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. 2008 Jul;20(7):1915-29.
doi: 10.1105/tpc.108.058529. Epub 2008 Jul 29.

XopD SUMO protease affects host transcription, promotes pathogen growth, and delays symptom development in xanthomonas-infected tomato leaves

Jung-Gun Kim  1 , Kyle W Taylor, Andrew Hotson, Mark Keegan, Eric A Schmelz, Mary Beth Mudgett
Affiliations

XopD SUMO protease affects host transcription, promotes pathogen growth, and delays symptom development in xanthomonas-infected tomato leaves

Jung-Gun Kim et al. Plant Cell. 2008 Jul.

Abstract

We demonstrate that XopD, a type III effector from Xanthomonas campestris pathovar vesicatoria (Xcv), suppresses symptom production during the late stages of infection in susceptible tomato (Solanum lycopersicum) leaves. XopD-dependent delay of tissue degeneration correlates with reduced chlorophyll loss, reduced salicylic acid levels, and changes in the mRNA abundance of senescence- and defense-associated genes despite high pathogen titers. Subsequent structure-function analyses led to the discovery that XopD is a DNA binding protein that alters host transcription. XopD contains a putative helix-loop-helix domain required for DNA binding and two conserved ERF-associated amphiphilic motifs required to repress salicylic acid- and jasmonic acid-induced gene transcription in planta. Taken together, these data reveal that XopD is a unique virulence factor in Xcv that alters host transcription, promotes pathogen multiplication, and delays the onset of leaf chlorosis and necrosis.

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Figures

Figure 1.
Figure 1.
XopD Is Required for Maximal Xcv Growth and to Delay the Development of Disease Symptoms in Infected Tomato Leaves. (A) Growth of Xcv (gray bars) and Xcv ΔxopD (red bars) strains in susceptible VF36 tomato. Leaves were hand-inoculated with a 105 cfu/mL suspension of bacteria. Data points represent mean log10 cfu per cm2 ± sd of three independent experiments. Error bars indicate sd of three independent experiments. The asterisk above the bars indicates statistically significant (t test, P < 0.05) differences between the bacterial numbers for Xcv and Xcv ΔxopD. (B) Phenotype of tomato leaves sampled in (A). Hole punches were used to quantify bacterial numbers depicted in (A). Leaves were photographed at 10 and 12 DAI. Similar phenotypes were observed in three independent experiments. (C) Expression of XopD in Xcv ΔxopD complements reduced pathogen growth and suppresses tissue necrosis in infected leaves. Susceptible VF36 tomato leaves were hand-inoculated with a 105 cfu/mL suspension of bacteria: Xcv (vector) containing pDSK519 (green bars), Xcv ΔxopD (vector) containing pDSK519 (yellow bars), and Xcv ΔxopD (XopD) containing pDSK519(xopD promoter-xopD(wt)-HA) (blue bars). Pathogen growth is shown in the left panel. Data points represent mean log10 cfu per cm2 ± sd of three independent experiments. The asterisk above the bars indicates statistically significant (t test, P < 0.05) differences between the bacterial numbers of Xcv (vector) and Xcv ΔxopD (vector). Leaf symptoms are shown in the right panel. Hole punches were used to quantify bacterial numbers depicted in the left panel. Leaves were photographed at 12 DAI. Similar phenotypes were observed in three independent experiments.
Figure 2.
Figure 2.
XopD Suppresses Senescence-Associated Responses during Infection. (A) XopD delays chlorophyll degradation in leaf tissue inoculated with Xcv. Susceptible VF36 tomato leaves were hand-inoculated with 10 mM MgCl2 buffer (square) or a 105 cfu/mL suspension of Xcv (circles) or Xcv ΔxopD (triangles). Total chlorophyll (μg/cm2) was extracted from the inoculated tissue. Error bars indicate sd. Different letters at each time point indicate statistically significant (one-way analysis of variance and Tukey's HSD test, P < 0.05) differences between the samples. (B) XopD alters the mRNA abundance of senescence- and defense-associated genes. Total RNA was isolated from tomato leaves hand-inoculated with105 cfu/mL of Xcv (white rectangles) or Xcv ΔxopD (black rectangles), respectively. Quantitative real-time RT-PCR was performed for four classes of tomato genes: (1) senescence/upregulated (SENU5); (2) senescence and defense/upregulated (SENU4); (3) defense/upregulated (Chi17); and (4) senescence/downregulated (SEND33 and Cab-1B) as well as the ethylene receptor gene ETR4. Relative expression levels at 6, 7, and 8 DAI are shown. Actin expression was used to normalize the expression value in each sample, and relative expression values were determined against the average value of the sample infected with wild-type Xcv at 6 DAI. The averages of the two independent experiments are shown. Error bars indicate sd.
Figure 3.
Figure 3.
XopD Reduces SA Levels during Infection. (A) XopD reduces SA levels in leaves inoculated with Xcv. Susceptible VF36 tomato leaves were hand-inoculated with buffer (white bars) or a 105 cfu/mL suspension of Xcv (gray bars) or Xcv ΔxopD (black bars). SA levels (i.e., pools of free SA and existing methyl salicylate) in infected tissue were quantified for 10 d. μg/g FW = μg of free and endogenous methyl salicylate per gram of fresh weight. Error bars indicate sd for two biological samples. Different letters above the day 6 bars indicate statistically significant (one-way analysis of variance and Tukey's HSD test, P < 0.05) differences between the samples. (B) Xcv ΔxopD growth is less restricted in NahG Moneymaker tomato leaves. Growth of Xcv (blue bars) and Xcv ΔxopD (green bars) strains in transgenic NahG tomato and that of Xcv (gray bars) and Xcv ΔxopD (red bars) strains in the wild-type Moneymaker tomato leaves are indicated. Leaves were hand-inoculated with a 105 cfu/mL suspension of bacteria. Data points represent mean log10 cfu per cm2 ± sd of three independent experiments. The asterisk above the bars indicates statistically significant (t test, P < 0.05) differences between the bacterial numbers of Xcv and Xcv ΔxopD in the wild-type Moneymaker tomato leaves. NahG leaves were not analyzed beyond 8 DAI because they were fully necrotic (see [C]) and died by 9 DAI. (C) XopD delays disease symptom production in wild-type Moneymaker and NahG tomato leaves inoculated with Xcv. Phenotype of leaves analyzed in (B) at 12 DAI (wild-type Moneymaker tomato) and 8 DAI (NahG Moneymaker tomato).
Figure 4.
Figure 4.
XopD Binds DNA and DNA Binding Is Required for Virulence in Tomato and Tissue Necrosis in N. benthamiana. (A) Schematic of the putative DNA binding domain of XopD protein (open rectangle). In red, the N-terminal DBD containing critical residues, V118 and L128 (VL), for DNA binding activity; and in black, the C-terminal SUMO protease domain containing catalytic core residues His, Asp, and Cys (HDC). (B) In vitro XopD DNA binding activity. Arabidopsis PDF1.2 promoter probes were used for EMSA by being incubated with an increasing concentration (0, 0.06, 0.12, and 0.25 μM) of purified GST, GST-XopD, GST-XopD(V118A), and GST-XopD(L128P) and then visualized by chemiluminescece. (C) XopD DBD is required for maximal Xcv growth in tomato. Leaves were hand-inoculated with a 1 × 105 cfu/mL suspension of Xcv ΔxopD expressing XopD(wt)-HA (gray bars), XopD(C470A)-HA (red bars), or XopD(V118P)-HA (blue bars). Data points represent mean log10 cfu per cm2 ± sd of three independent experiments. The asterisk above the bars indicates statistically significant (t test, P < 0.05) differences between the bacterial numbers of Xcv ΔxopD expressing XopD(wt)-HA and the XopD mutant proteins. (D) XopD protease activity and DBD are required to delay disease symptoms in VF36 leaves inoculated with Xcv (top panel). Representative phenotypes of tomato leaves inoculated in (C) after 11 and 12 d: wt, XopD(wt)-HA; C470A, XopD(C470A)-HA; V118P, XopD(V118P)-HA. Immunoblot analysis (bottom panel) of vector and protein expressed in Xcv. Xcv strains described in (C) were grown in XVM2 medium for 6 h with appropriate antibiotics. Total cellular lysate (∼5 × 107 cells) was examined by protein gel blot analysis using HA antisera. (E) Phenotype of XopD DNA binding mutants in N. benthamiana (top panel). Leaf was infiltrated with a suspension (OD600 = 0.5) of A. tumefaciens expressing the following: vector, vector control; wt, XopD(wt)-HA; V118P, XopD(V118P)-HA; and L128P, XopD(L128P)-HA. The leaf was photographed 6 DAI. Immunoblot analysis (bottom panel) of proteins transiently expressed in N. benthamiana leaves as shown in the top panel. Total protein (∼50μg) was extracted from tissue at 48 h after inoculation and then examined by protein gel blot analysis using HA antisera. These experiments were repeated three times with similar results.
Figure 5.
Figure 5.
XopD Has Two EAR Motifs Required for Virulence in Tomato and Tissue Necrosis in N. benthamiana. (A) Schematic view of XopD protein. The open rectangle represents XopD protein, and two EAR motifs, R1 and R2, are represented by red rectangles. The motifs are located between the N-terminal DBD and C-terminal SUMO protease domain containing catalytic core residues His, Asp, and Cys (HDC). (B) XopD EAR motifs are required for maximal Xcv growth in tomato. Susceptible VF36 tomato leaves were hand-inoculated with a 1 × 105 cfu/mL suspension of Xcv ΔxopD expressing XopD(wt)-HA (gray bars), XopD(C470A)-HA (red bars), XopDΔR1-HA (blue bars), XopDΔR2-HA (green bars), or XopDΔR1ΔR2-HA (yellow bars). Data points represent mean log10 cfu per cm2 ± sd of three independent experiments. The asterisk above the bars indicates statistically significant (t test, P < 0.05) differences between the bacterial numbers of Xcv ΔxopD expressing XopD(wt)-HA and the XopD mutant proteins. (C) XopD EAR motifs are required to suppress disease symptoms in tomato (top panel). Representative phenotype of VF36 tomato leaves inoculated in (B) after 12 d: wt, XopD(wt)-HA; C470A, XopD(C470A)-HA; ΔR1, XopDΔR1-HA; ΔR2, XopDΔR2-HA; ΔR1ΔR2, XopDΔR1ΔR2-HA. Immunoblot analysis (bottom panel) of proteins expressed in the bacteria. Xcv strains described in (B) were grown in XVM2 medium for 6 h with appropriate antibiotics. Total cellular lysate (∼5 × 107 cells) was examined by protein gel blot analysis using HA antisera. (D) XopDΔR1ΔR2-HA does not elicit necrosis in N. benthamiana (top panel). Leaf was infiltrated with the A. tumefaciens strains expressing the following: vector, vector control; wt, XopD(wt)-HA; C470A, XopD(C470A)-HA; ΔR1, XopDΔR1-HA; ΔR2, XopDΔR2-HA; ΔR1ΔR2, XopDΔR1ΔR2-HA. The phenotype was photographed 6 DAI. Immunoblot analysis (bottom panel) of proteins transiently expressed in N. benthamiana leaves as shown in top panel. Total protein (∼50μg) was extracted from tissue at 48 h after inoculation and then examined by protein gel blot analysis using HA antisera. These experiments were repeated three times with similar results.
Figure 6.
Figure 6.
XopD Represses Plant Defense Gene Transcription. (A) Schematic of transcription effector and reporter constructs coexpressed in N. benthamiana. Effectors (EYFP, XopD-HA, and mutant XopD-HA derivatives) were constitutively expressed using the cauliflower mosaic virus 35S promoter. The GUSplus reporter was fused to the ACT2, PR1, and PDF1.2 promoters. The nopaline synthase terminator (NOS-T) was used in all constructs. (B) XopD EAR motifs are required for repression of PR1 and PDF1.2 transcription. N. benthamiana leaves inoculated with each A. tumefaciens strain for 18 h were treated with buffer, SA (2 mM), or methyl jasmonate (MeJA; 100 μM). Samples were collected 12 h later and GUS activity (nmol of 4-methylumbelliferone [4-MU] mg−1 protein min−1) quantified. For each reporter, the following effectors were tested: EYFP, enhanced yellow fluorescent protein; WT, XopD-HA; C470A, XopD(C470A)-HA; V118P, XopD(V118P)-HA; ΔR1, XopDΔR1-HA; ΔR2, XopDΔR2-HA; ΔR1ΔR2, XopDΔR1ΔR2-HA. Bars represent buffer (white), SA (gray), and methyl jasmonate (black) treatment. Error bars indicate sd of three independent experiments.
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