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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 interacts with and degrades JAZ proteins in a COI1-independent manner.(A) HopX1 compromises the accumulation of JAZ5 in N. tabacum plants silenced for the NtCOI1 gene. Immunoblots showing JAZ5-HA accumulation in the presence of GFP-HopX1 or GFP alone, when co-expressed transiently in N. tabacum plants silenced for the NtCOI1 gene (line 18) or EV-transformed (line VC). CBB, Coomassie brilliant blue staining. This experiment was repeated three times with similar results. (B) HopX1 triggers the degradation of JAZΔJas proteins in a COI1-independent manner. N. benthamiana plants were transiently co-transformed with GFP-hopX1 or GFP alone, and the dominant-negative JAZ variants JAZ1ΔJas-HA, JAZ2ΔJas-HA, or JAZ7ΔJas-HA proteins as indicated. Protein stability was analyzed by immunoblot. This experiment was repeated twice times with similar results. (C) HopX1 interacts with JAZ repressors in PD assays. Immunoblots with anti-HA antibody of HopX1-HA or HopX1C179A-HA recovered after PD experiments using crude protein extracts from DEX:hopX1-HA (X1), DEX:hopX1C179A-HA (CA), or Col-0 (C) Arabidopsis plants, and resin-bound recombinant MBP or MBP-fused JAZ proteins (top). Input lanes show the level of expression of recombinant HopX1 proteins in transgenic and control plants. CBB staining shows the amount of recombinant JAZ-MBP or MBP proteins used in the resin (bottom). The results are representative of five independent experiments. (D) Schematic representation of the JAZ5 protein and its conserved domains. The NT, the ZIM, and the Jas domains are depicted and the corresponding JAZ5 fragments are represented. (E) HopX1 interacts with JAZ proteins through their conserved ZIM domains in PD assays. Immunoblot (anti-HA antibody) of HopX1-HA and HopX1C179A-HA recovered from PD reactions (using extracts of DEX:hopX1-HA [X1], DEX:hopX1C179A-HA [CA], or Col-0 [C] Arabidopsis plants) using MBP or MBP-fused JAZ5, JAZ51–91 (JAZ5 NT), JAZ592–163 (JAZ5 ZIM), or JAZ5164–274 (JAZ5 Jas) derivatives (top). The lower panels show the CBB staining of the input quantity of recombinant MBP proteins used on the column. The results are representative of three independent experiments. (F) Subcellular localization of HopX1 in plant cells. Confocal microscopy localization of transiently expressed GFP-HopX1 or GFP alone in N. benthamiana leaves 48 hours post-infiltration (green). Nuclei were stained with DAPI (blue). This experiment was repeated three times with similar results.
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pbio-1001792-g003: HopX1 interacts with and degrades JAZ proteins in a COI1-independent manner.(A) HopX1 compromises the accumulation of JAZ5 in N. tabacum plants silenced for the NtCOI1 gene. Immunoblots showing JAZ5-HA accumulation in the presence of GFP-HopX1 or GFP alone, when co-expressed transiently in N. tabacum plants silenced for the NtCOI1 gene (line 18) or EV-transformed (line VC). CBB, Coomassie brilliant blue staining. This experiment was repeated three times with similar results. (B) HopX1 triggers the degradation of JAZΔJas proteins in a COI1-independent manner. N. benthamiana plants were transiently co-transformed with GFP-hopX1 or GFP alone, and the dominant-negative JAZ variants JAZ1ΔJas-HA, JAZ2ΔJas-HA, or JAZ7ΔJas-HA proteins as indicated. Protein stability was analyzed by immunoblot. This experiment was repeated twice times with similar results. (C) HopX1 interacts with JAZ repressors in PD assays. Immunoblots with anti-HA antibody of HopX1-HA or HopX1C179A-HA recovered after PD experiments using crude protein extracts from DEX:hopX1-HA (X1), DEX:hopX1C179A-HA (CA), or Col-0 (C) Arabidopsis plants, and resin-bound recombinant MBP or MBP-fused JAZ proteins (top). Input lanes show the level of expression of recombinant HopX1 proteins in transgenic and control plants. CBB staining shows the amount of recombinant JAZ-MBP or MBP proteins used in the resin (bottom). The results are representative of five independent experiments. (D) Schematic representation of the JAZ5 protein and its conserved domains. The NT, the ZIM, and the Jas domains are depicted and the corresponding JAZ5 fragments are represented. (E) HopX1 interacts with JAZ proteins through their conserved ZIM domains in PD assays. Immunoblot (anti-HA antibody) of HopX1-HA and HopX1C179A-HA recovered from PD reactions (using extracts of DEX:hopX1-HA [X1], DEX:hopX1C179A-HA [CA], or Col-0 [C] Arabidopsis plants) using MBP or MBP-fused JAZ5, JAZ51–91 (JAZ5 NT), JAZ592–163 (JAZ5 ZIM), or JAZ5164–274 (JAZ5 Jas) derivatives (top). The lower panels show the CBB staining of the input quantity of recombinant MBP proteins used on the column. The results are representative of three independent experiments. (F) Subcellular localization of HopX1 in plant cells. Confocal microscopy localization of transiently expressed GFP-HopX1 or GFP alone in N. benthamiana leaves 48 hours post-infiltration (green). Nuclei were stained with DAPI (blue). This experiment was repeated three times with similar results.

Mentions: Increased JA-Ile levels promote binding of JAZs to SCFCOI1 and subsequent degradation of JAZ repressors via the ubiquitin/26S proteasome pathway [16]–[18]. Therefore, degradation of JAZs by HopX1 might be direct (through its protease activity) or an indirect effect mediated by JA-Ile synthesis and COI1. To investigate if JAZ degradation by HopX1 is direct or indirect, we first analyzed whether HopX1-induced degradation of JAZ5 was dependent on the 26S proteasome pathway by using the proteasomal inhibitor MG132. In HopX1 coexpression experiments in N. benthamiana, JAZ5 was not detected in the presence of MG132, indicating that degradation does not require the proteasome (Figure S6). We next checked whether HopX1-induced degradation of JAZ proteins occurred in a COI1-dependent or independent manner. To test this, we first used a stable transgenic N. tabacum line silenced for expression of the NtCOI1 gene [42]. Reverse transcription (RT)-PCR analysis confirmed that control N. tabacum plants (Line VC, transformed with EV) accumulated NtCOI1 mRNA, whereas NtCOI1 transcripts were undetectable in N. tabacum plants silenced for the NtCOI1 gene (Line L18) (Figure S7A). Interestingly, NtCOI1-silenced N. tabacum plants produced few seeds, a phenotype reminiscent of infertile Arabidopsis coi1-1 plants (Figure S7B) [19]. We next analyzed the ability of HopX1 to trigger JAZ5 degradation in both EV- and NtCOI1-silenced plants when transiently co-expressed in N. tabacum, a species that also allows facile transient gene expression assays [43]. Strikingly, GFP-HopX1 compromised the accumulation of JAZ5 in both EV- and NtCOI1-silenced plants to the same extent (Figure 3A). This suggests that HopX1 triggers the degradation of JAZ proteins in a COI1-independent manner.


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 interacts with and degrades JAZ proteins in a COI1-independent manner.(A) HopX1 compromises the accumulation of JAZ5 in N. tabacum plants silenced for the NtCOI1 gene. Immunoblots showing JAZ5-HA accumulation in the presence of GFP-HopX1 or GFP alone, when co-expressed transiently in N. tabacum plants silenced for the NtCOI1 gene (line 18) or EV-transformed (line VC). CBB, Coomassie brilliant blue staining. This experiment was repeated three times with similar results. (B) HopX1 triggers the degradation of JAZΔJas proteins in a COI1-independent manner. N. benthamiana plants were transiently co-transformed with GFP-hopX1 or GFP alone, and the dominant-negative JAZ variants JAZ1ΔJas-HA, JAZ2ΔJas-HA, or JAZ7ΔJas-HA proteins as indicated. Protein stability was analyzed by immunoblot. This experiment was repeated twice times with similar results. (C) HopX1 interacts with JAZ repressors in PD assays. Immunoblots with anti-HA antibody of HopX1-HA or HopX1C179A-HA recovered after PD experiments using crude protein extracts from DEX:hopX1-HA (X1), DEX:hopX1C179A-HA (CA), or Col-0 (C) Arabidopsis plants, and resin-bound recombinant MBP or MBP-fused JAZ proteins (top). Input lanes show the level of expression of recombinant HopX1 proteins in transgenic and control plants. CBB staining shows the amount of recombinant JAZ-MBP or MBP proteins used in the resin (bottom). The results are representative of five independent experiments. (D) Schematic representation of the JAZ5 protein and its conserved domains. The NT, the ZIM, and the Jas domains are depicted and the corresponding JAZ5 fragments are represented. (E) HopX1 interacts with JAZ proteins through their conserved ZIM domains in PD assays. Immunoblot (anti-HA antibody) of HopX1-HA and HopX1C179A-HA recovered from PD reactions (using extracts of DEX:hopX1-HA [X1], DEX:hopX1C179A-HA [CA], or Col-0 [C] Arabidopsis plants) using MBP or MBP-fused JAZ5, JAZ51–91 (JAZ5 NT), JAZ592–163 (JAZ5 ZIM), or JAZ5164–274 (JAZ5 Jas) derivatives (top). The lower panels show the CBB staining of the input quantity of recombinant MBP proteins used on the column. The results are representative of three independent experiments. (F) Subcellular localization of HopX1 in plant cells. Confocal microscopy localization of transiently expressed GFP-HopX1 or GFP alone in N. benthamiana leaves 48 hours post-infiltration (green). Nuclei were stained with DAPI (blue). This experiment was repeated three times with similar results.
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pbio-1001792-g003: HopX1 interacts with and degrades JAZ proteins in a COI1-independent manner.(A) HopX1 compromises the accumulation of JAZ5 in N. tabacum plants silenced for the NtCOI1 gene. Immunoblots showing JAZ5-HA accumulation in the presence of GFP-HopX1 or GFP alone, when co-expressed transiently in N. tabacum plants silenced for the NtCOI1 gene (line 18) or EV-transformed (line VC). CBB, Coomassie brilliant blue staining. This experiment was repeated three times with similar results. (B) HopX1 triggers the degradation of JAZΔJas proteins in a COI1-independent manner. N. benthamiana plants were transiently co-transformed with GFP-hopX1 or GFP alone, and the dominant-negative JAZ variants JAZ1ΔJas-HA, JAZ2ΔJas-HA, or JAZ7ΔJas-HA proteins as indicated. Protein stability was analyzed by immunoblot. This experiment was repeated twice times with similar results. (C) HopX1 interacts with JAZ repressors in PD assays. Immunoblots with anti-HA antibody of HopX1-HA or HopX1C179A-HA recovered after PD experiments using crude protein extracts from DEX:hopX1-HA (X1), DEX:hopX1C179A-HA (CA), or Col-0 (C) Arabidopsis plants, and resin-bound recombinant MBP or MBP-fused JAZ proteins (top). Input lanes show the level of expression of recombinant HopX1 proteins in transgenic and control plants. CBB staining shows the amount of recombinant JAZ-MBP or MBP proteins used in the resin (bottom). The results are representative of five independent experiments. (D) Schematic representation of the JAZ5 protein and its conserved domains. The NT, the ZIM, and the Jas domains are depicted and the corresponding JAZ5 fragments are represented. (E) HopX1 interacts with JAZ proteins through their conserved ZIM domains in PD assays. Immunoblot (anti-HA antibody) of HopX1-HA and HopX1C179A-HA recovered from PD reactions (using extracts of DEX:hopX1-HA [X1], DEX:hopX1C179A-HA [CA], or Col-0 [C] Arabidopsis plants) using MBP or MBP-fused JAZ5, JAZ51–91 (JAZ5 NT), JAZ592–163 (JAZ5 ZIM), or JAZ5164–274 (JAZ5 Jas) derivatives (top). The lower panels show the CBB staining of the input quantity of recombinant MBP proteins used on the column. The results are representative of three independent experiments. (F) Subcellular localization of HopX1 in plant cells. Confocal microscopy localization of transiently expressed GFP-HopX1 or GFP alone in N. benthamiana leaves 48 hours post-infiltration (green). Nuclei were stained with DAPI (blue). This experiment was repeated three times with similar results.
Mentions: Increased JA-Ile levels promote binding of JAZs to SCFCOI1 and subsequent degradation of JAZ repressors via the ubiquitin/26S proteasome pathway [16]–[18]. Therefore, degradation of JAZs by HopX1 might be direct (through its protease activity) or an indirect effect mediated by JA-Ile synthesis and COI1. To investigate if JAZ degradation by HopX1 is direct or indirect, we first analyzed whether HopX1-induced degradation of JAZ5 was dependent on the 26S proteasome pathway by using the proteasomal inhibitor MG132. In HopX1 coexpression experiments in N. benthamiana, JAZ5 was not detected in the presence of MG132, indicating that degradation does not require the proteasome (Figure S6). We next checked whether HopX1-induced degradation of JAZ proteins occurred in a COI1-dependent or independent manner. To test this, we first used a stable transgenic N. tabacum line silenced for expression of the NtCOI1 gene [42]. Reverse transcription (RT)-PCR analysis confirmed that control N. tabacum plants (Line VC, transformed with EV) accumulated NtCOI1 mRNA, whereas NtCOI1 transcripts were undetectable in N. tabacum plants silenced for the NtCOI1 gene (Line L18) (Figure S7A). Interestingly, NtCOI1-silenced N. tabacum plants produced few seeds, a phenotype reminiscent of infertile Arabidopsis coi1-1 plants (Figure S7B) [19]. We next analyzed the ability of HopX1 to trigger JAZ5 degradation in both EV- and NtCOI1-silenced plants when transiently co-expressed in N. tabacum, a species that also allows facile transient gene expression assays [43]. Strikingly, GFP-HopX1 compromised the accumulation of JAZ5 in both EV- and NtCOI1-silenced plants to the same extent (Figure 3A). This suggests that HopX1 triggers the degradation of JAZ proteins in a COI1-independent manner.

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