The bacterial effector HopX1 targets JAZ transcriptional repressors to activate jasmonate signaling and promote infection in Arabidopsis - PubMed (original) (raw)

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

Selena Gimenez-Ibanez et al. PLoS Biol. 2014.

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. HopX1 compromises the accumulation of JAZ proteins.

(A) HopX1 compromises the accumulation of JAZ5. Immunoblots showing JAZ5-HA accumulation in the presence of GFP-HopX1 when co-expressed transiently in N. benthamiana. Proteins were detected with anti-HA and anti-GFP antisera respectively. A non-specific band is shown as an internal loading control. CBB, Coomassie brilliant blue staining. This experiment was repeated four times with similar results. (B) HopX1 does not affect JAZ5 expression levels. RT-PCRs showing transgenic JAZ5 mRNA in N. benthamiana leaves transiently co-expressing JAZ5 with an EV control or GFP-HopX1. Actin8 was used as an amplification control. dpi, days post infiltration. This experiment was repeated twice with similar results. (C) HopX1 activity is not restricted to JAZ5, but targets all detectable JAZs. Immunoblots showing the accumulation of eight JAZ-HA proteins in the presence of an EV control or GFP-HopX1 when co-expressed transiently in N. benthamiana. This experiment was repeated twice with similar results. (D) HopX1 does not alter COI1 proteins levels. Immunoblots showing COI1-GFP accumulation in the presence of GFP-HopX1 when co-expressed transiently in N. benthamiana. This experiment was repeated twice with similar results. (E) HopX1 does not alter MYC2 proteins levels. Immunoblots showing MYC2-HA accumulation in the presence of GFP-HopX1 when co-expressed transiently in N. benthamiana. This experiment was repeated twice with similar results.

Figure 2

Figure 2. 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.

Figure 3

Figure 3. 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 JAZ1_Δ_Jas-HA, JAZ2_Δ_Jas-HA, or JAZ7_Δ_Jas-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:hopX1 C179A-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:hopX1 C179A-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.

Figure 4

Figure 4. HopX1 activates JA-dependent gene expression in Arabidopsis.

(A) Quantitative RT-PCR analysis of JAZ10 expression in Col-0 (EV) and stable transgenic Arabidopsis Aa–0 lines expressing the hopX1 or hopX1 C179A genes 36 hours after treatment with DEX or a mock solution. The measurements (three technical replicates) represent the expression level between mock (control) and DEX-treated plants relative to each Arabidopsis background. All samples were normalized against the housekeeping gene AtACT8. Error bars represent standard deviation (SD). The results are representative of four independent experiments. The results are representative of four independent experiments. (B) Phenotypes of stable transgenic Arabidopsis lines expressing the hopX1 or hopX1 C179A genes under the control of the DEX promoter. Pictures were taken six days after mock or DEX treatment. (C) Chlorotic phenotypes of stable transgenic Arabidopsis lines expressing the hopX1 or hopX1 C179A genes under the control of the DEX inducible promoter. Pictures were taken nine days after mock or DEX treatment. (D) Quantitative RT-PCR analysis of PR1 expression in stable transgenic Arabidopsis lines expressing the hopX1 or hopX1 C179A genes DEX-induced for 24 hours followed by a treatment with 1 mM SA or a mock solution for an additional 24 hours. The measurements (three technical replicates) represent the ratio of expression levels between control (non-DEX and non-SA treated plants) and treated plants in each Arabidopsis background. All samples were normalized against the housekeeping gene AtACT8. Error bars represent standard deviation (SD). The results are representative of two independent experiments. This experiment was repeated twice with similar results. (E) HopX1 mimics COR-induced susceptibility. Growth of a COR-deficient Pto DC3000 strain expressing hopX1, hopX1 C179A or an empty vector control on Arabidopsis Col-0 plants two days after spray inoculation with bacteria at 108 cfu/ml−1 supplemented with 2 µM of COR or a mock solution. Error bars indicate standard error of the mean (SEM). Red asterisks indicate statistically significant values compared to Pto DC3000 COR− carrying an EV treated in each condition (mock or COR treated) (Student's t test, **p<0.01). The results are representative of three independent experiments. (F) HopX1 promotes bacterial growth on Arabidopsis coi1 mutants. Growth of a COR-deficient Pto DC3000 strain expressing hopX1, hopX1 C179A, or an empty vector control on Arabidopsis coi1-30 plants two days after spray inoculation as in (E) supplemented with 2 µM of COR or a mock solution. Error bars indicate SEM. Red asterisks indicate statistically significant differences compared to Pto DC3000 COR- carrying an EV treated in each condition (mock or COR treated) (Student's t test, **p<0.01). The results are representative of two independent experiments. (G) Infection of Arabidopsis plants with Pto DC3000 COR− carrying HopX1 triggers JAZ1ΔJas degradation. Immunoblots showing JAZ1ΔJas accumulation in transgenic Col-0 plants expressing a JAZ1ΔJas-HA transgene under the control of the 35S promoter after mock treatment, or infection with Pto DC000, or Pto DC3000 COR− expressing hopX1, hopX1 C179A, or an empty vector. Non-transgenic Col-0 plants are included as a control. This experiment was repeated three times with similar results.

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S.G-I was supported by a “Juan de la Cierva” fellowship from the Spanish Ministry for Science and Innovation. This work was funded by the Spanish Ministry for Science and Innovation grants BIO2010-21739, CSD2007-00057 and EUI2008- 03666 to R.S. J.P.R is an Australian Research Council Future Fellow (FT0992129). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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