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. 2014 Jan;26(1):340-52.
doi: 10.1105/tpc.113.122499. Epub 2014 Jan 24.

ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during innate immune signaling in Arabidopsis

Jessica L Henty-Ridilla  1 , Jiejie Li, Brad Day, Christopher J Staiger
Affiliations

ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during innate immune signaling in Arabidopsis

Jessica L Henty-Ridilla et al. Plant Cell. 2014 Jan.

Abstract

Conserved microbe-associated molecular patterns (MAMPs) are sensed by pattern recognition receptors (PRRs) on cells of plants and animals. MAMP perception typically triggers rearrangements to actin cytoskeletal arrays during innate immune signaling. However, the signaling cascades linking PRR activation by MAMPs to cytoskeleton remodeling are not well characterized. Here, we developed a system to dissect, at high spatial and temporal resolution, the regulation of actin dynamics during innate immune signaling in plant cells. Within minutes of MAMP perception, we detected changes to single actin filament turnover in epidermal cells treated with bacterial and fungal MAMPs. These MAMP-induced alterations phenocopied an ACTIN DEPOLYMERIZING FACTOR4 (ADF4) knockout mutant. Moreover, actin arrays in the adf4 mutant were unresponsive to a bacterial MAMP, elf26, but responded normally to the fungal MAMP, chitin. Together, our data provide strong genetic and cytological evidence for the inhibition of ADF activity regulating actin remodeling during innate immune signaling. This work is the first to directly link an ADF/cofilin to the cytoskeletal rearrangements elicited directly after pathogen perception in plant or mammalian cells.

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Figures

Figure 1.
Figure 1.
Actin Filament Density Increases following elf26 Treatment. (A) to (C) VAEM images of hypocotyl epidermal cells expressing GFP-fABD2. Hypocotyls were treated with mock (A), 1 μM flg22 (B), or 1 μM elf26 (C) peptides for 5 min prior to imaging. Images are from individual representative hypocotyls following different treatments, with cells located near the cotyledon (top) at the left and cells located near the root (bottom) at the right. Bars = 10 μm. (D) Actin filament abundance, or percentage of occupancy, was measured and binned into three equal regions along the length of the hypocotyl. All epidermal cells treated with elf26 had significantly increased filament density throughout the hypocotyl when compared with mock-treated seedlings. However, cells treated with flg22 were not significantly different from mock. (E) The extent of actin filament bundling, or skewness, was measured from the same images used for (D). There was no significant difference between treatments with flg22, elf26, or mock. Values given are means ± se (n = 450 cells per region, from at least 30 hypocotyls). Asterisks represent significant differences by ANOVA, with Tukey HSD posthoc analysis (nd = not significantly different from mock; ***P < 0.001).
Figure 2.
Figure 2.
Recognition of elf26 by the EFR Receptor Complex Is Required for the Increase in Actin Abundance. (A) Arabidopsis knockout mutants define the early signaling steps required for increased actin abundance in hypocotyl epidermal cells. Filament abundance was measured in epidermal cells from homozygous signaling mutant seedlings expressing GFP-fABD2 following 5 min of treatment with 1 µM elf26 or mock. Columbia-0 expressing GFP-fABD2 was used as the wild-type control. Knockout lines for EFR and components of the PRR complex, BAK1 and BIK1, were used to define the host-signaling components required for eliciting the actin response. Wild-type and fls2 homozygous mutant seedlings exhibit enhanced filament abundance following 1 µM elf26 treatment, whereas knockout mutants for the EFR-PRR complex (i.e., efr-1, bak1-4, and bik1) did not have a measurable change compared with the mock control. (B) The extent of actin filament bundling was not significantly different from mock-treated controls. Images used for analysis in (A) were measured for filament bundling. Values given are means ±se (n = 300 cells per genotype from at least 30 hypocotyls). Asterisks represent significant differences by ANOVA, with Tukey HSD posthoc analysis (nd = not significantly different from mock; ***P < 0.001).
Figure 3.
Figure 3.
Filament Severing Is Significantly Reduced following elf26 Treatment. (A) Time-lapse series of VAEM images shows actin filament turnover in the cortical cytoplasm of a mock-treated epidermal cell. The highlighted filament (green dots) elongated at 1.72 µm s−1 before suffering several breaks (arrows). Actin filament bundles (stars) remained relatively stationary throughout the time series. (B) A representative growing filament (green dots) from a wild-type cell treated with 1 μM elf26 peptide for 5 min displayed fewer severing events (arrows) compared with the mock-treated cell. Micrographs in (A) and (B) were collected at 1.5-s intervals, and every other image is presented in each montage. Bars = 5 μm.
Figure 4.
Figure 4.
The adf4 Mutant Lacks Changes in Actin Filament Architecture and Dynamics following Treatment with elf26. (A) to (F) Actin filaments in mock-treated epidermal cells from the adf4 mutant (D) appeared to be significantly less abundant and more bundled compared with wild-type cells. Notably, the actin architecture in adf4 cells did not seem to change following treatment with elf26 peptide (E), unlike wild-type cells (B), where actin filaments appeared to be more abundant after MAMP treatment. In response to the fungal MAMP, chitin, cortical actin abundance increased in both the wild type (C) and the adf4 mutant (F). Bars = 5 µm. (G) Actin filament abundance was measured in epidermal cells at the base of wild-type and adf4 mutant hypocotyls after treatment with MAMPs for 5 min. Wild-type epidermal cells treated with 1 µM elf26 or 1 µM chitin had a significant increase in filament abundance compared with mock-treated seedlings. Actin filament abundance in cells from the adf4 mutant treated with 1 µM elf26 remained unchanged compared with mock-treated controls. However, actin filament abundance in the adf4 mutant was significantly elevated following treatment with chitin. (H) The extent of actin filament bundling was not altered following treatment with chitin in either wild-type or adf4 hypocotyl epidermal cells. The same images analyzed in (G) were measured for actin filament bundling. (I) to (K) Several parameters of actin filament turnover do not change in the adf4 mutant following elf26 treatment. The adf4 mutant had significantly enhanced filament lengths (I) and lifetimes (J) as well as a reduction in severing frequency (K) compared with the wild type. Whereas each of these parameters was significantly changed in wild-type seedlings upon treatment with elf26, these do not change in the adf4 mutant. (L) By contrast, there were significant increases in filament-filament annealing in the adf4 loss-of-function mutant compared with wild-type cells. Following elf26 treatment, both the wild type and the adf4 mutant respond with significantly enhanced filament–filament annealing. Values given are means ±se (n = 300 cells per treatment and genotype from at least 30 hypocotyls). Asterisks represent significant differences by ANOVA, with Tukey HSD posthoc analysis (nd = not significantly different from genotype-specific mock control; ***P < 0.001; † denotes significant differences between the wild type and adf4). For more details, see Supplemental Table 1.
Figure 5.
Figure 5.
Callose Deposition Fails to Occur in the adf4 Mutant following elf26 Treatment. (A) to (F) Callose deposits in epidermal cells from the base of wild-type hypocotyls appeared to increase following treatment with 1 µM elf26 (C) or 1 µM chitin (E) compared with mock control (A). Callose deposits in the adf4 mutant (B) appeared less abundant than in mock-treated WT cells (A). Furthermore, the number of callose deposits did not appear to increase in adf4 following 1 µM elf26 treatment (D), whereas they did increase following 1 µM chitin treatment (F). Bars = 20 µm. (G) Quantification of aniline blue-stained spots demonstrates that callose deposition was inhibited in the adf4 mutant following 1 µM elf26 treatment. Values given are means ±se (n = 50 images per treatment, from at least 30 hypocotyls). Asterisks represent significant differences by ANOVA, with Tukey HSD posthoc analysis (nd = not significantly different from mock; ***P < 0.001). (H) A model describing the role of ADF4 during innate immune signaling.
Figure 6.
Figure 6.
Transcriptional Activation in the CDPK Pathway Is Attenuated in adf4 following elf26 Treatment. Quantitative analysis of marker genes for early defense signaling in the MAPK and CDPK pathways. (A) to (C) A MAPK-specific reporter gene, FRK1 (A), as well as the MAPK dominant-pathway genes CYP81F2 (B) and WRKY33 (C), were induced in both wild-type and adf4 plants following treatment with elf26 or chitin. (D) and (E) PHI1, a CDPK-specific response gene (D), and the CDPK-synergistic pathway gene, NHL10 (E), were induced in the wild type following treatment with elf26; however, PHI1 and NHL10 induction was markedly reduced in the adf4 mutant. By contrast, both wild-type and adf4 plants responded with increased PHI1 and NHL10 transcripts following treatment with chitin. Expression of each defense signaling gene and the housekeeping gene GAPD were absent from controls lacking reverse transcriptase (data not shown). Mean values from triplicate biological samples and technical replications are plotted ±se, normalized to GAPD expression and presented as fold induction from mock. Defense gene expression was significantly increased following treatment with either elf26 or chitin on wild-type and the adf4 mutant seedlings compared with mock-treated controls (P < 0.001, ANOVA with Tukey HSD posthoc analysis).
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