The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns

Abstract

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.

Figure 1. Arabidopsis seedlings exhibit changes in actin organization in response to P. syringae pv. tomato DC3000.

Representative images of epidermal pavement cells from A. thaliana cotyledons expressing the actin reporter GFP-fABD2. Images show an apparent increase in filament abundance at 6 hpi when treated with Pseudomonas syringae pv. tomato DC3000 (B) or the T3SS-deficient mutant hrpH (C), compared to mock treated (A). At 24 hpi, an increase in the extent of filament bundling is observed following inoculation with DC3000 (E), compared to hrpH (F) and mock (D). Epidermal cells from 10 d-old light-grown seedlings were imaged with spinning disk confocal microscopy (SDCM), and micrographs shown are z-series projections compiled from 24 optical sections. Bar = 15 µm.

Figure 2. Changes in actin filament organization in response to inoculation with pathogenic and non-pathogenic P. syringae.

Actin architecture parameters for percent occupancy (A) and extent of filament bundling (B) were measured over time in response to P. syringae inoculation. Percent occupancy increases transiently in epidermal cells of DC3000-inoculated and hrpH-inoculated cotyledons at 0–18 hpi. Filament density returns to mock-treated levels at 18–21 hpi and then significantly decreases at 24–36 hpi in epidermal cells from DC3000-inoculated cotyledons. Seedlings treated with hrpH, however, do not show the same reduction in percent occupancy from 24 hpi onwards. The presence of actin filament bundles in DC3000-inoculated seedlings is significantly enhanced at 18–36 hpi. In contrast, hrpH-inoculated cotyledons show no change in filament bundling at any timepoint measured. Images were collected as described for Figure 1. Values given are means ± SE (n = 105–150 images per treatment, per timepoint, from n = 3 biological repeats). Significant differences by ANOVA, with Tukey HSD post-hoc analysis, are represented as follows: a, P≤0.05 between mock and treatment; b, P≤0.05 between DC3000 and treatment.

Figure 3. Diverse PTI-inducing microbes elicit an increase in actin filament density.

Actin architecture analysis of cotyledons inoculated with P. syringae pv. phaseolicola (Pph; [A]), Agrobacterium tumefaciens GV3101 (A. tum.; [C]), or Magnaporthe grisea (E) display a significant increase in actin filament density at 6–9 hpi. In contrast, bundling was not significantly different from mock-treated controls following Pph (B), A. tumefaciens (D), or M. grisea (F) inoculations. Values given are means ± SE (n = 150 images per treatment, from n = 3 biological repeats). Significant differences by ANOVA, with Tukey HSD post-hoc analysis, are represented as follows: a, P≤0.05 between mock and treatment; b, P≤0.05 between DC3000 and treatment.

Figure 4. Analysis of P. syringae mutants implicate a role for PTI in the filament density change.

Actin architecture analysis of epidermal cells treated with the T3SS-deficient mutant hrcC (A), the effectorless mutant D28E (C), the coronatine-deficient mutant COR- (E), and the flagellin deletion mutant ΔfliC (G) exhibit significantly enhanced actin filament density compared to mock-treated controls at 6–9 hpi. However, bundling analysis of the same images used in (A, C, E, & G) shows no significant change from mock-treated following hrcC (B), D28E (D), COR- (F), and ΔfliC (H) inoculation. Images were collected at 6–9 hpi. Values given are means ± SE (n = 150 images per treatment, from n = 3 biological repeats). Significant differences by ANOVA, with Tukey HSD post-hoc analysis, are represented as follows: a, P≤0.05 between mock and treatment; b, P≤0.05 between DC3000 and treatment.

Figure 5. Treatment with flg22 peptide is sufficient to increase filament density.

Twenty-four d-old plants that were hand-infiltrated with 1 µM flg22 peptide show significantly enhanced filament density (A), whereas the same concentration of either elf26 or A. tumefaciens flg (flg^At) peptide exhibit no change compared to mock controls. Bundling analysis (B) of the same images used in (A) shows no significant change for any MAMP peptide treatment. Epidermal pavement cells from rosette leaves were imaged by SDCM at 0–3 hpi and 24 optical sections were combined into a z-series projection. Values given are means ± SE (n = 150 images per treatment, from n = 3 biological repeats). Asterisks represent significant differences by ANOVA.

Figure 6. Arabidopsis knock-out mutants define the early signaling steps required for increased actin filament density.

Actin architecture analysis of epidermal cells was performed on 24 d-old Arabidopsis mutants that were hand-infiltrated with 1 µM flg22 peptide or chitin oligomers. Treatment with 1 µM flg22 (B & D) or 1 µM chitin (C & D) is sufficient to increase filament abundance in wild-type Columbia-0 plants, whereas mock-treatment does not elicit a change to actin architecture (A & D). Actin architecture analysis of mock (F & I)- or flg22 (G & I)-treated epidermal cells from flagellin-sensing 2 (fls2) knock-out mutants did not elicit a change in filament abundance, however, chitin-treatment (H & I) did. Similar to fls2, actin architecture in wild-type Ws-0 plants was not changed with mock (K & N)- or flg22-treatment (L & N), however treatment with chitin (M & N) was sufficient to increase filament density. Finally, actin architecture analysis of epidermal cells from brassinosteroid insensitive1-associated receptor like kinase1 (bak1-4) or botrytis induced kinase1 (bik1) homozygous mutant plants did not exhibit significantly different changes in filament density from mock (for bak1-4: [P & S]; for bik1: [U & X]) with either flg22 (for bak1-4: [Q & S]; for bik1: [V & X]) or chitin (for bak1-4: [R & S]; for bik1: [W & X]) treatments. No significant changes to bundling compared with mock respective controls were observed (E, J, O, T & Y). Epidermal pavement cells from rosette leaves were imaged by SDCM at 0–3 hpi and 24 optical sections were combined into a z-series projection. Values given are means ± SE (n = 150 images per treatment, from n = 3 biological repeats). Asterisks represent significant differences by ANOVA.

Figure 7. Disruption of the host-cell actin cytoskeleton promotes virulence.

The consequences of disrupting the host-cell actin cytoskeleton with latrunculin B (LatB) were measured by microscopy and by quantifying the amount of bacterial growth on co-infiltrated leaves. Representative images of epidermal pavement cells from Arabidopsis show an apparent increase in filament abundance at 6 hpi when treated with DC3000 or hrpH in the absence of LatB (A–C). However, actin arrays appear less abundant in epidermal cells of plants following treatment with 1 µM (D) or 10 µM LatB (G) but not treated with bacteria (i.e. mock). Further, the apparent increase in actin filament abundance appears severely reduced following co-infiltration of LatB and DC3000 (E & H) or hrpH (F & I). Significant decreases in actin filament abundance were measured for plants co-infiltrated with DC3000 or hrpH at various concentrations of LatB (J). Disruptions to the host-cell actin cytoskeleton elicited a significant increase in bacterial growth following co-infiltration with LatB and DC3000 or hrpH (K). Epidermal pavement cells from rosette leaves of 24 d-old wild-type Arabidopsis plants were imaged by SDCM at 6–9 hpi and 24 optical sections were combined into a z-series projection. Values given are means ± SE (n = 30 images per treatment). LatB co-infiltration experiments were repeated 3 times and the mean results are presented ± SE. Asterisks represent significant differences by ANOVA. nd, no significant difference compared to 0 µM treatment; *, P≤0.05; **, P≤0.001; ***, P≤0.0001.