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. 2012 Feb;8(2):e1002523.
doi: 10.1371/journal.ppat.1002523. Epub 2012 Feb 2.

A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion

Amy Huei-Yi Lee  1 , Brenden Hurley, Corinna Felsensteiner, Carmen Yea, Wenzislava Ckurshumova, Verena Bartetzko, Pauline W Wang, Van Quach, Jennifer D Lewis, Yulu C Liu, Frederik Börnke, Stephane Angers, Andrew Wilde, David S Guttman, Darrell Desveaux
Affiliations

A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion

Amy Huei-Yi Lee et al. PLoS Pathog. 2012 Feb.

Abstract

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HEK293T Cells expressing HopZ1a have altered morphology.
(A) HEK293T cells were transiently transfected with TAP-tagged HopZ1a and HopZ1a (C216A) for 24 h, followed by imaging of the transfected and untransfected cells. Arrows indicate cytoplasmic projections. (B) Immunoblot analysis of HEK293T cells transiently transfected with TAP-tagged HopZ1a and HopZ1a (C216A) with anti-HA antibody.
Figure 2
Figure 2. HopZ1a binds unassembled tubulin heterodimers and assembled tubulin polymers, the microtubules.
(A) HIS-HopZ1a, HIS-HopZ1a(C216A) and GST were immobilized on the surface of a Biacore CM5 sensor chip at the following response units (RU), 9953 RU, 8784 RU and 3800 RU, respectively. 500 µg/ml of bovine brain tubulin was flowed across the recombinant HopZ1a, HopZ1a(C216A) and GST -bound surface, generating a RU difference of 1049 RU, 1150 RU and −12.2 RU, respectively. The start and end of tubulin injection are indicated by arrows. (B) 6973 RU, 13598 RU or 18289 RU of HIS-HopZ1a was immobilized on a Biacore CM5 sensor chip. 500 µg/ml of bovine brain tubulin was flowed across the recombinant HopZ1a-bound surfaces, generating a RU difference of 59 RU, 324 RU and 615 RU, respectively. (C) Immunoblot analysis of HIS-HopZ1a and HIS-HopZ1a(C216A) proteins in a microtubule co-sedimentation assay detected with rabbit α-HIS antibody. In the absence of microtubules, HIS-HopZ1a and HIS-HopZ1a(C216A) proteins were found only in the supernatant (S) fractions. In the presence of microtubules, HIS-HopZ1a and HIS-HopZ1a(C216A) proteins were found predominantly in the pellet (P) fractions. (D) HopZ1a binds to tubulin in planta. 1.5 ml of taxol-treated clarified extracts from transgenic Arabidopsis expressing HA-tagged HopZ1a(C216A) or HopF2 were bound to 30 µl of α-HA antibody-coated agarose beads. Proteins were eluted from the α-HA beads by boiling the beads in 100 µl of Laemmli sample buffer. 5 µl, 2.5 µl and 1.25 µl of the resulting eluates (corresponding to 5%, 2.5% and 1.25% of eluates) were subjected to immunoblots using α-HA antibodies (top panel) or α-tubulin antibodies (bottom panel). In the top panel 20 ul of HopZ1a clarified extract and 5 ul of HopF2 clarified extract was loaded whereas in the bottom panel 3 ul of each clarified extract was loaded, The band intensities of HopZ1a, HopF2, and tubulin bands were quantified by ImageJ and shown at the bottom of each panel. The maximum band intensity observed in each blot is arbitrarily set at 1 and all other band intensities are shown relative to that value. The ratios of tubulin:HopZ1a(C216A) and tubulin:HopF2 are indicated at the bottom of the figure. (*) indicates non-specific, cross-reactive bands.
Figure 3
Figure 3. HopZ1a is an acetyltransferase activated by phytic acid and acetylates tubulin.
Purified HIS-HopZ1a (∼42 kDa), GST-HopZ1a (∼68 kDa) and HIS-HopZ1a(C216A) (∼42 kDa) proteins were incubated with or without 10 µg of tubulin heterodimers (∼55 kDa) or 100 nM phytic acid in the presence of 14C-labeled acetyl-CoA for 1 hour at 30°C. The acetyltransferase activity of HopZ1a is activated by phytic acid. Active HopZ1a autoacetylates in cis and acetylates tubulin. All samples were separated by 12% SDS-PAGE and the 14C-incorporation was analyzed by Phosphorimager.
Figure 4
Figure 4. The autoacetylation site of HopZ1a, K289, is important for the avirulence and virulence function of HopZ1a.
(A) The protein sequence of HopZ1a is aligned with HopZ1b, HopZ2 and PopP2 using Clustal W. The region flanking the conserved lysine residue is shown, with lysine 289 (in HopZ1a) indicated by a star. (B) Purified recombinant GST-HopZ1a, GST-HopZ1a(C216A) and GST-HopZ1a (K289R) proteins were incubated with tubulin heterodimers in the presence of 14C-labeled acetyl-CoA for 1 hour at 30°C. All samples were separated by 12% SDS-PAGE and the 14C-incorporation was analyzed by Phosphorimager. (C) Macroscopic HR of Arabidopsis Col-0 leaves infiltrated with 2×107 CFU/ml of PtoDC3000 expressing pUCP20-hopZ1a-HA (HopZ1a WT), pUCP20-hopZ1a(C216A)-HA [HopZ1a (C216A)] or pUCP20-hopZ1a(K289R)-HA [HopZ1a(K289R)]. (*) indicate HR. (D) Quantification of HR by electrolyte leakage of Arabidopsis Col-0 leaf discs after infiltration with 5×107 CFU/ml of PtoDC3000 expressing empty vector (EV), pUCP20-hopZ1a-HA (HopZ1a WT), pUCP20-hopZ1a(C216A)-HA [HopZ1a (C216A)], or pUCP20-hopZ1a(K289R)-HA [HopZ1a(K289R)]. Error bars represent standard error and (*) indicate statistically significant differences (2-tailed student t-test, p<0.01). The experiment was repeated twice with similar results. (E) P. syringae (Pci0788-9) growth assay in Arabidopsis. Pci0788-9 carrying pUCP20-hopZ1a-HA (HopZ1a WT) grew significantly better than Pci0788-9 carrying pUCP20-hopZ1a(K289R)-HA [HopZ1a(K289R)] or empty vector (EV) on day 3. The bacterial growth difference between HopZ1a WT and HopZ1a K289R or EV was statistically significant [as indicated by (*), 2-tailed student t-test, p<0.01]. Error bars represent standard error. Experiments were repeated three times and the data from one representative experiment is presented.
Figure 5
Figure 5. Effects of HopZ1a on the microtubule networks.
Confocal microscopy images of five-day-old GFP-MAP4 (A) and GFP-AtEB1 (B) seedlings infected with PtoDC3000 expressing empty vector pUCP20, pUCP20-hopZ1a-HA, pUCP20-hopZ1a(C216A)-HA, or pDSK519-avrRpt2 for ∼16 hours. Scale bar = 25 µm. (A) Quantification of the GFP fluorescence of GFP-MAP4 from 61 uninfected cells, 57 PtoDC3000-infected cells, 61 PtoDC3000(HopZ1a)-infected cells, 71 PtoDC3000(HopZ1aC216A)-infected cells and 67 PtoDC3000(AvrRpt2)-infected cells. (B) Quantification of the GFP fluorescence of GFP-AtEB1 from 37 uninfected cells, 82 PtoDC3000-infected cells, 127 PtoDC3000(HopZ1a)-infected cells, 90 PtoDC3000(HopZ1aC216A)-infected cells and 122 PtoDC3000(AvrRpt2)-infected cells. Error bars indicate standard error. [(*) indicate statistical significance. P = 0.05, Fisher's PLSD posthoc test.]
Figure 6
Figure 6. Microtubule destruction promotes P. syringae growth.
P. syringae growth assay in Arabidopsis Col-0. In the presence of microtubule inhibitor, oryzalin, PtoDC3000 (DC) grew significantly better after three days, while P. syringae (DC hrcC) without a functional TTSS did not. The bacterial growth difference between DC in the presence or absence of oryzalin was statistically significant [as indicated by (*), 2-tailed student t-test, p = 0.008]. Experiments were repeated three times and the data from one representative experiment is presented.
Figure 7
Figure 7. HopZ1a blocks the plant secretory pathway.
Secretion assay in N. benthaminana. Extracellular fluid was isolated at 24 h post-inoculation from N. benthamiana expressing secGFP infiltrated with Agrobacterium carrying HopZ1a-myc [HopZ1a], HopZ1a(C216A)-myc [HopZ1a(C/A)], and AtSYP121-Sp2-myc [AtSYP121]. The expression of secGFP in the extracellular or intracellular fractions was analyzed by immunoblots using an anti-GFP serum; the expression of effectors was analyzed with anti-myc antibody. Transient expression of HopZ1a-myc, but not the catalytic mutant HopZ1a(C216A)-myc, reduced the accumulation of secGFP in the apoplastic fluid of N. benthamiana.
Figure 8
Figure 8. HopZ1a inhibits cell wall-based defense.
(A) zar1-1/Dex:hopZ1a and zar1-1/Dex:hopZ1a(C216A) transgenic leaves were sprayed with water (−DEX) or 30 µM dexamethasone to induce HopZ1a protein expression (+DEX) for 24 h. Leaves were then syringe-infiltrated with 10 µM of flg22 for 24 h, followed by clearing and staining with 0.01% Aniline blue for callose. Expression of HopZ1a (+DEX), but not HopZ1a(C216A), suppressed flg22-induced callose deposition. (B) Quantification of callose depositions of 12 images per treatment. Error bars indicate standard error.
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