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. 2012;8(11):e1003006.
doi: 10.1371/journal.ppat.1003006. Epub 2012 Nov 8.

Arabidopsis Actin-Depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics

Katie Porter  1 , Masaki Shimono, Miaoying Tian, Brad Day
Affiliations

Arabidopsis Actin-Depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics

Katie Porter et al. PLoS Pathog. 2012.

Abstract

The primary role of Actin-Depolymerizing Factors (ADFs) is to sever filamentous actin, generating pointed ends, which in turn are incorporated into newly formed filaments, thus supporting stochastic actin dynamics. Arabidopsis ADF4 was recently shown to be required for the activation of resistance in Arabidopsis following infection with the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst) expressing the effector protein AvrPphB. Herein, we demonstrate that the expression of RPS5, the cognate resistance protein of AvrPphB, was dramatically reduced in the adf4 mutant, suggesting a link between actin cytoskeletal dynamics and the transcriptional regulation of R-protein activation. By examining the PTI (PAMP Triggered Immunity) response in the adf4 mutant when challenged with Pst expressing AvrPphB, we observed a significant reduction in the expression of the PTI-specific target gene FRK1 (Flg22-Induced Receptor Kinase 1). These data are in agreement with recent observations demonstrating a requirement for RPS5 in PTI-signaling in the presence of AvrPphB. Furthermore, MAPK (Mitogen-Activated Protein Kinase)-signaling was significantly reduced in the adf4 mutant, while no such reduction was observed in the rps5-1 point mutation under similar conditions. Isoelectric focusing confirmed phosphorylation of ADF4 at serine-6, and additional in planta analyses of ADF4's role in immune signaling demonstrates that nuclear localization is phosphorylation independent, while localization to the actin cytoskeleton is linked to ADF4 phosphorylation. Taken together, these data suggest a novel role for ADF4 in controlling gene-for-gene resistance activation, as well as MAPK-signaling, via the coordinated regulation of actin cytoskeletal dynamics and R-gene transcription.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ADF4 is required for RPS5 mRNA accumulation and resistance to Pseudomonas syringae expressing the cysteine protease effector AvrPphB.
Time-course of mRNA accumulation of (A) RPS5 and (C) PBS1 in Col-0 and adf4 mutant plants following dip inoculation with Pst AvrPphB. (B) Expression levels of RPS5 in Col-0, pbs1, adf4/g:ADF4, and adf3. (D) RPS5 mRNA accumulation in Col-0 and rps5-1, comparing each to their basal untreated levels at 24 hpi with Pst AvrPphB. Error bars represent mean ± SEM from two technical replicates of two independent biological repeats (n = 4). Statistical significance was determined using two-way ANOVA as compared to Col-0, with Bonferroni post test, where *p<0.05 and ***p<0.001. hpi = hours post inoculation.
Figure 2
Figure 2. Flg22-induced receptor kinase 1 expression in the adf4 mutant is reduced when the effector protein AvrPphB is expressed in planta.
Relative expression levels of FRK1 mRNA in Col-0, adf4, and rps5-1 plants when treated with (A) 10 µM flg22, (B) mock inoculated with MgCl2 by hand infiltration (C) Pst AvrPphB, or (D) the hrpH − (Pst hrpH −). Error bars represent mean ± SEM from two technical replicates of two independent biological repeats (n = 4). Statistical significance was determined using two-way ANOVA, as compared to Col-0, with Bonferroni post test where *p<0.05 and **p<0.005. hpi = hours post-inoculation.
Figure 3
Figure 3. Mitogen Activated Protein Kinase (MAPK) phosphorylation is reduced in the adf4 mutant in the presence of AvrPphB.
(A) Percent maximal phosphorylation of the MPK3/6 TEY motif in Col-0 and the adf4 mutant, +/− AvrPphB, followed by 1 µM flg22 treatment. (B) Percent maximal phosphorylation of the MPK3/6 TEY motif in Col-0 and the rps5-1 mutant, +/− AvrPphB, followed by 1 µM flg22 treatment. AvrPphB expression was induced at 48 h pre-treatment with 100 µM estradiol in Col-0, adf4 and rps5-1 mutant plants containing an estradiol-inducible AvrPphB transgene (pER8:AvrPphB). Statistical significance was determined using two-way ANOVA as compared to Col-0 untreated, with Bonferroni post test, where *p<0.05, **p<0.005, n = 3. (C) Western blot analysis of MPK3/6 TEY phosphorylation.
Figure 4
Figure 4. Phosphorylation of ADF4 is required for RPS5 mRNA expression.
(A) Western blot of isoelectric focusing (IEF) and SDS PAGE analysis of wild type ADF4 (upper) and phospho-null ADF4_S6A (lower). Arrows indicate direction of IEF and SDS PAGE. (B) The relative expression levels of RPS5 were determined by qRT-PCR. (C) HR phenotypes at 22 hours after bacterial infiltration (upper), disease phenotypes at 4 dpi (lower). (D) Enumeration of bacterial growth at 0 and 4 dpi. HR and bacterial population experiments were repeated at least 3 times. Error bars, representing mean ± SEM, were calculated from two (A; n = 4) or three (D; n = 9) technical replicates of two independent biological repeats. Statistical significance was determined using two-way ANOVA, comparing adf4 to Col-0, with Bonferroni post test, where *p<0.05; ***p<0.001. hpi = hours post inoculation; dpi = days post inoculation.
Figure 5
Figure 5. Confocal microscopy demonstrates phosphorylation of ADF4 affects cytoskeletal localization, but not nuclear localization.
(A) Laser-scanning confocal microscopy of adf4, adf4/35S:ADF4, adf4/35S:ADF4_S6A and adf4/35S:ADF4_S6D isolated nuclei; DAPI stained nuclei (blue), immunochemistry FITC (green), and overlay. Bar = 2 µm. (B) Images of transiently expressed fABD2-GFP (green), dsRed- ADF4 _S6A/_S6D (red), and overlay in Nicotiana benthamiana taken by laser-scanning confocal microscopy. Bar = 5 µm. (C) Graphical representation of the overlay coefficient according to Manders (R) and the co-localization coefficients m1 and m2. Error bars, representing mean ± SEM, were calculated from two biological repeats (n = 40). Overlap coefficient (R) is considered to be co-localized when #R = 0.6 to 1.0, and co-localization coefficients indicate co-localization when *m1>0.5 and *m2>0.5.
Figure 6
Figure 6. Proposed model illustrating the virulence and avirulence function of the bacterial cysteine protease AvrPphB through an ADF4-dependent mechanism.
Following delivery of AvrPphB into the plant cells by Pst via the T3SS, AvrPphB targets multiple innate immune signaling pathways, including: 1) PTI, via the cleavage of BIK1 kinase; 2) ETI, via the cleavage of the kinase PBS1, a guardee of the resistance protein RPS5. We propose a potential role for AvrPphB in the modulation of actin cytoskeletal dynamics via the targeting of an unknown kinase responsible for the phosphorylation of ADF4 that ultimately results in reduced expression of RPS5, as well as specific down-regulation of MAP kinase signaling. ADF4 translocation into the nucleus is independent of phosphorylation status, however, F-actin co-localization and RPS5 gene expression are dependent upon the phosphorylation of ADF4.
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References

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