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

The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as “non-self” features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as “non-self” features or induce a “modified-self” state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2Pto also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2Pto were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2Pto interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2Pto. In support of this hypothesis, HopF2Pto interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2Pto did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2Pto and that a pathogenic advantage can be conveyed by TTSEs that target RIN4

The peptide binding preferences of each PDZ domain of Baz determined by phage display screens.

<p>(<b>A</b>) Peptides identified to interact with the PDZ domains of Baz. For PDZ1 and PDZ3 screens 1 and 2 were fully independent. Individual peptides are shown with their C-termini to the right. Sequence logos with weighted amino acid positions are shown below. Colours represent the following: black, hydrophobic; green; polar; red, negative; blue positive. (<b>B</b>) A Coomassie brilliant blue-stained 10% SDS-PAGE gel showing that each GST fusion protein used in the phage display had a similar stability. Equimolar amounts were loaded. The gel also shows that GST-Baz PDZ1-3 was equally stable (further analyses shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086412#pone-0086412-t001" target="_blank">Table 1</a>). (<b>C</b>) Peptides previously identified to interact with <i>C. elegans</i> Par-3 PDZ2 and PDZ3 and human Par-3 PDZ3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086412#pone.0086412-Tonikian1" target="_blank">[28]</a>. Position P₀ residues with identity to those in the corresponding Baz PDZ domain-interacting peptides are shown in red. For peptides with position P₀ matches, matching internal residues are also indicated in red until the identity with a Baz peptide ends.</p

Peptide binding strengths of Baz PDZ domains alone or in tandem.

*<p>Replicates used separately purified proteins and separately prepared peptides.</p

Quantification of Baz PDZ domain-peptide interactions by surface plasmon resonance.

<p>(<b>A</b>) Peptides used in direct interaction assays. They include a common linker that was also found in the peptide library for the phage display screen, and they were biotinylated at their N-termini. One peptide was selected for each of the PDZ domains from those identified in the phage display screen (Peptide 1 for PDZ1, etc). (<b>B</b>) Quantifications of binding between each peptide and each PDZ domain at the indicated peptide concentrations by SPR. PDZ1 and PDZ3 showed strong selective binding to their respective peptides. PDZ2 showed weaker binding but still preferred Peptide 2. The data represent the means±SD for four runs with the same protein and peptide samples. The experiment was replicated with separately purified proteins and separately prepared peptides with similar results (data not shown). (<b>C</b>) SPR response curves for different concentrations of Peptide 1 binding to immobilized GST-PDZ1. The peptide injection and wash times are indicated. The red lines are fits of the curves used to determine the K_D values shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086412#pone-0086412-t001" target="_blank">Table 1</a>. (<b>D–E</b>) The equivalent analyses as in (<b>C</b>) but for Peptide 2-PDZ2 and Peptide 3-PDZ3 binding, respectively. Note the weaker interactions of Peptide 2-PDZ2.</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

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

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

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