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. 2013 Oct;9(10):e1003715.
doi: 10.1371/journal.ppat.1003715. Epub 2013 Oct 31.

Bacterial effector activates jasmonate signaling by directly targeting JAZ transcriptional repressors

Shushu Jiang  1 , Jian Yao, Ka-Wai Ma, Huanbin Zhou, Jikui Song, Sheng Yang He, Wenbo Ma
Affiliations

Bacterial effector activates jasmonate signaling by directly targeting JAZ transcriptional repressors

Shushu Jiang et al. PLoS Pathog. 2013 Oct.

Abstract

Gram-negative bacterial pathogens deliver a variety of virulence proteins through the type III secretion system (T3SS) directly into the host cytoplasm. These type III secreted effectors (T3SEs) play an essential role in bacterial infection, mainly by targeting host immunity. However, the molecular basis of their functionalities remains largely enigmatic. Here, we show that the Pseudomonas syringae T3SE HopZ1a, a member of the widely distributed YopJ effector family, directly interacts with jasmonate ZIM-domain (JAZ) proteins through the conserved Jas domain in plant hosts. JAZs are transcription repressors of jasmonate (JA)-responsive genes and major components of the jasmonate receptor complex. Upon interaction, JAZs can be acetylated by HopZ1a through a putative acetyltransferase activity. Importantly, P. syringae producing the wild-type, but not a catalytic mutant of HopZ1a, promotes the degradation of HopZ1-interacting JAZs and activates JA signaling during bacterial infection. Furthermore, HopZ1a could partially rescue the virulence defect of a P. syringae mutant that lacks the production of coronatine, a JA-mimicking phytotoxin produced by a few P. syringae strains. These results highlight a novel example by which a bacterial effector directly manipulates the core regulators of phytohormone signaling to facilitate infection. The targeting of JAZ repressors by both coronatine toxin and HopZ1 effector suggests that the JA receptor complex is potentially a major hub of host targets for bacterial pathogens.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HopZ1a interacts with the soybean protein GmJAZ1.
(A) HopZ1a and GmJAZ1 interact in vitro. GmJAZ1-HA and GST-HopZ1a proteins were expressed in E. coli. Precipitation of GmJAZ1 with HopZ1a was determined by western blots before (Input) and after affinity purification (Pull-down) using anti-HA antibody. The protein abundances of GST, GST-HopZ1a and GST-HopZ1a(C216A) on the affinity resins after washes were detected by Coomassie blue staining. (B) Bimolecular fluorescence complementation analysis showing HopZ1a-GmJAZ1 interactions in plant cells. HopZ1a(C216A)-nYFP and GmJAZ1-cYFP were co-expressed in N. benthamiana using Agrobacterium-mediated transient expression. Leaves co-infiltrated with Agrobacterium carrying GmJAZ1-cYFP+nYFP or cYFP+HopZ1a(C216A)-nYFP were used as negative controls. Fluorescence was detected by confocal microscopy from the infiltrated tissues at 48 hpi. DAPI was used to stain the nuclei. These experiments were repeated three times with similar results.
Figure 2
Figure 2. HopZ1a promotes GmJAZ1 degradation in planta.
(A) HopZ1a induces the degradation of GmJAZ1 when the proteins were co-expressed in N. benthamiana. GmJAZ1-FLAG and HopZ1a-HA were transiently expressed in N. benthamiana and the abundance of GmJAZ1 was detected by western blots at 20 hpi. The same protein gel was stained with Coomassie blue to show equal loading. (B) HopZ1a induces GmJAZ1 degradation using a semi-in vitro assay. GmJAZ1-FLAG and 3×FLAG-HopZ1a were transiently expressed in N. benthamiana individually. Total proteins were extracted from the infiltrated leaves 20 hours post Agro-infiltration, mixed in equal volume, and incubated at 4°C for six hours. The abundance of GmJAZ1-FLAG was then analyzed by western blots. These experiments were repeated three times with similar results.
Figure 3
Figure 3. HopZ1a interacts with AtJAZs.
(A) HopZ1a interacts with AtJAZs in vitro. Precipitation of MBP-AtJAZ-HIS with GST-HopZ1a was detected by western blots before (Input) and after affinity purification (Pull-down) using anti-HIS antibody. (B) Bimolecular fluorescence complementation analysis showing the interaction between HopZ1a and AtJAZ6 in planta. HopZ1a(C216A)-nYFP and AtJAZ6-cYFP were co-expressed in N. benthamiana. Fluorescence in the infiltrated leaves was monitored by confocal microscopy at 48 hours post Agro-infiltration. DAPI was used to stain the nuclei. These experiments were repeated three times with similar results.
Figure 4
Figure 4. JAZs are acetylation substrates of HopZ1a.
(A) HopZ1a acetylates GmJAZ1 in vitro. Tag-free HopZ1a and HopZ1a(C216A), and HIS-GmJAZ1 were purified from E. coli and subjected to in vitro acetylation assays. The acetylated proteins were detected by autoradiography after exposure at −80°C for five days. (B) HopZ1a acetylates MBP-AtJAZ6-HIS in vitro. (C) The mutant AtJAZ6ΔJas no longer interacts with HopZ1a. Purified HopZ1a was incubated with MBP-AtJAZ6-HIS or MBP-AtJAZ6ΔJas-HIS in the in vitro pull-down assay. (D) HopZ1a no longer acetylates AtJAZ6ΔJas. (E) HopZ1a does not trigger the degradation of AtJAZ6ΔJas. AtJAZ6ΔJas-YFP-HA and 3×FLAG-HopZ1a were co-expressed in N. benthamiana. The abundance of AtJAZ6ΔJas was detected by western blots. All the in vitro acetylation experiments were repeated at least three times with similar results. The in vitro pull-down and degradation experiments were repeated twice with similar results.
Figure 5
Figure 5. HopZ1a triggers the degradation of AtJAZ1 during bacterial infection.
(A) HopZ1a, but not AvrRpt2, promotes the degradation of AtJAZ1 in the Arabidopsis ecotype Col-0 (wild-type) during bacterial infection. Six-week 35S-HA-AtJAZ1Arabidopsis transgenic plants were infiltrated with PtoDC3000, PtoDC3118 carrying the empty pUCP18 vector (EV), or PtoDC3118 expressing HopZ1a, HopZ1a(C216A) or AvrRpt2. (B) HopZ1a promotes the degradation of AtJAZ1 in zar1-1 Arabidopsis plants. Six week-old 35S-HA-AtJAZ1 zar1-1 Arabidopsis plants were inoculated with PtoDC3000, PtoDC3118 carrying the empty pUCP18 vector (EV), or PtoDC3118 expressing HopZ1a or HopZ1a(C216A). (C) HopZ1a-mediated degradation of AtJAZ1 is dependent on COI1. 35S-HA-AtJAZ1, coi1-30 Arabidopsis plants were inoculated with PtoDC3000, PtoDC3118 carrying the empty pUCP18 vector (EV), or PtoDC3118 expressing HopZ1a or HopZ1a(C216A). Bacterial infection assays were conducted using inoculums at OD600 = 0.2 (approximately 2×108 cfu/mL). The abundance of AtJAZ1 was determined by western blots using anti-HA antibody at 6 hpi. The protein gels were stained with Coomassie blue as loading controls. These experiments were repeated three times with similar results.
Figure 6
Figure 6. HopZ1a activates JA signaling during bacterial infection.
Arabidopsis zar1-1 mutant plants were inoculated with PtoDC3000 or PtoDC3118 carrying the empty pUCP18 vector (EV), HopZ1a or HopZ1a(C216A). The transcript levels of the JA-responsive genes AtJAZ9 and AtJAZ10, as well as the SA biosynthetic gene AtICS1 were determined by quantitative RT-PCR. (A) HopZ1a induces the expression of JA-responsive genes in Arabidopsis. The abundances of AtJAZ9 and AtJAZ10 transcripts were examined at 6 hpi using AtActin as the internal standard. Relative expression levels were determined by comparing the normalized AtJAZ9 or AtJAZ10 transcripts between infected and mock-treated (leaves infiltrated with 10 mM MgSO4) samples. (B) HopZ1a reduces the expression of AtICS1 in Arabidopsis. AtICS1 transcript level was analyzed at 9 hpi using AtUBQ5 as the internal standard. Values are means ± standard deviations (as error bars) (n = 5). All experiments were repeated at least five times with similar results. The expression of HopZ1a in P. syringae was confirmed by western blots (Fig. S8).
Figure 7
Figure 7. HopZ1a partially complements the virulence function of coronatine in bacteria growth.
(A) HopZ1a promotes the multiplication of PtoDC3118 in Arabidopsis. Arabidopsis zar1-1 plants were dip-inoculated with PtoDC3000 carrying pUCP20tk (EV), pUCP20tk::hopZ1a-HA, or PtoDC3118 carrying pUCP18 (EV), pUCP18::hopZ1a-HA or pUCP18::hopZ1a(C216A)-HA at OD600 = 0.2 (approximately 2×108 cfu/mL). Bacterial populations were determined at 0 and 3 days post inoculation. The average colony forming units per square centimeter (cfu/cm2) and standard deviations (as error bars) are presented. Different letters at the top of the bars represent data with statistically significant differences (two tailed t-test p<0.01). (B) COI1 is required for the virulence activity of HopZ1a. coi1-1, zar1-1 double mutant plants were dip-inoculated with PtoDC3118 carrying pUCP18 (EV), pUCP18::hopZ1a-HA or pUCP18::hopZ1a(C216A)-HA. Bacterial multiplications were examined at 0 and 3 days post inoculation. The average colony forming units per square centimeter (cfu/cm2) and standard deviations (as error bars) are presented. The expression of HopZ1a or HopZ1a(C216A) in P. syringae was confirmed by western blots (Fig. S8). These experiments were repeated at least five times with similar results.
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