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, 7 (2), 164-75

Pseudomonas Syringae Effector Protein AvrB Perturbs Arabidopsis Hormone Signaling by Activating MAP Kinase 4

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Pseudomonas Syringae Effector Protein AvrB Perturbs Arabidopsis Hormone Signaling by Activating MAP Kinase 4

Haitao Cui et al. Cell Host Microbe.

Abstract

Pathogenic microbes often modulate phytohormone physiology in the host to their advantage. We previously showed that the Pseudomonas syringae effector protein AvrB perturbs hormone signaling, as exemplified by upregulated expression of jasmonic acid response genes, and enhances plant susceptibility. Here we show that these effects of AvrB require the Arabidopsis mitogen-activated protein kinase MAP kinase 4 (MPK4), HSP90 chaperone components, and the AvrB-interacting protein, RIN4. AvrB interacts with MPK4 and the HSP90 chaperone, and AvrB induces MPK4 activation in a manner promoted by HSP90; RIN4 likely acts downstream of MPK4. These findings link Arabidopsis proteins MPK4, HSP90, and RIN4 into a pathway that P. syringae AvrB activates for the benefit of the bacterium, perturbing hormone signaling and enhancing plant susceptibility.

Figures

Figure 1
Figure 1. HSP90 Chaperone Components, but Not TAO1, Are Required for AvrB-Induced Susceptibility and PDF1.2 Expression
(A) GDA inhibits AvrB-3xFLAG transgene-induced susceptibility. (B) TAO1 is not required for AvrB-induced susceptibility. Plants of the indicated genotypes were pre-treated with estradiol, infiltrated with hrcC mutant bacteria in the presence of GDA or buffer (DMSO), and bacterial populations within leaves determined at the indicated times (A and B). (C) GDA abolishes PDF1.2 induction by the AvrB-3xFLAG transgene. (D) TAO1 is not required for PDF1.2 induction by the AvrB-3xFLAG transgene. (E) GDA inhibits PDF1.2 induction by bacterially delivered AvrB. (F) RAR1 is required for PDF1.2 induction by bacterially delivered AvrB. Arabidopsis plants of the indicated genotype were infiltrated with estradiol for 24 hours (C and D) or the indicated bacterial strains (E and F) for 6 and 9 hours (E) or 6 hours (F), and RNA isolated for quantitative RT PCR. For experiments in C and E, GDA or buffer (DMSO) was co-infiltrated into the leaves. The PDF1.2 expression was determined by real-time RT-PCR. Data in are representative of three independent experiments with similar results.
Figure 2
Figure 2. AvrB Interacts with the HSP90 Chaperone
(A) AvrB directly interacts with RAR1. An equal amount of AvrB-His recombinant protein was incubated with bacterial lysates containing GST or GST-tagged RAR1, SGT1b, or HSP90. The presence of AvrB-His in the GST pull-down was detected by immunoblot using an anti-His antibody. Coomassie Brilliant Blue (CBB) staining shows the amounts of GST-fusion proteins. (B) CHORD1 domain is sufficient for interaction with AvrB. GST-tagged full-length and truncated RAR1 were incubated with equal amount of His-AvrB in a GST pull-down assay, the presence of His-AvrB in the protein complex was detected by anti-His immunoblot. (C) AvrB interacts with HSP90 in plants. Arabidopsis rpm1 plants with (+) or without (−) the AvrB-3xFLAG transgene were induced with estradiol, and protein extract was immunoprecipitated with an agarose-conjugated anti-FLAG antibody. Amounts of HSP90 proteins in the immune complex were determined by immunoblot using anti-HSP90 antibodies. (D) RAR1 is required for AvrB-HSP90 interaction in vivo. Co-immunoprecipitation assay for AvrB-HSP90 interaction was conducted as in (C) using Arabidopsis rpm1 (RAR1) or rpm1/rar1-29 (rar1) plants containing (+) or lacking (−) the AvrB-3xFLAG transgene.
Figure 3
Figure 3. AvrB Directly Interacts with and Induces Phosphorylation of MPK4
(A) AvrB-3xFLAG induces MPK4 phosphorylation. Specific anti-MPK antibodies were used to immunoprecipitate MPK4 and MPK6 proteins from estradiol-treated rpm1 plants with (+) or without (−) the estradiol-inducible AvrB-3xFLAG transgene, and the level of dual phosphorylation on MPK4 and MPK6 was determined by immunoblot using anti-phospho-ERK1 antibodies. Equal loading of immunoprecipitated proteins was confirmed by immunoblot using anti-MPK4 and anti-MPK6 antibodies. (B) and (C) Bacterially-delivered AvrB induces MPK4 phosphorylation. Arabidopsis rpm1 plants were inoculated with 108 CFU/ml DC3682 carrying an empty vector (pDSK), WT avrB, avrBT125A, avrBR266G or avrBD297A mutant plasmids for 6 hrs, and the phosphorylation state of the immunoprecipitated MPK4 was determined by immunoblot using anti-phospho-ERK1 antibodies.
Figure 4
Figure 4. MPK4 Is Required for AvrB-Induced PDF1.2 Expression and Susceptibility
(A) The avrBT125A and avrBR266G mutations abolish JA-signaling activity. Arabidopsis rpm1 plants were inoculated with DC3682 carrying an empty vector (pDSK), WT avrB, avrBT125A, or avrBR266G. RNA was isolated 6 hr post inoculation, and the expression of PDF1.2 was determined by real-time RT-PCR. (B) MPK4 is required for PDF1.2 induction by the AvrB-3xFLAG transgene. Plants of the indicated genotypes were treated with estradiol for 24 hrs, and RNA was isolated for real-time RT-PCR. (C) The AvrB-3xFLAG transgene does not induce susceptibility in the mpk4 mutant. Plants of the indicated genotypes were pre-treated with estradiol, inoculated with the P. syringae hrcC mutant strain, and the bacterial population in the leaf was determined at the indicated times. The data shown are representative of two independent experiments with similar results.
Figure 5
Figure 5. AvrB Interacts with MPK4
(A) AvrB interacts with MPK4 in vitro. Equal amounts of AvrB-His or AvrBD297A-His were incubated with GST or GST-MPK4, and the presence of AvrB after GST pull-down was detected by immunoblot using anti-His antibody. (B) AvrB-3xFLAG interacts with MPK4 in plants in a RAR1-dependent manner. RAR1/rpm1 (RAR1) or rar1-29/rpm1 (rar1) plants with or without the AvrB-3xFLAG transgene were induced with estradiol, and protein extracts were immunoprecipitated with agarose-conjugated anti-FLAG antibody. The presence of MPK4 in the immune complex was detected by immunoblot using anti-MPK4 antibodies. (C) RAR1 is required for AvrB-3xFLAG-induced MPK4 phosphorylation. Plants of the indicated genotypes were induced with estradiol, and the phosphorylation state of immunoprecipitated MPK4 was determined by immunoblot using anti-phospho-ERK1 antibodies.
Figure 6
Figure 6. HSP90 Promotes MPK4 Activation
(A) MPK4 does not interact with RAR1 in vitro. AvrB-His and GST-RAR1 were expressed in E. coli and purified proteins used in a pull-down assay. The presence of GST-RAR1 was detected by immunoblot using anti-RAR1 antibodies. The AvrB-His protein was used as a positive control. A His-tagged human WD-repeat protein EED (Han et al., 2007) was included as a negative control. (B) MPK4 interacts with SGT1b and HSP90 in vitro. GST-MPK4, SGT1b-His, and HSP90-His proteins were expressed in E. coli. The presence of GST-MPK4 was detected by immunoblot using anti-MPK4 antibodies. (C) MPK4 interacts with HSP90 in plants. Protein extracts from transgenic plants carrying NP-MPK4-3xFLAG were immunoprecipitated with an agarose-conjugated anti-FLAG antibody, and the presence of HSP90 was determined by immunoblot using anti-HSP90 antibodies. The presence of MPK4-3xFLAG was determined by immunoblot using anti-MPK4 antibodies. (D) GDA inhibits PAMP-induced MPK4 activation. WT Arabidopsis plants (Col-0) were infiltrated with hrcC bacteria in the presence of GDA or buffer (DMSO). Kinase activity of the immunoprecipitated MPK4 protein was determined using an in vitro kinase assay employing myelin basic protein (MBP) as a substrate. To assess MPK4 activation in protoplasts, MPK4-3xFLAG was expressed in protoplasts prepared from WT plants, treated with GDA or buffer (DMSO), and induced with flg22. The MPK activity of the immunoprecipitated MPK4-3xFLAG was determined using the in vitro kinase assay with MBP as a substrate. (E) GDA inhibits MeJA-induced PDF1.2 expression. WT plants were pretreated with GDA or buffer (DMSO) prior to the application of MeJA. PDF1.2 expression was determined using real-time RT-PCR.
Figure 7
Figure 7. RIN4 Mediates PDF1.2 Induction Down-Stream of MPK4
(A) RIN4 is required for PDF1.2 induction by bacterially delivered AvrB. Plants of the indicated genotypes were infiltrated with the indicated bacterial strains and RNA was isolated 6 hr later for gene expression analysis. (B) RIN4 is required for JA-induced PDF1.2 expression. Plants of the indicated genotypes were treated with MeJA at the indicated times before RNA isolaton. (C) Overexpression of RIN4 constitutively activates PDF1.2 expression. WT or RIN4 transgenic (RIN4-ox) plants were treated with dexmethosome as described (Kim et al., 2005) for the indicated times before RNA was isolated for PDF1.2 expression analysis. (D) RIN4 interacts with MPK4 in vitro. Recombinant GST-MPK4 protein was incubated with bacterial lysates containing RIN4-His or CK-His (negative control as in Fig. 7A). (E) RIN4 interacts with MPK4 in plants. Protein extract from transgenic plants carrying NP-MPK4-3xFLAG was immunoprecipitated with an agarose-conjugated anti-FLAG antibody, and the presence of RIN4 in the immune complex was determined by immunoblot using anti-RIN4 antibodies. The presence of MPK4-3xFLAG was determined by immunoblot using anti-FLAG antibodies. Arrow head indicates MPK4-3xFLAG, whereas asterisk indicates IgG heavy chain from the anti-FLAG antibody used in immunoprecipitation. (F) MPK4 phosphorylates RIN4 in vitro. MPK4-3xFLAG was stimulated with (+) or without (−) flg22 in protoplasts, immunoprecipitated with anti-FLAG antibody, and the isolated MPK4-3xFLAG protein was incubated with recombinant RIN4 protein in an in vitro kinase assay. RIN4 phosphorylation (p-RIN4) was detected by autoradiography. CBB stain indicates amount of RIN4 protein in the gel.

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