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. 2016 Mar 25;291(13):6813-30.
doi: 10.1074/jbc.M115.678953. Epub 2016 Jan 28.

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

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RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

Shoko Fujiwara et al. J Biol Chem. .
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Abstract

The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.

Keywords: Saccharomyces cerevisiae; plant defense; redox regulation; thioredoxin; type III secretion system (T3SS).

Figures

FIGURE 1.
FIGURE 1.
Expression of RipAY, but not other ChaC family proteins from various organisms, causes growth inhibition of yeast. A, phylogenetic analysis of ChaC domain containing proteins from various organisms. The organisms, locus tag/gene name, and GenbankTM accession numbers (in parentheses) are as follows: E. coli ChaC (L28709.1); R. solanacearum RSc0782 (AL646052.1) and RSp1022/RipAY (AL646053.1); A. citrulli Aave_2801 (CP000512.1) and Aave_4606 (CP000512.1); P. syringae PSPT_05239 (AE016853.1); S. cerevisiae YER163c/Gcg1 (NM_001179053.3); Aspergillus oryzae ChaC (XM_001818295.2); Homo sapiens ChaC1 (NM_001142776.1) and ChaC2 (NM_001008708.2); Mus musculus ChaC1 (NM_026929.4) and ChaC2 (NM_026527.3); Xenopus tropicalis ChaC1 (NM_001130284.1) and ChaC2 (NM_001017137.2); Gallus gallus ChaC1 (NM_001199656.1) and ChaC2 (AJ720918.1); Danio rerio ChaC1 (NM_001110126.1) and ChaC2 (BC154137.1); A. thaliana AT5G26220/GGCT2;1 (BT005234.1), AT4G31290/GGCT2;2 (BT006411.1), and AT1G44790/GGCT2;3 (BT006192.1). B, schematic representation of putative domain organization of ChaC domain-containing proteins from various organisms. C, yeast growth inhibition assay showing serial dilutions of S. cerevisiae BY4743 cells grown under inducing (galactose) or noninducing (glucose) conditions that are carrying plasmids expressing the indicated C-terminal GFP-tagged proteins under the control of the GAL1 promoter. The cells were grown at 26 °C for 2 days for SD (−Ura) and 3 days for SGal (−Ura). D and E, detections of the ChaC domain-containing proteins expressed in yeast. Yeast cells carrying the plasmids expressing GFP-tagged RipAY or other ChaC proteins under the control of the GAL1 promoter were grown in SD (−Ura) liquid medium to mid-log phase and then shifted to SGal (−Ura) liquid medium to induce the protein expression, further cultured for 12 h, and analyzed by immunoblotting with anti-GFP antibody (D) or fluorescence microscopy with native GFP fluorescence (E). Glucose 6-phosphate dehydrogenase (G6PDH) was used as a loading control. Scale bar, 5 μm. DIC, differential interference contrast.
FIGURE 2.
FIGURE 2.
Expression of RipAY in yeast causes growth inhibition by depletion of intracellular glutathione through its conserved ChaC domain. A, the GSH catabolism by a classical γ-glutamyl transpeptidase- and GGCT-dependent pathway and a novel ChaC family-dependent pathway. GGT, γ-glutamyl transpeptidase. B, the RipAY protein was modeled using the Phyre2 server. The homology model obtained was superimposed on the crystal structure of GGCT (C7orf24, Protein Data Bank codes 2PN7 and 2RBH) using the graphics program CueMol. Shown is superimposition of homology-modeled RipAY structure (green) on GGCT structure (blue). C, putative active site residues of RipAY (Y129LSL and Glu216; green) were superimposed on corresponding active site residues of GGCT (Y22GSN and Glu98; blue). D, multiple alignment of amino acid sequences in the regions of putative substrate binding and catalytic sites of the class I and II ChaC proteins. The conserved amino acids, Y129XSL and Glu216, are the putative substrate binding site and catalytic glutamate residue for GGCT, respectively. E, Protein A-tagged Gcg1, RipAY WT, and active site mutant, E216Q, were expressed in a glutathione-degrading enzyme-deficient strain (dug3Δ ecm38Δ gcg1Δ triple mutant) of yeast cells and immunopurified with IgG-beads. The GGCT activity of the beads immobilized with Gcg1, RipAYWT, or RipAYE216Q proteins was measured by a Dug1-coupled method as described under “Experimental Procedures.” EV, empty vector. F, the proteins extracted from the beads shown in E were resolved on SDS-PAGE followed by immunoblotting using a rabbit IgG. G, mutations in putative substrate binding and catalytic sites of RipAY restore the yeast growth inhibition caused by expression of RipAY. Yeast cells carrying empty vector or GAL1 expression vector of WT or the indicated mutant proteins of RipAY were spotted on SD (−Ura) and SGal (−Ura) plates and cultured for 2 days and 3 days, respectively. H, yeast cells carrying GAL1 expression plasmid of GFP-tagged WT or the indicated mutant proteins of RipAY were grown in SD (−Ura) liquid medium to mid-log phase and then shifted to SGal (−Ura) liquid medium to induce the protein expression, further cultured for 12 h, and analyzed by immunoblotting with anti-GFP antibody. I, mutations in putative substrate binding and catalytic sites of RipAY restore the reduction of intracellular GSH level in yeast caused by expression of RipAY. The total GSH levels of the cell lysates from yeast cells expressing the indicated proteins were measured as described under “Experimental Procedures.” Values represent the mean ± S.E. (error bars) (n ≥ 3).
FIGURE 3.
FIGURE 3.
Increased uptake of GSH does not rescue the growth inhibition effect caused by expression of RipAY. A, yeast wild-type and gsh1Δ/gsh1Δ homozygote cells carrying the indicated plasmids were spotted on SD (−Ura) (OFF) or SGal (−Leu, −Ura) (ON) plates supplemented with the indicated concentration of GSH. EV, empty vector. Cells were incubated at 26 °C for 2 days for SD (−Leu, −Ura) or 3 days for SGal (−Leu, −Ura). B, yeast cells carrying the indicated plasmids were spotted on SD (−Leu, −Ura) (RipAY expression: OFF) or SGal (−Leu, −Ura) (RipAY expression: ON) plates supplemented with the indicated concentration of GSH. Cells were incubated at 26 °C for 2 days for SD (−Leu, −Ura) or 3 days for SGal (−Leu, −Ura). RipAY or HGT1 were expressed under the control of a galactose-inducible GAL1 promoter or a strong constitutive TEF1 promoter, respectively. C, yeast cells carrying the indicated plasmids growing exponentially in SD (−Leu, −Ura) liquid medium were transferred to SGal (−Leu, −Ura) liquid medium and cultured at 26 °C for 19 h, and then GSH (100 μm) was supplemented at 30 min before termination of the culture. D, yeast cells carrying indicated plasmids growing exponentially in SD (−Leu, −Ura) liquid medium were transferred to SGal (−Leu, −Ura) liquid medium supplemented with or without 100 μm GSH and cultured at 26 °C for 19 h. The total GSH levels of the cell lysates from yeast cells expressing indicated proteins were measured. Values represent the mean ± S.E. (error bars) (n = 4 for C and n = 3 for D).
FIGURE 4.
FIGURE 4.
The GSH level of eggplant leaves inoculated with R. solanacearum wild-type, RipAY-deficient, and T3SS-deficient strains. A, necrotic lesions on eggplant leaf 3 days postinoculation with R. solanacearum WT strain (WT), RipAY-deficient strain (ΔripAY), or T3SS-deficient strain (ΔhrcU). Mock, inoculation of buffer without bacteria as a control. B, the GSH level of total lysate extracted from eggplant leaves inoculated with the indicated R. solanacearum strains at 1 day postinoculation was measured as described under “Experimental Procedures.” Values represent the mean ± S.E. (error bars) (n ≥ 7). ****, p < 0.0001, one-way analysis of variance post hoc test (Turkey's multiple comparison test).
FIGURE 5.
FIGURE 5.
RipAY exhibits GGCT activity in the presence of eukaryotic factor(s). A, purification of recombinant Gcg1 and RipAY proteins. Recombinant His6-tagged Gcg1 and RipAY were expressed in E. coli and purified using nickel-nitrilotriacetic acid affinity chromatography. Purified proteins were analyzed on SDS-PAGE with Coomassie Blue staining. B, measurement of GGCT activity of recombinant Gcg1 and RipAY purified from E. coli. 0.1 μg of Gcg1 and 1 μg of RipAY were incubated with 5 mm glutathione, the reactions were terminated at the indicated time points, the released Cys-Gly dipeptides were digested with Cys-Gly dipeptidase Dug1, and then the released Cys was measured by acidic ninhydrin. C, effect of concentration of the yeast protein extract on activation of GGCT activity of RipAY. The beads bound with RipAYWT or RipAYE216Q proteins expressed in E. coli were preincubated with different concentrations of native yeast protein extracted from a glutathione-degrading enzyme-deficient strain (dug3Δ ecm38Δ gcg1Δ triple mutant) for 2 h at 4 °C and washed with the same buffer three times, and then GGCT activity of the beads was measured by the Dug1-coupled method. D, the Protein A-tagged RipAYWT and RipAYE216Q proteins extracted from the beads shown in C were resolved on SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining. E, the GGCT activity of bacterially expressed RipAY-His6 protein incubated with 10 μg of protein of the cell lysates from R. solanacearum, S. cerevisiae, human cultured cell HeLa, A. thaliana, or eggplant S. melongena cells was measured by a Dug1-coupled method. Values represent the mean ± S.E. (error bars) (n ≥ 3).
FIGURE 6.
FIGURE 6.
Identification of thioredoxin as a eukaryotic activator for GGCT activity of RipAY. A, biochemical purification of the eukaryotic activator of RipAYfrom yeast protein extracts by HiPrep DEAE FF 16/10 ion exchange, HiPrep Butyl FF 16/10 hydrophobic interaction, and HiPrep 16/60 Sephacryl S-100 HR gel filtration chromatography. B, gel filtration fractions surrounding activity were loaded onto a Tricine SDS-polyacrylamide gel, visualized by silver staining. C, gel bands of fractions 37–41 stained with Coomassie Brilliant Blue were collected and in-gel digested, and following LC-MS/MS analysis, yeast thioredoxin Trx1 and Trx2 proteins were identified as the activator. Amino acid sequences of S. cerevisiae Trx1 and Trx2 and their corresponding tryptic peptides identified by LC-MS/MS analysis are shown.
FIGURE 7.
FIGURE 7.
Yeast thioredoxins can activate a GGCT activity of RipAY both in vivo and in vitro. A, S. cerevisiae WT, trx3Δ single mutant, trx1/2Δ double mutant,and trx1/2/3Δ triple mutant cells carrying plasmid expressing RipAY or Gcg1 under the control of the Tet-off promoter were grown in noninducing (+Dox) or inducing (−Dox) conditions. Yeast cells transformed with corresponding empty vector were used as a control. The cells were grown at 26 °C for 3 days for SD (−Ura, +Dox) and 4 days for SD (−Ura, −Dox). B, the GGCT activity of bacterially expressed RipAY-His6 protein incubated with 10 μg of protein of the cell lysates from the indicated S. cerevisiae thioredoxin mutant cells was measured by the Dug1-coupled method. Values represent the mean ± S.E. (error bars) (n ≥ 3). C, bacterially expressed S. cerevisiae Trx1-His6 protein can activate the GGCT activity of bacterially expressed RipAY-His6 protein.
FIGURE 8.
FIGURE 8.
Plant thioredoxins can bind to RipAY and activate GGCT activity of RipAY in an isoform-specific manner. A, yeast two-hybrid assay for interaction between RipAY and various thioredoxins on the plate with different stringency conditions. RipAY WT (RipAYWT) and a mutant form of RipAY (RipAYCS), in which the sole cysteine residue at position 333 was substituted by serine, were used as bait. R. solanacearum TrxA (RsTrxA), S. cerevisiae Trx1 (ScTrx1), A. thaliana Trx-h2, -h3, and -h5 (AtTrx-h2, AtTrx-h3, and AtTrx-h5), and a mutant form of Trx-h5 (AtTrx-h5CS), in which active site cysteine residues at position 39 and 42 were substituted by serine, were used as prey. B, yeast cells carrying empty vector or GAL1 expression vector of WT, active site mutant E216Q, or C333S mutant of RipAY were spotted on SD (−Ura) and SGal (−Ura) plates and cultured for 2 days and 3 days, respectively. C, GGCT activity of the beads bound with Protein A-tagged RipAYWT, RipAYE216Q, or RipAYC333S proteins expressed in E. coli incubated with 1.6 μm AtTrx-h5-His6 protein was measured by the Dug1-coupled method. Values represent the mean ± S.E. (error bars) (n = 3). D, Protein A-tagged RipAYWT, RipAYE216Q, and RipAYC333S proteins extracted from the beads shown in C were resolved on SDS-PAGE and visualized by Coomassie Brilliant Blue staining. E, effect of thioredoxin isoforms on activation of GGCT activity of RipAY. The GGCT activity of RipAY incubated with various concentrations of thioredoxin isoforms was measured by the Dug1-coupled method. Values represent the mean ± S.E. (n ≥ 3). F, phylogenetic analysis of thioredoxin isoforms from various organisms. The organisms, locus tag/gene name, and GenbankTM accession numbers (in parentheses) are as follows: R. solanacearum, RSc1188/RsTrxA (AL646052.1) and RSc0779/RsTrx (AL646052.1); E. coli, EcTrxA (M26133.1) and EcTrxC (U8594.1); H. sapiens, HsTxn1 (JQ313905.1) and HsTxn2 (DQ891579.2); S. cerevisiae, YLR043C/ScTrx1 (NM_001181930.1), YGR209C/ScTrx2 (NM_001181338.3), and YCR083C/ScTrx3 (NM_001178789.1); A. thaliana, At3g51030/AtTrx-h1 (NM_114963.4), At5g39950/AtTrx-h2 (AY113052.1), At5g42980/AtTrx-h3 (AY065098.1), At1g19730/AtTrx-h4 (BT004710.1), At1g45145/AtTrx-h5 (AY040028.1), At1g59730/AtTrx-h7 (BT0031540.1), At1g69880/AtTrx-h8 (BT003670.1), and At3g08710/AtTrx-h9 (BT0011728.1).
FIGURE 9.
FIGURE 9.
Enzyme kinetic analysis of activated RipAY and Gcg1. Shown is a Michaelis-Menten plot of Trx-h5-activated RipAY (A) and Gcg1 (B) toward glutathione. Gcg1 (0.8 μm) or RipAY (0.04 μm) in the presence of Trx-h5 (8 μm) was used for determination of kinetic parameters. Different concentrations of glutathione were used ranging from 0.5 to 20 mm. Dug1-coupled assay was used for the study as described under “Experimental Procedures.” Data of three independent experiments were analyzed by non-linear regression using GraphPad prism version 6.0. Error bars, S.E.
FIGURE 10.
FIGURE 10.
A model depicting how the RipAY T3SS effector is activated by host eukaryotic thioredoxins to trigger GGCT activity. RipAY was injected into a host plant cell as an inactive form, and then its GGCT activity was stimulated by host eukaryotic thioredoxins, such as Trx-h5, to degrade glutathione and other unknown γ-glutamyl compound(s). 5-OP, 5-oxoproline.

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