‐His) (light grey bar), Xcv ΔxopD (XopDXccB100) containing pVSP61(lacZ promoter‐XopDXccB100‐His) (grey bar), Xcv ΔxopD (XopDvc1) containing pVSP61(lacZ promoter‐XopDvc1‐His) (dark grey bar) or Xcv ΔxopD (XopDvc2) containing pVSP61(lacZ promoter‐XopDvc2‐His) (black bar). Data points represent mean log10 colony‐forming units (cfu)/cm2± standard deviation (SD) of three tomato plants. Error bars indicate SD. The asterisks above the bars indicate statistically significant (t‐test, P < 0.05) differences between the bacterial numbers for Xcv (XopD) and Xcv ΔxopD (vector) or Xcv ΔxopD (XopDXccB100). (C) Phenotype of the Xcv‐infected tomato leaves sampled in (B). Hole punches were used for the quantification of the bacterial numbers depicted in (B). Leaves were photographed at 11 and 13 days post‐inoculation. Similar phenotypes were observed in three independent experiments.
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