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. 2013 Apr;161(4):2049-61.
doi: 10.1104/pp.112.209023. Epub 2013 Feb 8.

Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins

Fabian Giska  1 , Malgorzata Lichocka, Marcin Piechocki, Michał Dadlez, Elmon Schmelzer, Jacek Hennig, Magdalena Krzymowska
Affiliations

Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins

Fabian Giska et al. Plant Physiol. 2013 Apr.

Abstract

HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.

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Figures

Figure 1.
Figure 1.
The 14-3-3 binding site is conserved in HopQ1, the TTSS effector from P. syringae, and XopQ, its xenolog from Xanthomonas spp. The canonical mode-1 14-3-3-interacting motif is shaded in gray, and the putative phospho-Ser is highlighted in red. [See online article for color version of this figure.]
Figure 2.
Figure 2.
The predicted 14-3-3 binding motif of HopQ1 is phosphorylated by plant kinases. A and B, HopQ1-Strep tag II fusion protein expressed in N. benthamiana leaves was affinity purified and subjected to LC-MS/MS analyses. C and D, HopQ1-6xHis expressed in bacteria was incubated with total protein extracts from N. benthamiana and then affinity purified and analyzed by LC-MS/MS. A and C show the fragmentation spectra with peak assignment to b, y, b-H3PO4, and y-H3PO4, with “–P” denoting loss of an H3PO4 group. Major signals of the MS/MS spectra are identified by their corresponding fragment tags of the b, y series, but also of the y-H3PO4 and b-H3PO4 series, which were expected in the case of a phosphorylated peptide. B and D show the peptide sequences with daughter ions of the b, y, b-H3PO4, and y-H3PO4 series found in the spectra. In the peptide derived from HopQ1 phosphorylated in vitro (C and D), the presence of the full series of b2 to b10 fragments and the expected b-H3PO4 series allows for unequivocal localization of the phosphate at Ser-51. In the peptide derived from HopQ1 expressed in planta (A and B), the majority of b and b-H3PO4 pairs are also present. In addition, the presence of strong y, y-PO3, and y-H3PO4 signals indicates that the site of phosphorylation is Ser-51, not Ser-49.
Figure 3.
Figure 3.
Ser-51 plays a critical role in the phosphorylation of HopQ1. Recombinant HopQ1 variants with a C-terminal 6xHis epitope were incubated with leaf protein extracts from N. benthamiana in buffer containing [γ-32P]ATP. Samples were resolved by SDS-PAGE and analyzed by autoradiography. Only residual phosphorylation was detected for HopQ1 mutated at position 51. As a loading control, the Coomassie Brilliant Blue (CBB)-stained gel is shown in the bottom panel. This experiment was performed three times with similar results.
Figure 4.
Figure 4.
HopQ1 binds to 14-3-3a in a phosphorylation-dependent manner. Representative gel filtration runs on a Superdex 200 column are shown. Recombinant HopQ1 with a C-terminal 6xHis epitope was incubated with recombinant CPK3 prior to binding to 14-3-3a (orange trace). As a control, nonphosphorylated HopQ1 was used (green trace). Under the conditions used (5 mm DTT), HopQ1 exists as a monomer (elution volumes of 14.68 and 14.69). 14-3-3a was eluted as dimers (peaks 13.63 and 13.72). Recombinant CPK3 was removed from the reaction by affinity capture on a GST column. The experiment was performed twice with similar results.
Figure 5.
Figure 5.
Subcellular localization of HopQ1 variants. Confocal images show representative N. benthamiana leaf epidermal cells (top panels) or mesophyll cells (bottom panels) transiently expressing either wild-type HopQ1-eYFP (left panels) or HopQ1-S51A-eYFP (right panels). White arrowheads indicate the nuclei. The photographs were taken 72 h after agroinfiltration. For each variant, approximately 50 transformed cells were examined. Bars = 10 µm.
Figure 6.
Figure 6.
Coexpression of 14-3-3a and HopQ1 affects the nuclear-cytoplasmic partitioning of the binding partners. A, N. benthamiana leaves were coinfiltrated with A. tumefaciens strains carrying constructs encoding 14-3-3a-mRFP and variants of HopQ1 fused to eYFP. For each variant, approximately 50 transformed cells were examined. B, The same constructs were transiently coexpressed in bean epidermal cells via particle bombardment. In both plant species, 14-3-3a-mRFP localized to the cytoplasm and nucleus. Coexpression with wild-type HopQ1-eYFP resulted in the relocation of 14-3-3a-mRFP from the nucleus to the cytoplasm. A noninteracting form of HopQ1 (HopQ1-S51A-eYFP) shows highly increased nuclear accumulation and does not alter the localization of 14-3-3a-mRFP. For each variant, approximately 20 transformed cells were examined. White arrowheads indicate the nuclei. Bars = 10 µm.
Figure 7.
Figure 7.
Interaction with 14-3-3 proteins increases HopQ1 stability in plants. A, The presence of Ser-51 affects HopQ1 steady-state level in planta. Wild-type HopQ1 or HopQ1-S51A proteins carrying C-terminal 3xHA epitopes were transiently coexpressed with GFP in N. benthamiana leaves. Crude protein extracts (20 μg of proteins per lane) were isolated from leaves 48 h after agroinfiltration. HopQ1 variants were detected by immunoblot analysis using an antibody specific to HA. As an expression control, the level of GFP was checked using anti-GFP antibody. The experiment was repeated twice. B, Modification of the 14-3-3 binding motif does not change HopQ1 stability in P. syringae. C-terminally His-tagged HopQ1 variants were expressed in P. syringae pv tabaci DAPP-PG677. Crude protein extracts prepared from overnight bacterial cultures were fractionated on 12.5% SDS-PAGE and subjected to immunoblot analysis using a specific anti-His antibody. Equal protein loading is shown by Ponceau Red staining. The experiment was repeated twice. C, R18, the inhibitor of 14-3-3 binding, affects HopQ1 stability in plant extracts. Wild-type HopQ1 protein carrying a C-terminal Flag epitope was transiently expressed in N. benthamiana leaves. Crude protein extract was supplemented with recombinant 14-3-3a-Strep II protein isolated from E. coli or R18 peptide at various concentrations, as indicated. HopQ1 was detected by immunoblot analysis using specific primary antibodies. The level of 14-3-3a was monitored using Strep-Tactin AP conjugate. Equal protein loading is shown by Ponceau Red staining. D, Application of 14-3-3a reverts the effect of R18 on HopQ1 stability. Wild-type HopQ1 protein carrying a C-terminal Flag epitope was transiently expressed in N. benthamiana leaves. Crude protein extract was supplemented with R18 and increasing amounts of recombinant 14-3-3a-Strep II protein isolated from E. coli, as indicated. HopQ1 was detected by immunoblot analysis using specific primary antibodies. The level of 14-3-3a was monitored using Strep-Tactin AP conjugate. Equal protein loading is shown by Ponceau Red staining. E, Assembled in vitro complex of HopQ1-6xHis and 14-3-3a-Strep II was incubated with bean crude protein extract in the absence or presence of R18. HopQ1 and 14-3-3a were detected by immunoblot analyses using anti-His antibodies or Strep-Tactin AP conjugate, respectively. Equal protein loading is shown by Ponceau Red staining. F, In vitro phosphorylated HopQ1-6xHis was added to bean crude extract and incubated in the presence of 14-3-3a or BSA. HopQ1 and 14-3-3a were detected by immunoblot analysis using anti-His antibodies or Strep-Tactin AP conjugate, respectively. Equal protein loading is shown by Ponceau Red staining.
Figure 8.
Figure 8.
HopQ1 interaction with 14-3-3s is not critical for its perception by host plants. A, Binary vectors encoding either wild-type HopQ1 or HopQ1-S51A were introduced via agroinfiltration into tobacco leaves. The hypersensitive response developed in the infiltrated area within 48 h in response to both A. tumefaciens strains. In contrast, no macroscopic signs of tissue necrotization developed in control leaves expressing GFP. B, N. benthamiana plants were inoculated with PsyB728a strains carrying pBBR1-MCS2 derivatives, which express HopQ1, HopQ1-S51A, or mCherry protein, as a control. Disease symptoms developed only in control plants treated with PsyB728a encoding mCherry protein. The photographs were taken 10 d post inoculation. The experiment was performed twice, with similar results.
Figure 9.
Figure 9.
Assessment of virulence properties of the HopQ1 effector mutated to eliminate 14-3-3 binding. Bean leaves were inoculated with P. syringae pv tomato DC3000D28E (approximately 105 cfu mL−1) strains expressing HopQ1 or HopQ1-S51A. Immediately prior to infiltration, bacteria were mixed in a 1:1 ratio. Two and 6 d post inoculation (dpi), two leaf discs per plant were cut out from the infiltrated zones, ground in sterile 10 mm MgCl2, diluted, and plated on KB medium. Bacterial strains were distinguished by a selectable marker. The CI was calculated as the ratio of bacteria expressing the wild-type HopQ1 to bacteria expressing the mutated HopQ1 isolated from plant leaf and normalized to the input titers of the bacteria. Asterisks indicate that the index is significantly different from 1, as established using Student’s t test (P < 0.01). The experiment was performed three times with similar results. [See online article for color version of this figure.]
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