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. 2016 Jul;171(3):2239-55.
doi: 10.1104/pp.16.01593. Epub 2016 May 23.

The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection

Masaki Shimono  1 , Yi-Ju Lu  1 , Katie Porter  1 , Brian H Kvitko  1 , Jessica Henty-Ridilla  1 , Allison Creason  1 , Sheng Yang He  1 , Jeff H Chang  1 , Christopher J Staiger  1 , Brad Day  2
Affiliations

The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection

Masaki Shimono et al. Plant Physiol. 2016 Jul.

Abstract

The plant cytoskeleton underpins the function of a multitude of cellular mechanisms, including those associated with developmental- and stress-associated signaling processes. In recent years, the actin cytoskeleton has been demonstrated to play a key role in plant immune signaling, including a recent demonstration that pathogens target actin filaments to block plant defense and immunity. Herein, we quantified spatial changes in host actin filament organization after infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating that the type-III effector HopG1 is required for pathogen-induced changes to actin filament architecture and host disease symptom development during infection. Using a suite of pathogen effector deletion constructs, coupled with high-resolution microscopy, we found that deletion of hopG1 from Pst DC3000 resulted in a reduction in actin bundling and a concomitant increase in the density of filament arrays in Arabidopsis, both of which correlate with host disease symptom development. As a mechanism underpinning this activity, we further show that the HopG1 effector interacts with an Arabidopsis mitochondrial-localized kinesin motor protein. Kinesin mutant plants show reduced disease symptoms after pathogen infection, which can be complemented by actin-modifying agents. In total, our results support a model in which HopG1 induces changes in the organization of the actin cytoskeleton as part of its virulence function in promoting disease symptom development.

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Figures

Figure 1.
Figure 1.
Modulation of host actin cytoskeletal organization by P. syringae is T3SS system-dependent. Actin filament organization in 10-d-old Col-0/GFP-fABD2 cotyledons after pathogen inoculation was monitored at 24 h by laser scanning confocal microscopy. A, Pst DC3000; B, Pst DC3000 D28E; C, Pst DC3000 ΔhrcC. Seedlings were inoculated with Pst DC3000 strains at a concentration of 3 × 107 colony-forming units (cfu) ml−1. Micrographs shown are representative of collected images. Error bars represent mean ± se from the skewness and density values of three independent biological replicates (n = 87–90 z-series projections per treatment). Statistical significance was determined using a Student’s t-test, as compared to mock treatment. **, P < 0.001. Bar = 20 μm.
Figure 2.
Figure 2.
P. syringae T3E polymutants display differential activities with regard to manipulation of the Arabidopsis actin cytoskeleton. A, Type three-secretion system effector Pst DC3000 polymutants show a differential growth response in Col-0. Bacterial growth of Pst DC3000 and the suite of Pst DC3000 effector polymutants were enumerated at 24 h after dip-inoculation with Pst DC3000 polymutant strains at a concentration of 3 × 107 cfu ml−1. Shaded boxes indicate the presence of the described effector gene clusters. Error bars, representing mean ± se, were calculated from five (n = 15) biological replicates for Pst DC3000, and from two (n = 6) for each of the Pst DC3000 polymutants. Bacterial growth was enumerated based on cfu per mg fresh weight. Statistical significance was determined using a one-way ANOVA, Tukey test; P < 0.01. B, Pst DC3000 in planta growth correlates with the degree of modulation of host actin architecture. Actin skewness and density values were determined in Col-0/GFP-fABD2 seedlings at 24 hpi. Representative micrographs (left) from n = 80–90 are shown. Quantification of actin bundling (center) and density (right) indicates a bacterial density-dependent alteration in host actin cytoskeleton architecture after inoculation. Wild-type Col-0 plants and Col-0/GFP-fABD2 plants were dip-inoculated with Pst DC3000 at concentrations of 3 × 107 cfu ml−1, 3 × 106 cfu ml−1, and 106 cfu ml−1. Mock = MgCl2 dip-inoculated. **, P < 0.001. Bar = 20 μm. C, In planta bacterial growth enumeration of Pst DC3000 D28E and the ∆hrcC mutant. Bacterial growth was enumerated at 24 hpi with Pst DC3000 at three separate initial inoculation concentrations; concentrations of 3 × 107 cfu ml−1, 3 × 106 cfu ml−1, and 106 cfu ml−1. Pst DC3000 D28E and the ∆hrcC mutant were inoculated at 3 × 107 cfu ml−1. Bacterial growth assays were repeated twice. Error bars, representing mean ± se, were calculated from three (n = 9) technical replicates. Statistical significance was determined using a one-way ANOVA; P < 0.01.
Figure 3.
Figure 3.
Actin bundling in Arabidopsis is suppressed by the Pst DC3000 cluster IX polymutant. Actin filament architecture in 10-d-old Col-0/GFP-fABD2 cotyledons was quantified at 24 h after dip-inoculation with the Pst DC3000 polymutants. A, CUCPB5459 (ΔI, II, IV, IX, X); B, ΔIX (CUCPB5529); and C, ΔCEL (CUCPB5115). Seedlings were dip-inoculated with Pst DC3000 strains at a concentration of 3 × 107 cfu ml−1 for analyses shown in A and B. In C, seedlings were dip-inoculated with Pst DC3000 at a concentration of 106 cfu ml−1 and with Pst DC3000 ΔCEL at 3 × 107 cfu ml−1. Representative confocal micrographs are shown. Values for actin skewness and density are shown as relative comparisons to Pst DC3000-inoculated plants. Error bars represent mean ± se from the skewness and density values of three independent biological repeats (n = 80–90). Statistical significance was determined using a Student’s t-test, as compared to Pst DC3000 treatment. *, P < 0.01; **, P < 0.001. Bar = 20 μm.
Figure 4.
Figure 4.
The T3E HopG1 induces actin bundling and a concomitant reduction in actin filament array density in Arabidopsis epidermal cells. Changes in actin filament organization in 10-d-old Col-0/GFP-fABD2 cotyledons was quantified at 24 h after dip-inoculation with Pst DC3000 and each of the cluster IX single effector mutant-complemented CUCPB5529 (i.e. cluster ΔIX) lines. A, Illustration of the cluster IX region of Pst DC3000. Scale bar at right = 1 kb. B, ΔIX + phopAO1; C, ΔIX + phopV1; D, ΔIX + phopAA1-2; E, ΔIX + phopQ1-1; and F, ΔIX + phopG1. Representative micrographs are shown. Initial pathogen inoculums for Pst DC3000 were 3 × 107 cfu ml−1. Actin skewness and density values were determined in 10-d-old Col-0/GFP-fABD2 seedlings at 24 h after dip-inoculation with Pst DC3000. Error bars represent mean ± se from the skewness and density values of three independent biological repeats (n = 80–90). Statistical significance was determined using a Student’s t-test, as compared to Pst DC3000 treatment. *, P < 0.01; **, P < 0.001. Bar = 20 μm.
Figure 5.
Figure 5.
Pathogen-induced actin bundling is suppressed in the hopG1 single mutant. Actin filament organization in 10-d-old Col-0/GFP-fABD2 cotyledons was observed at 24 h after dip-inoculation with the Pst DC3000, ΔhopG1, and Pst DC3000 ΔhopG1 + phopG1. A, Pst DC3000 ΔhopG1 and B, Pst DC3000 ΔhopG1 + phopG1. Pst DC3000 strains were inoculated at a concentration of 3 × 107 cfu ml−1. Representative micrographs are shown. Actin skewness and density values were determined in 10-d-old Col-0/GFP-fABD2 seedlings at 24 h after dip-inoculation with Pst DC3000. Error bars represent mean ± se from the skewness and density values of seven independent biological repeats (n = 202–204) for A and three independent biological repeats (n = 71–74) for B. Statistical significance was determined using a Student’s t-test, as compared to Pst DC3000 wild type. **P < 0.001. Bar = 20 μm. C, In planta bacterial growth enumeration of Pst DC3000, the Pst DC3000 ΔhopG1 mutant, and the Pst DC3000 ΔhopG1 mutant complemented with hopG1 (i.e. ΔhopG1 phopG1). Bacterial growth was enumerated at 24 h after dip-inoculation at 3 × 107 cfu mL−1. Bacterial growth assays were repeated twice. Error bars, representing mean ± se, were calculated from three (n = 6) technical replicates. Statistical significance was determined using a one-way ANOVA, Tukey test; P < 0.01.
Figure 6.
Figure 6.
HopG1 is required for Pst DC3000-induced actin-dependent chlorosis. A, Pst DC3000 induces chlorosis in Arabidopsis at 3 dpi. Inoculation of 10-d-old wild-type Col-0 seedlings with Pst DC3000, Pst DC3000 ΔIX, Pst DC3000 ΔIX phopG1, and Pst DC3000 ΔhopG1 at a concentration of 3 × 107 cfu ml−1 revealed a requirement for HopG1 in chlorosis elicitation. B, Actin stabilization inhibits HopG1-induced chlorosis. Five-week-old plants were co-infiltrated with either MgCl2, cytochalasin-d, or jasplakinolide and Pst DC3000, the cluster IX polymutant (± HopG1), or Pst DC3000 ΔhopG1; plants were monitored for the induction of senescence-associated chlorosis at 3 dpi. Leaves inoculated with wild-type Pst DC3000 alone showed visible signs of chlorosis at 3 dpi. C, Bacterial growth assays of Pst DC3000 and the cluster IX variants 10-d-old Col-0 plants at 0–3 dpi. Pst DC3000 strains were inoculated at a concentration of 3 × 107 cfu ml−1. Bacterial growth assays were repeated at least three times. Error bars, representing mean ± se, were calculated from three (n = 9) technical replicates of three independent biological repeats. Bacterial growth was enumerated based on cfu per mg fresh weight of plant tissue. D, The actin-stabilizing and barbed-end binding agents, jasplakinolide and cytochalasin-d, respectively, do not affect the growth of Pst DC3000 or the cluster IX mutant variants in wild-type Col-0. Five-week-old Col-0 leaves were co-infiltrated with cytochalasin-d or jasplakinolide, and Pst DC3000 strains at a concentration of 2 × 105 cfu mL−1, and bacterial growth was enumerated at 3 dpi. Bacterial growth assays were repeated three times. Error bars, representing mean ± se, were calculated from three (n = 9) technical replicates of three independent biological repeats. Statistical significance was determined using a one-way ANOVA, Tukey test; P < 0.01. CD, cytochalasin-d; f.w., fresh weight; Jasp, jasplakinolide; Mock, MgCl2.
Figure 7.
Figure 7.
HopG1 induces host disease symptoms through the function of a mitochondrial-localized kinesin. A, The Pst DC3000 T3E HopG1 interacts with the N-terminal half of an Arabidopsis kinesin (At4g39050). Epitope-tagged fusions to the open-reading frame of HopG1 (T7) and the first 978 nucleotides of kinesin (HA) were transiently expressed (40 h) in N. benthamiana and protein extracts were isolated and analyzed for a specific interaction between HopG1 and kinesin using co-immunoprecipitation assays. B, Kinesin and HopG1 are associated with the host actin cytoskeleton. Epitope-tagged fusions to the first 978 nucleotides of kinesin (HA) and the open-reading frame of HopG1 (T7) were transiently expressed (40 h) in N. benthamiana and total protein extracts were analyzed by co-immunoprecipitation analysis for a specific interaction among kinesin, HopG1, and the actin cytoskeleton (left blot), and an interaction between kinesin and the actin cytoskeleton (right blot). C, A mitochondrial-localized kinesin is required for Pst DC3000-induced symptom development. Five-week-old plants were co-infiltrated with either MgCl2, cytochalasin-d, or jasplakinolide and Pst DC3000 at a concentration of 2 × 105 cfu ml−1, and plants were monitored for the induction of chlorosis. Wild-type Col-0 plants inoculated with Pst DC3000 showed visible signs of disease at 3 dpi. When plants were co-inoculated with Pst DC3000 and cytochalasin-d, pathogen-induced chlorosis phenotype was enhanced; in the presence of jasplakinolide, the chlorosis phenotype was reduced. In the Arabidopsis kinesin mutant, inoculation with Pst DC3000 showed a significantly reduced chlorosis phenotype (compared to wild-type Col-0), while co-inoculation with Pst DC3000 and cytochalasin-d resulted in the elicitation of a pronounced disease phenotype. Co-inoculation of the kinesin mutant with Pst DC3000 and jasplakinolide showed a marked reduction in disease symptoms. D, Bacterial growth assays of Pst DC3000 in wild-type Col-0 and the kinesin mutant. Pst DC3000 strains were inoculated at a concentration of 3 × 107 cfu ml−1. Bacterial growth assays were performed two times with similar results. Error bars, representing mean ± se, were calculated from three (n = 6) technical replicates of two independent biological repeats. In planta pathogen growth was enumerated based on cfu per cm2 plant tissue. Statistical significance was assessed using a Student’s t-test. E, The actin-stabilizing and barbed-end binding agents, jasplakinolide and cytochalasin-d, respectively, do not affect the in planta growth of Pst DC3000 in wild-type Col-0 or the kinesin mutant. Five-week-old wild-type Col-0 leaves were co-infiltrated with cytochalasin-d or jasplakinolide and Pst DC3000 (2 × 105 cfu ml−1), and bacterial growth was enumerated at 3 dpi. Bacterial growth assays were performed two times. Error bars, representing mean ± se, were calculated from three (n = 6) technical replicates of two independent biological repeats. Statistical significance was determined using a one-way ANOVA, Tukey test; P < 0.01. CD, cytochalasin-d; f.w., fresh weight; Jasp, jasplakinolide; Mock, MgCl2.
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