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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 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.
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pbio-1001792-g001: 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.

Mentions: To identify Pta 11528 effector proteins that could target JAZs, we analyzed a Pta 11528 effector library constructed in a binary vector within the T-DNA region, in which individual genes are expressed from the 35S promoter as genetic fusions to three C-terminal hemagglutinin (HA) epitope tags (Table S1). We transiently co-expressed JAZ5-HA with individual effector genes from this library or an empty vector (EV) control in N. benthamiana by agroinfiltration. Using this approach, we identified HopX1 as a Pta 11528 effector capable of compromising JAZ5 accumulation (Figure S2). To confirm this result and to exclude a potential effect of the HA tag, we developed an independent form of HopX1 with an N-terminal green fluorescent protein (GFP) fusion. We then transiently co-expressed an EV construct or GFP-hopX1 under the control of the 35S promoter with 35S:JAZ5-HA in N. benthamiana leaf tissue. Western blot analysis showed that GFP-HopX1 accumulated in N. benthamiana (Figure 1A). Similar to previous results, JAZ5-HA protein was detectable when co-expressed with the EV control but not (or only weakly) with GFP-HopX1, despite the fact that GFP-hopX1 co-expression did not affect JAZ5-HA mRNA expression levels (Figure 1B). These results indicate that HopX1 compromised the accumulation of JAZ5 protein when transiently co-expressed in N. benthamiana without affecting gene expression levels.


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 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.
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Related In: Results  -  Collection

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pbio-1001792-g001: 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.
Mentions: To identify Pta 11528 effector proteins that could target JAZs, we analyzed a Pta 11528 effector library constructed in a binary vector within the T-DNA region, in which individual genes are expressed from the 35S promoter as genetic fusions to three C-terminal hemagglutinin (HA) epitope tags (Table S1). We transiently co-expressed JAZ5-HA with individual effector genes from this library or an empty vector (EV) control in N. benthamiana by agroinfiltration. Using this approach, we identified HopX1 as a Pta 11528 effector capable of compromising JAZ5 accumulation (Figure S2). To confirm this result and to exclude a potential effect of the HA tag, we developed an independent form of HopX1 with an N-terminal green fluorescent protein (GFP) fusion. We then transiently co-expressed an EV construct or GFP-hopX1 under the control of the 35S promoter with 35S:JAZ5-HA in N. benthamiana leaf tissue. Western blot analysis showed that GFP-HopX1 accumulated in N. benthamiana (Figure 1A). Similar to previous results, JAZ5-HA protein was detectable when co-expressed with the EV control but not (or only weakly) with GFP-HopX1, despite the fact that GFP-hopX1 co-expression did not affect JAZ5-HA mRNA expression levels (Figure 1B). These results indicate that HopX1 compromised the accumulation of JAZ5 protein when transiently co-expressed in N. benthamiana without affecting gene expression levels.

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