A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

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

Microtubule destruction promotes <i>P. syringae</i> growth.

<p><i>P. syringae</i> growth assay in Arabidopsis Col-0. In the presence of microtubule inhibitor, oryzalin, <i>Pto</i>DC3000 (DC) grew significantly better after three days, while <i>P. syringae</i> (DC <i>hrcC</i>) 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.</p

Effects of HopZ1a on the microtubule networks.

<p>Confocal microscopy images of five-day-old GFP-MAP4 (<b>A</b>) and GFP-AtEB1 (<b>B</b>) seedlings infected with <i>Pto</i>DC3000 expressing empty vector <i>pUCP20</i>, <i>pUCP20-hopZ1a-HA</i>, <i>pUCP20-hopZ1a(C216A)-HA</i>, or <i>pDSK519-avrRpt2</i> for ∼16 hours. Scale bar = 25 µm. (<b>A</b>) Quantification of the GFP fluorescence of GFP-MAP4 from 61 uninfected cells, 57 <i>Pto</i>DC3000-infected cells, 61 <i>Pto</i>DC3000(HopZ1a)-infected cells, 71 <i>Pto</i>DC3000(HopZ1aC216A)-infected cells and 67 <i>Pto</i>DC3000(AvrRpt2)-infected cells. (<b>B</b>) Quantification of the GFP fluorescence of GFP-AtEB1 from 37 uninfected cells, 82 <i>Pto</i>DC3000-infected cells, 127 <i>Pto</i>DC3000(HopZ1a)-infected cells, 90 <i>Pto</i>DC3000(HopZ1aC216A)-infected cells and 122 <i>Pto</i>DC3000(AvrRpt2)-infected cells. Error bars indicate standard error. [(*) indicate statistical significance. P = 0.05, Fisher's PLSD posthoc test.]</p

HopZ1a is an acetyltransferase activated by phytic acid and acetylates tubulin.

<p>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 <i>in cis</i> and acetylates tubulin. All samples were separated by 12% SDS-PAGE and the ¹⁴C-incorporation was analyzed by Phosphorimager.</p

HopZ1a inhibits cell wall-based defense.

<p>(<b>A</b>) <i>zar1-1/Dex:hopZ1a</i> and <i>zar1-1/Dex:hopZ1a(C216A)</i> 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>B</b>) Quantification of callose depositions of 12 images per treatment. Error bars indicate standard error.</p

The autoacetylation site of HopZ1a, K289, is important for the avirulence and virulence function of HopZ1a.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) Macroscopic HR of Arabidopsis Col-0 leaves infiltrated with 2×10⁷ CFU/ml of <i>Pto</i>DC3000 expressing <i>pUCP20-hopZ1a-HA</i> (HopZ1a WT), <i>pUCP20-hopZ1a(C216A)-HA</i> [HopZ1a (C216A)] or <i>pUCP20-hopZ1a(K289R)-HA</i> [HopZ1a(K289R)]. (*) indicate HR. (<b>D</b>) Quantification of HR by electrolyte leakage of Arabidopsis Col-0 leaf discs after infiltration with 5×10⁷ CFU/ml of <i>Pto</i>DC3000 expressing empty vector (EV), <i>pUCP20-hopZ1a-HA</i> (HopZ1a WT), <i>pUCP20-hopZ1a(C216A)-HA</i> [HopZ1a (C216A)], or <i>pUCP20-hopZ1a(K289R)-HA</i> [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. (<b>E</b>) <i>P. syringae</i> (<i>Pci</i>0788-9) growth assay in Arabidopsis. <i>Pci</i>0788-9 carrying <i>pUCP20-hopZ1a-HA</i> (HopZ1a WT) grew significantly better than <i>Pci</i>0788-9 carrying <i>pUCP20-hopZ1a(K289R)-HA</i> [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.</p