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. 2014 Feb 20;10(2):e1003952.
doi: 10.1371/journal.ppat.1003952. eCollection 2014 Feb.

AvrBsT acetylates Arabidopsis ACIP1, a protein that associates with microtubules and is required for immunity

Mi Sun Cheong  1 , Angela Kirik  2 , Jung-Gun Kim  1 , Kenneth Frame  1 , Viktor Kirik  2 , Mary Beth Mudgett  1
Affiliations

AvrBsT acetylates Arabidopsis ACIP1, a protein that associates with microtubules and is required for immunity

Mi Sun Cheong et al. PLoS Pathog. .

Abstract

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 Arabidopsis thaliana 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 ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato 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 in planta alters ACIP1's defense function, which is linked to the activation of ETI.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. AvrBsT interacts with Arabidopsis ACIP1.
(A) Yeast two-hybrid assay showing AvrBsT binding to Arabidopsis 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 in vitro. GST or GST-AvrBsT was incubated with E. coli 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.
Figure 2
Figure 2. AvrBsT is an acetyltransferase that specifically acetylates ACIP1.
(A) AvrBsT auto-acetylation activity in vitro. Acetylation reactions using GST and GST-AvrBsT (wild-type, C222A, H154A, and K282R) proteins. (B) AvrBsT trans-acetylates ACIP1 in vitro. 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.
Figure 3
Figure 3. Mutation of K282 attenuates AvrBsT-triggered resistance.
(A) Growth of Pst DC3000 in Arabidopsis 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 t-test, **p<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.
Figure 4
Figure 4. Members of Arabidopsis ACIP family are required for AvrBsT-triggered ETI.
(A) Increased growth of Pst DC3000 and Pst DC3000 AvrBsT in Pi-0 ACIP 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 t-test, *p<0.05, **p<0.01). Experiment was repeated three times with similar results. Inset: Immunoblot analysis of protein extracted from Pi-0 and Pi-0 ACIP 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 ACIP 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 t-test, *p<0.05). Experiment was repeated three times with similar results.
Figure 5
Figure 5. Members of Arabidopsis ACIP family are required for PTI.
(A) Flg22-stimulated oxidative burst response in Pi-0 and Pi-0 ACIP 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 t-test, **p<0.01). (B) Flg22-stimulated PTI marker gene induction in Pi-0 and Pi-0 ACIP RNAi leaves. Leaves of three plants were infiltrated with water (control) or 100 nM flg22 and then pooled for RNA extraction. WRKY22 and WRKY29 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 Pst DC3000 ΔhrcU in Pi-0 (black bars) and Pi-0 ACIP 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 ΔhrcU-stimulated PTI marker gene induction in Pi-0 and Pi-0 ACIP RNAi lines. Leaves were infiltrated with a 2×108 cells/mL suspension of Pst DC3000 (grey bar), Pst DC3000 (ΔhrcU) (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 t-test,*p<0.05,**p<0.01, ***p<0.001). Similar results were obtained in three independent experiments for (A–C), and two independent experiments for (D).
Figure 6
Figure 6. GFP-ACIP1 co-localizes with microtubules.
(A) Single plane images of periclinal surface of epidermal cells of 4-day old etiolated Pi-0 PACIP1::GFP-ACIP1/P35S::mCHERRY-TUA5 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 PACIP1::GFP-ACIP1 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.
Figure 7
Figure 7. AvrBsT alters GFP-ACIP1's localization in a catalytic-dependent manner.
Pi-0 PACIP1::GFP-ACIP1 leaves were inoculated with (A) 1 mM MgCl2 or a 3×108 cells/mL suspension of (B) Pst DC3000 vector, (C) Pst DC3000 ΔhrcU, (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.
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