AvrBsT Acetylates <i>Arabidopsis</i> ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity

<div><p>Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in <i>Arabidopsis thaliana</i> Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for <i>ACETYLATED INTERACTING PROTEIN1</i>), an unknown protein from <i>Arabidopsis</i>. Genetic studies revealed that <i>Arabidopsis</i> ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during <i>Pseudomonas syringae</i> pathovar <i>tomato</i> DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation <i>in planta</i> alters ACIP1's defense function, which is linked to the activation of ETI.</p></div

Members of <i>Arabidopsis</i> ACIP family are required for AvrBsT-triggered ETI.

<p>(A) Increased growth of Pst DC3000 and Pst DC3000 AvrBsT in Pi-0 <i>ACIP</i> RNAi line #1 (red bars) and line #29 (blue bars) compared to wild-type Pi-0 (black bars). Leaves were syringe-infiltrated with a 1×10⁵ cells/mL suspension of bacteria. Titers were assessed at 0 and 3 days post-inoculation (DPI). Data are mean cfu/cm² ± SD (n = 4). Asterisks indicate statistically significant differences from Pi-0 (student <i>t</i>-test, *<i>p</i><0.05, **<i>p</i><0.01). Experiment was repeated three times with similar results. Inset: Immunoblot analysis of protein extracted from Pi-0 and Pi-0 <i>ACIP</i> RNAi leaves using anti-ACIP1 sera. Black dot, non-specific band (NS); arrowhead, detected ∼20 kDa protein band expected to correspond to ACIP1, ACIP-L1, and/or ACIP-L3. STD, molecular weight standard in kDa. Ponceau S-stained Rubisco large subunit was used as loading control. (B) AvrBsT-elicited HR phenotype in Pi-0 and Pi-0 <i>ACIP</i> RNAi lines. Leaves were infiltrated with a 3×10⁸ cells/mL suspension of Pst DC3000 alone (vector) or Pst DC3000 AvrBsT (AvrBsT). Photograph was taken at 9 hours post-inoculation (HPI). Number of leaves exhibiting confluent HR at 10 HPI out of 25 inoculated leaves is shown at right. (C) Quantification of electrolyte leakage in the leaves described in (B) at 10 HPI. Error bars represent SD (n = 10). Asterisks indicate statistically significant differences from Pi-0 (student's <i>t</i>-test, *p<0.05). Experiment was repeated three times with similar results.</p

AvrBsT alters GFP-ACIP1's localization in a catalytic-dependent manner.

<p>Pi-0 <i>P_ACIP1</i>::<i>GFP-ACIP1</i> leaves were inoculated with (A) 1 mM MgCl₂ or a 3×10⁸ cells/mL suspension of (B) Pst DC3000 vector, (C) Pst DC3000 Δ<i>hrcU</i>, (D) Pst DC3000 AvrBsT, (E) Pst DC3000 AvrBsT(H154A), (F) Pst DC3000 AvrBsT(K282R), or (G) Pst DC3000 AvrRpt2. Spinning disk confocal images were recorded at 6–7 HPI. Bar = 10 µm. Similar results were obtained in more than 3 independent experiments.</p

Mutation of K282 attenuates AvrBsT-triggered resistance.

<p>(A) Growth of Pst DC3000 in <i>Arabidopsis</i> Pi-0 leaves. Leaves were syringe infiltrated with a 1×10⁵ cells/mL suspension of bacteria: Pst DC3000 carrying vector (black bars), AvrBsT (white bars), AvrBsT(H154A) (dark grey bars) or AvrBsT(K282R) (light grey bars). Titers were assessed at 0 and 3 days post-inoculation. Data are mean cfu/cm² ± SD (n = 6). Asterisks indicate statistically significant differences from Pi-0 (student's <i>t</i>-test, **<i>p</i><0.01). Similar results were obtained in three independent experiments. (B) HR phenotypes in Pi-0 leaves. Leaves were infiltrated with a 3×10⁸ cells/mL suspension of Pst DC3000 carrying vector, AvrBsT or AvrBsT(K282R). Photograph was taken at 12 hours post-inoculation (HPI). Number of leaves exhibiting confluent HR at 10 HPI out of 18 inoculated leaves is shown at bottom.</p

AvrBsT is an acetyltransferase that specifically acetylates ACIP1.

<p>(A) AvrBsT auto-acetylation activity <i>in vitro</i>. Acetylation reactions using GST and GST-AvrBsT (wild-type, C222A, H154A, and K282R) proteins. (B) AvrBsT trans-acetylates ACIP1 <i>in vitro</i>. Acetylation reactions using GST-ACIP1 or GST with GST-AvrBsT, GST-AvrBsT(C222A), GST-AvrBT(H154A) or GST-AvrBsT(K282R). (C) Substrate specificity of AvrBsT and HopZ1a. Acetylation reactions using GST-ACIP1 or GST with GST-HopZ1a or GST-AvrBsT. For acetylation reactions, proteins were incubated with 0.4 µCi ¹⁴C-acetyl CoA and 100 nM inositol hexakisphosphate (IP₆) for 30 min at RT. Proteins were then separated by SDS-PAGE. Gels were stained with Coomassie and then analyzed by autoradiography. GST and GST-HopZ1a were used as negative and positive acetyltransferase enzyme controls, respectively. Acetylated proteins (GST-HopZ1a-^AC, GST-AvrBsT-^AC, and GST-ACIP1-^AC) are labeled in the autoradiograph. STD, molecular weight standard in kDa. GST = 28 kDa; GST-HopZ1a = 70 kDa; GST-AvrBsT = 65 kDa; GST-ACIP1 = 50 kDa. Similar results were obtained in three independent experiments.</p

AvrBsT interacts with <i>Arabidopsis</i> ACIP1.

<p>(A) Yeast two-hybrid assay showing AvrBsT binding to <i>Arabidopsis</i> ACIP1. Yeast strain AH109 carrying pXDGATcy86 (vector) or pXDGATcy86(AvrBsT) were independently transformed with the pGADT7 (vector) or pGADT7 containing ACIP1. pXDGATcy86 contains the GAL4 DNA-binding domain (BD) and pGADT7 contains the GAL4 activation domain (AD). Strains were spotted on nonselective (SD – LW) and selective (SD –LWH +1 mM 3-AT) media and then incubated at 30°C for 3 d. (B) GST-AvrBsT affinity purification of His6-ACIP1 <i>in vitro</i>. GST or GST-AvrBsT was incubated with <i>E. coli</i> extracts containing His6-ACIP1. Proteins were purified by using glutathione sepharose and analyzed by immunoblot (IB) analysis using anti-GST and anti-His sera. Protein input is shown on left and pull-down on right. Expected protein MW: GST = 28 kDa; GST-AvrBsT = 65 kDa; and His6-ACIP1 = 28 kDa. +, protein expressed; −, vector control. STD, molecular weight standard. Similar phenotypes were observed in at least 3 independent experiments.</p

Members of <i>Arabidopsis</i> ACIP family are required for PTI.

<p>(A) Flg22-stimulated oxidative burst response in Pi-0 and Pi-0 <i>ACIP</i> RNAi leaves. RLU = relative luminescence unit. Error bars represent SD (n = 9). Response in both RNAi lines (#1 and #29) was significantly different from that in Pi-0 between time interval 28–32 minutes (student's <i>t</i>-test, **<i>p</i><0.01). (B) Flg22-stimulated PTI marker gene induction in Pi-0 and Pi-0 <i>ACIP</i> RNAi leaves. Leaves of three plants were infiltrated with water (control) or 100 nM flg22 and then pooled for RNA extraction. <i>WRKY22</i> and <i>WRKY29</i> mRNA levels were quantified by qPCR. UBQ5 was used to normalize the expression value for each sample. Relative expression (mean ± SD; n = 4) is shown. (C) Growth of <i>Pst</i> DC3000 <i>ΔhrcU</i> in Pi-0 (black bars) and Pi-0 <i>ACIP</i> RNAi leaves (red and blue bars). Leaves were inoculated with a 1×10⁵ cells/mL suspension of bacteria. Titers were assessed at 0 and 4 DPI. Data are mean cfu/cm² ± SD (n = 4). (D) Pst DC3000 <i>ΔhrcU</i>-stimulated PTI marker gene induction in Pi-0 and Pi-0 <i>ACIP</i> RNAi lines. Leaves were infiltrated with a 2×10⁸ cells/mL suspension of Pst DC3000 (grey bar), Pst DC3000 (<i>ΔhrcU</i>) (black bar) or 1 mM MgCl₂ (white bar). Samples were collected at 6 HPI and then analyzed as described in (B). Asterisks indicate statistically significant differences from Pi-0 (student's <i>t</i>-test,*<i>p</i><0.05,**<i>p</i><0.01, ***p<0.001). Similar results were obtained in three independent experiments for (A–C), and two independent experiments for (D).</p

GFP-ACIP1 co-localizes with microtubules.

<p>(A) Single plane images of periclinal surface of epidermal cells of 4-day old etiolated Pi-0 <i>P_ACIP1</i>::<i>GFP-ACIP1/P_35S::mCHERRY-TUA5</i> hypocotyl cells. Arrowheads show GFP-ACIP1 punctae that are not associated with microtubules. (B) Localization of GFP-ACIP1 in 4-day old etiolated Pi-0 <i>P_ACIP1</i>::<i>GFP-ACIP1</i> hypocotyls treated with MeOH or MeOH +10 µM oryzalin (top panels), or DMSO or DMSO +1 µM latrunculin B (bottom panels). Cells were imaged using confocal microscopy. Bars = 10 µm.</p

Regulation of Cell Wall-Bound Invertase in Pepper Leaves by <em>Xanthomonas campestris</em> pv. <em>vesicatoria</em> Type Three Effectors

<div><p><em>Xanthomonas campestris</em> pv. <em>vesicatoria (Xcv)</em> possess a type 3 secretion system (T3SS) to deliver effector proteins into its <em>Solanaceous</em> host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that <em>Xcv</em> may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient <em>Xcv</em> strain showed a higher level of cw-Inv mRNA and enzyme activity relative to <em>Xcv</em> wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the <em>pathogenesis-related</em> gene <em>PRQ.</em> These results suggest that <em>Xcv</em> suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify <em>Xcv</em> T3Es that regulate cw-Inv activity, a screen was performed with eighteen <em>Xcv</em> strains, each deficient in an individual T3E. Seven <em>Xcv</em> T3E deletion strains caused a significant change in cw-Inv activity compared to <em>Xcv</em> wild type. Among them, <em>Xcv</em> lacking the <em>xopB</em> gene (<em>Xcv</em> Δ<em>xopB</em>) caused the most prominent increase in cw-Inv activity. Deletion of <em>xopB</em> increased the mRNA abundance of <em>PRQ</em> in <em>Xcv</em> Δ<em>xopB-</em>infected pepper leaves, but not of <em>Pti5</em> and <em>Acre31,</em> two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited <em>Xcv</em>-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity <em>in planta</em> to suppress sugar-enhanced defense responses during <em>Xcv</em> infection.</p> </div

Reduced <i>TFT1</i> mRNA expression in VIGS tomato leaves correlates with reduced PTI marker mRNA abundance in response to Xcv infection.

<p>Relative mRNA levels for four PTI marker genes (<i>PTI5</i>, <i>GRAS4</i>, <i>WRKY28</i>, and <i>LRR22</i>) in 4 control (TRV2) and 4 <i>TFT1</i>-silenced (TRV2-TFT1) tomato lines. Leaflets on the same branch were inoculated with 1×10⁵ CFU/mL of Xcv or Xcv <i>ΔhrpF</i>. Total RNA isolated from inoculated leaves at 6 HPI was used for Q-PCR. <i>Actin</i> mRNA expression was used to normalize the expression value in each sample. Error bars indicate SD for four plants. Asterisk indicates significant difference (<i>t</i> test, P<0.05) in the infected TRV2-TFT1 lines compared to the similarly infected TRV2 lines.</p