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. 2013 Apr;161(4):2062-74.
doi: 10.1104/pp.112.211748. Epub 2013 Feb 15.

The Pseudomonas syringae effector HopQ1 promotes bacterial virulence and interacts with tomato 14-3-3 proteins in a phosphorylation-dependent manner

Wei Li  1 , Koste A Yadeta, James Mitch Elmore, Gitta Coaker
Affiliations

The Pseudomonas syringae effector HopQ1 promotes bacterial virulence and interacts with tomato 14-3-3 proteins in a phosphorylation-dependent manner

Wei Li et al. Plant Physiol. 2013 Apr.

Abstract

A key virulence strategy of bacterial pathogens is the delivery of multiple pathogen effector proteins into host cells during infection. The Hrp outer protein Q (HopQ1) effector from Pseudomonas syringae pv tomato (Pto) strain DC3000 is conserved across multiple bacterial plant pathogens. Here, we investigated the virulence function and host targets of HopQ1 in tomato (Solanum lycopersicum). Transgenic tomato lines expressing dexamethasone-inducible HopQ1 exhibited enhanced disease susceptibility to virulent Pto DC3000, the Pto ΔhrcC mutant, and decreased expression of a pathogen-associated molecular pattern-triggered marker gene after bacterial inoculation. HopQ1-interacting proteins were coimmunoprecipitated and identified by mass spectrometry. HopQ1 can associate with multiple tomato 14-3-3 proteins, including TFT1 and TFT5. HopQ1 is phosphorylated in tomato, and four phosphorylated peptides were identified by mass spectrometry. HopQ1 possesses a conserved mode I 14-3-3 binding motif whose serine-51 residue is phosphorylated in tomato and regulates its association with TFT1 and TFT5. Confocal microscopy and fractionation reveal that HopQ1 exhibits nucleocytoplasmic localization, while HopQ1 dephosphorylation mimics exhibit more pronounced nuclear localization. HopQ1 delivered from Pto DC3000 was found to promote bacterial virulence in the tomato genotype Rio Grande 76R. However, the HopQ1(S51A) mutant delivered from Pto DC3000 was unable to promote pathogen virulence. Taken together, our data demonstrate that HopQ1 enhances bacterial virulence and associates with tomato 14-3-3 proteins in a phosphorylation-dependent manner that influences HopQ1's subcellular localization and virulence-promoting activities in planta.

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Figures

Figure 1.
Figure 1.
Transgenic tomato plants expressing HopQ1 exhibit enhanced disease susceptibility to Pto. T4 homozygous transgenic tomato plants expressing Dex-inducible HopQ1-3xFLAG or GFP were sprayed with 30 μm Dex 24 h before syringe infiltration with Pto DC3000. A, Growth curve illustrating bacterial population sizes 4 d post inoculation with Pto DC3000 at a concentration of 1 × 105 cfu mL−1. B, Disease symptoms 4 d post inoculation with Pto DC3000. C, Growth curve illustrating bacterial population sizes 4 d post inoculation with Pto DC3000 ΔhrcC at a concentration of 1 × 106 cfu mL−1. For growth curves in A and C, values represent means ± sd (n = 6). The data shown are representative of three independent experiments with similar results. Statistical differences were detected by a two-tailed Student’s t test (α = 0.01). D, Anti-FLAG western blot illustrating HopQ1 protein expression. [See online article for color version of this figure.]
Figure 2.
Figure 2.
HopQ1 suppresses mRNA levels of the GRAS2 marker gene during infection. T4 homozygous transgenic tomato plants expressing Dex-inducible HopQ1-3xFLAG or GFP were sprayed with 30 μm Dex 24 h before syringe infiltration with 2 × 108 cfu mL−1 Pto DC3000 ΔhrcC or 10 mm MgCl2. Total RNA was isolated 6 h post inoculation. qRT-PCR was performed for the GRAS2 PTI marker gene. Actin expression was used to normalize the expression value of each sample. Values represent means ± sd (n = 3). The data shown are representative of three independent experiments with similar results. Statistical differences were detected by a two-tailed Student’s t test (α = 0.01). RQ, Relative quantification.
Figure 3.
Figure 3.
HopQ1’s 14-3-3 binding motif is widely conserved in homologous effectors and is phosphorylated in tomato. A, Multiple sequence alignment of the N terminus of Pto DC3000 HopQ1 and homologs from Xanthomonas spp. and Pseudomonas spp. Phosphorylated residues of HopQ1 are highlighted with asterisks. The mode I 14-3-3 binding site (RS/TXpSXP) is indicated with a line above the motif. Psph, P. syringae pv phaseolicola 1448A; Psm, P. syringae pv maculicola ES4326; Xg, Xanthomonas gardneri ATCC19865; Xoo, Xanthomonas oryzae pv oryzicola BLS256; Xcc, X. campestris pv campestris B100. B, T4 homozygous transgenic tomato plants expressing Dex-inducible HopQ1-3xFLAG or GFP were sprayed with 30 μm Dex 24 h before harvesting. Two grams of tomato leaf tissue was used for anti-FLAG immunoprecipitations. HopQ1 phosphorylated peptides were identified by mass spectrometry. The y and b ion series are labeled for each spectra, and the phosphorylated amino acids are highlighted in red. C, Manually annotated spectra matching Ser-51 phosphorylation. [See online article for color version of this figure.]
Figure 4.
Figure 4.
HopQ1 associates with the tomato 14-3-3 proteins TFT1 and TFT5 using a split-luciferase complementation assay. A, Split-luciferase complementation assay between HopQ1-NLuc, TFT1-CLuc, TFT5-CLuc, and controls. Binary vectors containing split-luciferase constructs were expressed in N. benthamiana using A. tumefaciens-mediated transient expression. SGT1b-Nluc and CLuc-RAR1 as well as HopQ1-NLuc and CLuc-Rin4 were used as positive and negative controls, respectively. Forty hours post inoculation, 1 mm luciferin was infiltrated and the resulting bioluminescence image was captured. B, Western blot demonstrating the expression of HopQ1-NLuc. N. benthamiana leaf tissue was harvested 24 h post inoculation.
Figure 5.
Figure 5.
Mutation of HopQ1’s 14-3-3 binding motif affects its association with tomato 14-3-3 proteins. A, HopQ1-3xFLAG and GFP-3xFLAG were transiently expressed with TFT1-HA and TFT5-HA in N. benthamiana using A. tumefaciens-mediated transient expression. Forty hours post inoculation, tissue was harvested and TFT1-HA and TFT5-HA were immunoprecipitated with HA antisera (IP). Associated proteins were detected by immunoblot analyses. B, HopQ1(S51A)-3xFLAG, HopQ1(M5)-3xFLAG, and the HopQ1(65–447) truncation were transiently expressed with TFT1-HA and TFT5-HA in N. benthamiana for immunoprecipitations as described in A.
Figure 6.
Figure 6.
HopQ1’s nucleocytoplasmic localization is influenced by its phosphorylation status. Confocal laser scanning microscopy is shown for N. benthamiana plant leaves transiently expressing HopQ1-GFP and GFP-tagged TFTs. A, HopQ1-GFP, TFT1-GFP, and TFT5-GFP were expressed in N. benthamiana, and confocal images of epidermal cells were taken 48 h post infiltration. B, HopQ1-GFP localization after coexpression with TFT1-HA or TFT5-HA in N. benthamiana. Images were taken as described in A. C, HopQ1(S51A)-GFP and HopQ1(M5)-GFP localization in N. benthamiana epidermal cells. Images were taken as described in A. D, The Pto DC3000 cluster IV deletion transformed with empty pBBR1 vector or pBBR1 expressing HopQ1-3xFLAG or HopQ1(S51A)-3xFLAG was vacuum infiltrated into tomato ‘Moneymaker’ at a concentration of 1 × 108 cfu mL−1. Twelve hours post infiltration, tissue was harvested, nuclei were isolated, and fractions were subjected to anti-FLAG western blotting. Nuclei enrichment was detected by anti-histone H3 western blotting. Nuclei purity was detected by the chloroplast-specific PSII membrane protein PsbO using anti-PsbO western blotting. V, Empty pBBR1 vector.
Figure 7.
Figure 7.
HopQ1’s phosphorylation status does not affect its ability to elicit an HR in tobacco. A, HopQ1 induces an HR in tobacco. Dex-inducible HopQ1-3xFLAG or GFP was expressed in tobacco using A. tumefaciens-mediated transient expression. Thirty micromolars of Dex was applied 24 h post infiltration, and photographs were taken 72 h post infiltration. B, Western blots probed with anti-FLAG showing expression levels of all constructs 40 h post infiltration. [See online article for color version of this figure.]
Figure 8.
Figure 8.
The HopQ1(S51A) dephosphorylation mimic cannot promote bacterial virulence in transgenic tomato plants. A, Transgenic tomato ‘Moneymaker’ plants expressing Dex-inducible HopQ1-3xFLAG, HopQ1(S51A)-3xFLAG, or GFP were sprayed with 30 μm Dex 16 h before syringe infiltration with Pto DC3000. The growth curve illustrates bacterial population sizes 4 d post inoculation with Pto DC3000. Values represent means ± sd (n = 6). The data shown are representative of three independent experiments with similar results. Statistical differences were detected by a two-tailed Student’s t test. B, Anti-FLAG western blot illustrating HopQ1 protein expression in all transgenic lines.
Figure 9.
Figure 9.
Pto DC3000-delivered HopQ1, but not HopQ1(S51A), can promote bacterial virulence in tomato ‘Rio Grande 76R’. A, The Pto DC3000 Δhopq1 deletion exhibits reduced bacterial virulence during ETI. Tomato ‘Rio Grande 76R’ plants were syringe infiltrated with 1 × 105 cfu mL−1 Pto DC3000 or Pto DC3000 Δhopq1. Growth curves illustrating bacterial population sizes are shown 3 and 5 d post inoculation. B, Expression of HopQ1from the broad-host-range vector pBBR1 can complement the Pto DC3000 cluster IV deletion lacking the HopQ1, HopD1, and HopR1 effectors. Tomato ‘Rio Grande 76R’ plants were syringe infiltrated with 1 × 105 cfu mL−1 bacteria, and growth curves were determined 4 d post inoculation. C, The Pto DC3000 cluster IV deletion transformed with empty pBBR1 vector, or pBBR1 expressing HopQ1-3xFLAG, HopQ1(S51A)-3xFLAG, or HopQ1(M5)-3xFLAG, were grown in hrp-inducing minimal medium for 16 h at 18°C. The resulting bacterial pellet and precipitated secreted proteins were subjected to an anti-FLAG western blot to detect protein expression. D, Expression of HopQ1(S51A) from the broad-host-range vector pBBR1-MCS5 cannot complement the Pto DC3000 cluster IV deletion. Growth curves were conducted as described in B. For all graphs, values represent means ± sd (n = 6). The data shown are representative of three independent experiments with similar results. Statistical differences were detected by a two-tailed Student’s t test. EV, Empty vector.
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