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The bacterial effector HopX1 targets JAZ transcriptional repressors to activate jasmonate signaling and promote infection in Arabidopsis.

Gimenez-Ibanez S, Boter M, Fernández-Barbero G, Chini A, Rathjen JP, Solano R - PLoS Biol. (2014)

Bottom Line: Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors.Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity.HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid, Spain.

ABSTRACT
Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.

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HopX1 encodes a putative cysteine protease and this activity is required for HopX1-mediated degradation of JAZs.(A) HopX1 has protease activity in vitro on the general substrate casein when immunoprecipitated from transgenic Arabidopsis plants expressing the transgene. HopX1 or HopX1C179A-HA purified under non-denaturing conditions from transgenic Arabidopsis plants incubated with fluorescein isothiocyanate (FITC)-labeled casein. Trypsin was used as a positive control. As a negative control, we included wild-type Col-0 plants (EV) subjected to the same immunoprecipitation procedure as for the transgenic plants. Immunoblots showing HopX1-HA and HopX1C179A-HA effector inputs are also shown. The results are representative of three independent experiments performed with three independent immunoprecipitations of HopX1-HA and HopX1C179A-HA from transgenic Arabidopisis plants. (B) HopX1 has protease activity on JAZs when immunoprecipitated from transgenic Arabidopsis plants expressing the transgene. The immunoblot shows MBP-JAZ5 accumulation after incubation with immunoprecipitated HopX1-HA or HopX1C179A-HA from transgenic Arabidopsis plants in the presence or not of protease inhibitors. As a negative control, we included wild-type Col-0 plants (EV) subject to the same immunoprecipitation procedure as for the transgenic plants. The results are representative of three independent experiments performed as in (A). (C) Degradation of JAZ5 by HopX1 requires the cysteine-based catalytic triad of a putative protease in vivo. The immunoblots show JAZ5-HA accumulation in the presence of GFP-HopX1, GFP-HopX1C179A or GFP alone when co-expressed transiently in N. benthamiana. This experiment was repeated three times with similar results.
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pbio-1001792-g002: HopX1 encodes a putative cysteine protease and this activity is required for HopX1-mediated degradation of JAZs.(A) HopX1 has protease activity in vitro on the general substrate casein when immunoprecipitated from transgenic Arabidopsis plants expressing the transgene. HopX1 or HopX1C179A-HA purified under non-denaturing conditions from transgenic Arabidopsis plants incubated with fluorescein isothiocyanate (FITC)-labeled casein. Trypsin was used as a positive control. As a negative control, we included wild-type Col-0 plants (EV) subjected to the same immunoprecipitation procedure as for the transgenic plants. Immunoblots showing HopX1-HA and HopX1C179A-HA effector inputs are also shown. The results are representative of three independent experiments performed with three independent immunoprecipitations of HopX1-HA and HopX1C179A-HA from transgenic Arabidopisis plants. (B) HopX1 has protease activity on JAZs when immunoprecipitated from transgenic Arabidopsis plants expressing the transgene. The immunoblot shows MBP-JAZ5 accumulation after incubation with immunoprecipitated HopX1-HA or HopX1C179A-HA from transgenic Arabidopsis plants in the presence or not of protease inhibitors. As a negative control, we included wild-type Col-0 plants (EV) subject to the same immunoprecipitation procedure as for the transgenic plants. The results are representative of three independent experiments performed as in (A). (C) Degradation of JAZ5 by HopX1 requires the cysteine-based catalytic triad of a putative protease in vivo. The immunoblots show JAZ5-HA accumulation in the presence of GFP-HopX1, GFP-HopX1C179A or GFP alone when co-expressed transiently in N. benthamiana. This experiment was repeated three times with similar results.

Mentions: To determine if HopX1 has cysteine protease activity in vitro, we used a kit designed for the detection of protease activity (serine, aspartic, cysteine, and metalloproteinases) using fluorometry based on the hydrolysis of a labeled casein general substrate [39]. As previously described by Nimchuck and colleagues [38], we did not detect any protease activity when purified recombinant HopX1 protein fused to maltose binding protein (MBP) was incubated with the casein-labeled substrate in vitro, indicating that this recombinant protein may be inactive or that it might lack a co-factor (Figure S4B). However, we detected significant protease activity when the casein substrate was incubated with HopX1-HA immunopurified directly from stable transgenic Arabidopsis plants expressing the hopX1 gene from a dexamethasone-inducible promoter (DEX) (Figure 2A), suggesting that HopX1 expressed in planta has protease activity. To test whether this activity of HopX1 required its conserved cysteine protease catalytic triad, we substituted the conserved Cys-179 residue within this domain for an alanine residue to generate the HopX1C179A mutant. HopX1C179A-HA inmunoprecipitated from transgenic Arabidopsis plants did not show any proteolytic activity compared to negative controls. The trypsin enzyme used as a positive control in these experiments showed much higher activity on the casein substrate than HopX1 (Figure 2A). These data indicate that HopX1 has protease activity, but seems to operate suboptimally on a general substrate in vitro. To test whether HopX1 may have evolved specific substrate selectivity, we incubated inmunoprecipitated HopX1 and HopX1C179A from Arabidopsis with recombinant MBP-JAZ5 expressed and purified from Escherichia coli cells with or without protease inhibitors. The amount of MBP-JAZ5 diminished significantly when incubated with HopX1 but not with HopX1C179A or buffer in the absence of protease inhibitors, but not in its presence (Figure 2B). Thus, HopX1, but not HopX1C179A, is capable of inducing JAZ5 degradation in vitro suggesting that the effector indeed acts as a protease on the JAZ5 substrate.


The bacterial effector HopX1 targets JAZ transcriptional repressors to activate jasmonate signaling and promote infection in Arabidopsis.

Gimenez-Ibanez S, Boter M, Fernández-Barbero G, Chini A, Rathjen JP, Solano R - PLoS Biol. (2014)

HopX1 encodes a putative cysteine protease and this activity is required for HopX1-mediated degradation of JAZs.(A) HopX1 has protease activity in vitro on the general substrate casein when immunoprecipitated from transgenic Arabidopsis plants expressing the transgene. HopX1 or HopX1C179A-HA purified under non-denaturing conditions from transgenic Arabidopsis plants incubated with fluorescein isothiocyanate (FITC)-labeled casein. Trypsin was used as a positive control. As a negative control, we included wild-type Col-0 plants (EV) subjected to the same immunoprecipitation procedure as for the transgenic plants. Immunoblots showing HopX1-HA and HopX1C179A-HA effector inputs are also shown. The results are representative of three independent experiments performed with three independent immunoprecipitations of HopX1-HA and HopX1C179A-HA from transgenic Arabidopisis plants. (B) HopX1 has protease activity on JAZs when immunoprecipitated from transgenic Arabidopsis plants expressing the transgene. The immunoblot shows MBP-JAZ5 accumulation after incubation with immunoprecipitated HopX1-HA or HopX1C179A-HA from transgenic Arabidopsis plants in the presence or not of protease inhibitors. As a negative control, we included wild-type Col-0 plants (EV) subject to the same immunoprecipitation procedure as for the transgenic plants. The results are representative of three independent experiments performed as in (A). (C) Degradation of JAZ5 by HopX1 requires the cysteine-based catalytic triad of a putative protease in vivo. The immunoblots show JAZ5-HA accumulation in the presence of GFP-HopX1, GFP-HopX1C179A or GFP alone when co-expressed transiently in N. benthamiana. This experiment was repeated three times with similar results.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928049&req=5

pbio-1001792-g002: HopX1 encodes a putative cysteine protease and this activity is required for HopX1-mediated degradation of JAZs.(A) HopX1 has protease activity in vitro on the general substrate casein when immunoprecipitated from transgenic Arabidopsis plants expressing the transgene. HopX1 or HopX1C179A-HA purified under non-denaturing conditions from transgenic Arabidopsis plants incubated with fluorescein isothiocyanate (FITC)-labeled casein. Trypsin was used as a positive control. As a negative control, we included wild-type Col-0 plants (EV) subjected to the same immunoprecipitation procedure as for the transgenic plants. Immunoblots showing HopX1-HA and HopX1C179A-HA effector inputs are also shown. The results are representative of three independent experiments performed with three independent immunoprecipitations of HopX1-HA and HopX1C179A-HA from transgenic Arabidopisis plants. (B) HopX1 has protease activity on JAZs when immunoprecipitated from transgenic Arabidopsis plants expressing the transgene. The immunoblot shows MBP-JAZ5 accumulation after incubation with immunoprecipitated HopX1-HA or HopX1C179A-HA from transgenic Arabidopsis plants in the presence or not of protease inhibitors. As a negative control, we included wild-type Col-0 plants (EV) subject to the same immunoprecipitation procedure as for the transgenic plants. The results are representative of three independent experiments performed as in (A). (C) Degradation of JAZ5 by HopX1 requires the cysteine-based catalytic triad of a putative protease in vivo. The immunoblots show JAZ5-HA accumulation in the presence of GFP-HopX1, GFP-HopX1C179A or GFP alone when co-expressed transiently in N. benthamiana. This experiment was repeated three times with similar results.
Mentions: To determine if HopX1 has cysteine protease activity in vitro, we used a kit designed for the detection of protease activity (serine, aspartic, cysteine, and metalloproteinases) using fluorometry based on the hydrolysis of a labeled casein general substrate [39]. As previously described by Nimchuck and colleagues [38], we did not detect any protease activity when purified recombinant HopX1 protein fused to maltose binding protein (MBP) was incubated with the casein-labeled substrate in vitro, indicating that this recombinant protein may be inactive or that it might lack a co-factor (Figure S4B). However, we detected significant protease activity when the casein substrate was incubated with HopX1-HA immunopurified directly from stable transgenic Arabidopsis plants expressing the hopX1 gene from a dexamethasone-inducible promoter (DEX) (Figure 2A), suggesting that HopX1 expressed in planta has protease activity. To test whether this activity of HopX1 required its conserved cysteine protease catalytic triad, we substituted the conserved Cys-179 residue within this domain for an alanine residue to generate the HopX1C179A mutant. HopX1C179A-HA inmunoprecipitated from transgenic Arabidopsis plants did not show any proteolytic activity compared to negative controls. The trypsin enzyme used as a positive control in these experiments showed much higher activity on the casein substrate than HopX1 (Figure 2A). These data indicate that HopX1 has protease activity, but seems to operate suboptimally on a general substrate in vitro. To test whether HopX1 may have evolved specific substrate selectivity, we incubated inmunoprecipitated HopX1 and HopX1C179A from Arabidopsis with recombinant MBP-JAZ5 expressed and purified from Escherichia coli cells with or without protease inhibitors. The amount of MBP-JAZ5 diminished significantly when incubated with HopX1 but not with HopX1C179A or buffer in the absence of protease inhibitors, but not in its presence (Figure 2B). Thus, HopX1, but not HopX1C179A, is capable of inducing JAZ5 degradation in vitro suggesting that the effector indeed acts as a protease on the JAZ5 substrate.

Bottom Line: Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors.Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity.HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Madrid, Spain.

ABSTRACT
Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.

Show MeSH
Related in: MedlinePlus