Arabidopsis formin 2 regulates cell-to-cell trafficking by capping and stabilizing actin filaments at plasmodesmata
- Tsinghua University, China
- Institute of Botany, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
Figures
Figure 1 with 2 supplements
AtFH2 localizes to plasmodesmata and its PD localization is determined by the TM-containing N-terminus.
(A) Images of AtFH2-mCherry and callose (stained with aniline blue) in Arabidopsis epidermal pavement cells and the merged image. (B) Images of AtFH2-mCherry and PDLP1-eGFP in Arabidopsis epidermal …
Figure 1—figure supplement 1
No overt developmental defect is detected in seedlings and adult plants of atfh2 mutants.
(A) Physical structure of the AtFH2 gene. Exons, introns and untranslated regions are represented by black boxes, black lines, and grey boxes, respectively. Two T-DNA insertion lines GK_066D02 and …
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Figure 1—figure supplement 1—source data 1
Quantification of relative expression of AtFH2 plotted in Figure 1—figure Supplement 1C.
- https://doi.org/10.7554/eLife.36316.005
Figure 1—figure supplement 2
AtFH2 localizes to cell borders and its localization pattern does not depend on the presence of an intact actin cytoskeleton.
(A) Transgenic expression of AtFH2-eGFP in Arabidopsis. eGFP fluorescence is visible as punctate spots on the boundaries of Arabidopsis epidermal pavement cells. (B) AtFH2-eGFP (green) localizes at …
Figure 2 with 4 supplements
Loss of function of AtFH2 increases SEL of PD.
(A) Diagram of the eGFP diffusion assay in Arabidopsis epidermal pavement cells. The edge of the initial cell expressing eGFP, cells in the first diffusion layer and cells in the second diffusion …
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Figure 2—source data 1
Quantification of eGFP diffusion plotted in Figure 2B.
- https://doi.org/10.7554/eLife.36316.022
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Figure 2—source data 2
Quantification of eGFP diffusion plotted in Figure 2C.
- https://doi.org/10.7554/eLife.36316.023
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Figure 2—source data 3
Quantification of eGFP diffusion plotted in Figure 2D.
- https://doi.org/10.7554/eLife.36316.024
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Figure 2—source data 4
Quantification of CMV MP transporting plotted in Figure 2E.
- https://doi.org/10.7554/eLife.36316.025
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Figure 2—source data 5
Quantification of Fny-CMV infection plants plotted in Figure 2H.
- https://doi.org/10.7554/eLife.36316.026
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Figure 2—source data 6
Quantification of relative CMV MP RNA accumulation plotted in Figure 2I.
- https://doi.org/10.7554/eLife.36316.027
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Figure 2—source data 7
Quantification of leaf length plotted in Figure 2J.
- https://doi.org/10.7554/eLife.36316.028
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Figure 2—source data 8
Quantification of leaf length plotted in Figure 2K.
- https://doi.org/10.7554/eLife.36316.029
Figure 2—figure supplement 1
Loss of function of AtFH1 and/or AtFH2 increases SEL of PD.
(A) eGFP, HEDL-mCherry and the merged image. A non-mobile fluorescent protein, HDEL-mCherry, was used to indicate the bombarded cell. Bar = 10 μm. (B) Quantification of eGFP diffusion cell layers in …
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Figure 2—figure supplement 1—source data 1
Quantification of eGFP diffusion plotted in Figure 2—Figure Supplement 1B.
- https://doi.org/10.7554/eLife.36316.009
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Figure 2—figure supplement 1—source data 2
Quantification of eGFP diffusion plotted in Figure 2—Figure Supplement 1C.
- https://doi.org/10.7554/eLife.36316.010
Figure 2—figure supplement 2
No overt defect of cytoplasmic streaming is detected in hypocotyl cells of atfh2 mutants.
(A) Time-lapse images of plastid movement in the hypocotyl cells of wild type and atfh2-1 Arabidopsis. White arrows indicate the plastid. Bar = 10 μm. (B) Average velocity of plastids in the …
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Figure 2—figure supplement 2—source data 1
Quantification of cytoplasmic streaming plotted in Figure 2—Figure Supplement 1B.
- https://doi.org/10.7554/eLife.36316.012
Figure 2—figure supplement 3
Overexpression of AtFH2 does not affect the overall organization of actin filaments and cell-to-cell trafficking.
(A) Determination of the level of AtFH2 transcripts in WT, atfh2 mutants and AtFH2 OE plants by qRT-PCR. AtFH2 OE represents Arabidopsis plants expressing pCAMBIA1301-35S:AtFH2-mCherry. The …
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Figure 2—figure supplement 3—source data 1
Quantification of relative expression of AtFH2 plotted in Figure 2—Figure Supplement 3A.
- https://doi.org/10.7554/eLife.36316.014
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Figure 2—figure supplement 3—source data 2
Quantification of actin filament density plotted in Figure 2—Figure Supplement 3D.
- https://doi.org/10.7554/eLife.36316.015
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Figure 2—figure supplement 3—source data 3
Quantification of eGFP diffusion plotted in Figure 2—Figure Supplement 3E.
- https://doi.org/10.7554/eLife.36316.016
Figure 2—figure supplement 4
The symptoms of Fny-CMV infection in Arabidopsis plants.
(A) Arabidopsis leaves showing symptoms of viral infection after inoculation with Fny-CMV. Compared to plants treated with water (mock), the leaves of the Fny-CMV-treated plants start to become …
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Figure 2—figure supplement 4—source data 1
Quantification of leaf length plotted in Figure 2—figure supplement 4C.
- https://doi.org/10.7554/eLife.36316.018
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Figure 2—figure supplement 4—source data 2
Quantification of leaf length plotted in Figure 2—figure supplement 4D.
- https://doi.org/10.7554/eLife.36316.019
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Figure 2—figure supplement 4—source data 3
Quantification of Fny-CMV infection plants plotted in Figure 2—figure supplement 4E.
- https://doi.org/10.7554/eLife.36316.020
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Figure 2—figure supplement 4—source data 4
Quantification of relative CMV MP RNA accumulationin Figure 2—figure supplement 4F.
- https://doi.org/10.7554/eLife.36316.021
Figure 3 with 1 supplement
Determination of the intracellular localization of Arabidopsis Class I formins.
(A) Phylogenetic analysis of Class I Arabidopsis formins. The phylogenetic tree was made by MEGA 4.0 based on neighbor joining analysis. Numbers on branches represent the bootstrap values. (B–I) …
Figure 3—figure supplement 1
Several Class I rice formins localize to plasmodesmata.
(A) Phylogenetic analysis of Class I Oryza sativa Japonica formins. Phylogenetic tree was made by MEGA 4.0 based on neighbor joining analysis. Numbers on branches represent the bootstrap values. …
Figure 4 with 2 supplements
AtFH1 and AtFH2 redundantly regulate PD permeability.
(A) Images of eGFP diffusion in leaf epidermal pavement cells of WT, atfh1, atfh2 and atfh1 atfh2 plants. The edge of the cell that initially expressed eGFP is marked by a red line. The edge of …
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Figure 4—source data 1
Quantification of eGFP diffusion plotted in Figure 4B.
- https://doi.org/10.7554/eLife.36316.042
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Figure 4—source data 2
Quantification of eGFP diffusion plotted in Figure 4C.
- https://doi.org/10.7554/eLife.36316.043
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Figure 4—source data 3
Quantification of CMV MP transporting plotted in Figure 4D.
- https://doi.org/10.7554/eLife.36316.044
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Figure 4—source data 4
Quantification of Fny-CMV infection plants plotted in Figure 4G.
- https://doi.org/10.7554/eLife.36316.045
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Figure 4—source data 5
Quantification of leaf length plotted in Figure 4H.
- https://doi.org/10.7554/eLife.36316.046
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Figure 4—source data 6
Quantification of leaf length plotted in Figure 4I.
- https://doi.org/10.7554/eLife.36316.047
Figure 4—figure supplement 1
Characterization of atfh1 and atfh1 atfh2 mutants.
(A) Physical structure of the AtFH1 gene. Exons, introns and untranslated regions are represented by black boxes, black lines, and grey boxes, respectively. Salk_143939 was designated as atfh1-3. …
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Figure 4—figure supplement 1—source data 1
Quantification of relative expression of AtFH1 plotted in Figure 4—figure supplement 1C.
- https://doi.org/10.7554/eLife.36316.034
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Figure 4—figure supplement 1—source data 2
Quantification of circularity plotted in Figure 4—figure supplement 1D.
- https://doi.org/10.7554/eLife.36316.035
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Figure 4—figure supplement 1—source data 3
Quantification of eGFP diffusion plotted in Figure 4—figure supplement 1E.
- https://doi.org/10.7554/eLife.36316.036
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Figure 4—figure supplement 1—source data 4
Quantification of relative expression of AtFH1 plotted in Figure 4—figure supplement 1F.
- https://doi.org/10.7554/eLife.36316.037
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Figure 4—figure supplement 1—source data 5
Quantification of relative expression of AtFH2 plotted in Figure 4—figure supplement 1G.
- https://doi.org/10.7554/eLife.36316.038
Figure 4—figure supplement 2
Callose accumulation is not affected in atfh1, atfh2 and atfh1 atfh2 mutants.
(A) Confocal micrographs of leaf tissue from wild-type Col-0 plants, atfh1 plants, atfh2 plants and atfh1 atfh2 plants stained with 0.1% aniline blue to reveal callose accumulation. Images were …
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Figure 4—figure supplement 2—source data 1
Quantification of the size of aniline blue-stained foci in Figure 4—figure supplement 2B.
- https://doi.org/10.7554/eLife.36316.040
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Figure 4—figure supplement 2—source data 2
Quantification of the gray values of aniline blue-stained foci in Figure 4—figure supplement 2C.
- https://doi.org/10.7554/eLife.36316.041
Figure 5
AtFH2 fails to nucleate actin assembly from actin or actin bound to profilin, and slightly inhibits spontaneous actin polymerization in vitro.
(A) Schematic representation of the domain organization of AtFH2 and the fragments used for the generation of recombinant truncated AtFH2 proteins. (B) Purified recombinant truncated AtFH2 proteins. …
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Figure 5—source data 1
Quantification of number of actin filaments plotted in Figure 5E.
- https://doi.org/10.7554/eLife.36316.049
Figure 6
AtFH2 caps the barbed end of actin filaments and stabilizes them from dilution-mediated actin depolymerization.
(A) AtFH2-ΔN inhibits actin barbed end elongation in a dose-dependent manner. Pre-formed F-actin seeds (0.8 μM) were incubated with different concentrations of AtFH2-ΔN for 5 min at room …
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Figure 6—source data 1
Quantification of of the dissociation constant (Kd) of AtFH2 plotted in Figure 6B.
- https://doi.org/10.7554/eLife.36316.051
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Figure 6—source data 2
Quantification of the length of actin filaments plotted in Figure 6D.
- https://doi.org/10.7554/eLife.36316.052
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Figure 6—source data 3
Quantification of the elongation rates of actin filaments plotted in Figure 6F.
- https://doi.org/10.7554/eLife.36316.053
Figure 7
Interaction of AtFH2 with actin filaments is required for its function in regulating the permeability of PD.
(A) Protein sequence alignment of the FH2 domains in AtFH2 and three other well-characterized formins. Ile519 (I) and Lys672 (K) in AtFH2 (red triangles) correspond to Ile1431 and Lys1601 in Bni1p. …
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Figure 7—source data 1
Quantification of eGFP diffusion plotted in Figure 7G.
- https://doi.org/10.7554/eLife.36316.055
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Figure 7—source data 2
Quantification of Fny-CMV infection plants plotted in Figure 7H.
- https://doi.org/10.7554/eLife.36316.056
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | AtFH1 | PMID: 14671023 | TAIR: At3g25500 | |
| Gene (Arabidopsis thaliana) | AtFH2 | this paper | TAIR: At2g43800 | |
| Gene (Arabidopsis thaliana) | AtFH4 | PMID: 16313636 | TAIR: At1g24150 | |
| Gene (Arabidopsis thaliana) | AtFH5 | PMID: 15765105 | TAIR: At5g54650 | |
| Gene (Arabidopsis thaliana) | AtFH6 | PMID: 15319477 | TAIR: At5g67470 | |
| Gene (Arabidopsis thaliana) | AtFH8 | PMID: 16313636 | TAIR: At1g70140 | |
| Gene (Arabidopsis thaliana) | AtFH9 | NA | TAIR: At5g48360 | |
| Gene (Arabidopsis thaliana) | AtFH10 | NA | TAIR: At3g07540 | |
| Gene (Arabidopsis thaliana) | AtFH11 | NA | TAIR: At3g05470 | |
| Gene (Oryza sativa) | OsFH8 | PMID: 15256004 | RAP-DB: Os03g0204100 | |
| Gene (Oryza sativa) | OsFH11 | PMID: 15256004 | RAP-DB: Os07g0545500 | |
| Gene (Oryza sativa) | OsFH15 | PMID: 15256004 | RAP-DB: Os09g0517600 | |
| Gene (Oryza sativa) | OsFH16 | PMID: 15256004 | RAP-DB: Os02g0739100 | |
| Genetic reagent (Arabidopsis thaliana) | atfh2-1 | this paper | GK_066D02 | |
| Genetic reagent (Arabidopsis thaliana) | atfh2-2 | this paper | GK_396H03 | |
| Genetic reagent (Arabidopsis thaliana) | atfh1-1 | PMID: 23202131 | Salk_032981 | |
| Genetic reagent (Arabidopsis thaliana) | atfh1-3 | this paper | Salk_143939 | |
| Strain, strain background (Escherichia coli) | DH5α | PMID: 6345791 | ||
| Strain, strain background (Agrobacterium tumefaciens) | GV3101 | other | widely distributed | |
| Strain, strain background (Escherichia coli) | Tuner (DE3) (pLysS) | other | Novagen, Schwalbach, Germany | |
| Transfected construct | pdGN (vector) | PMID: 16126836 | ||
| Transfected construct | pdGN-35S:HDEL- mCherry | this paper | vector-promoter: protein construct | |
| Transfected construct | pCambia1301 (vector) | other | GenBank: AF234297.1 | binary vector |
| Transfected construct | pBI101 (vector) | other | GenBank: U12639.1 | binary vector |
| Biological sample (Arabidopsis thaliana) | PDLP1pro:PDLP1- GFP | PMID: 18215111 | ||
| Biological sample (Arabidopsis thaliana) | PDCB1pro:YFP- PDCB1 | PMID: 19223515 | ||
| Biological sample (Arabidopsis thaliana) | AFH2p:AFH2- eGFP; atfh2 | this paper | promoter:eGFP fusion protein; atfh2-1 mutant background | |
| Biological sample (Arabidopsis thaliana) | AFH2p:AFH2- mCherry; atfh2 | this paper | promoter:eGFP fusion protein; atfh2-1 mutant background | |
| Biological sample (Arabidopsis thaliana) | 35S:AFH2- mCherry; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:AtFH1(N)- eGFP; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:AtFH4(N)- eGFP; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:AtFH5-eGFP; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:AtFH6(N)- eGFP; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:AtFH8(N)- eGFP; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:AtFH9(N)- eGFP; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:AtFH10(N)- eGFP; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:AtFH11(N)- eGFP; Col-0 | this paper | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | 35S:GFP-ABD2; Col-0 | PMID: 15557099 | promoter:eGFP fusion protein; Col-0 background | |
| Biological sample (Arabidopsis thaliana) | AFH2p:AFH2M; atfh2 | this paper | promoter:recombinant protein; atfh2-1 background | |
| Recombinant DNA reagent | pGD-CMV MP-mCherry | PMID: 20435906 | ||
| Recombinant DNA reagent | pBI101-35S:AtFH2N175- eGFP-NOS | this paper | vector-promoter:protein construct | |
| Recombinant DNA reagent | pBI101- 35S:AtFH2N282- eGFP-NOS | this paper | vector-promoter:protein construct | |
| Recombinant DNA reagent | pBI101-35S: AtFH2N437-eGFP- NOS | this paper | vector-promoter:protein construct | |
| Recombinant DNA reagent | pBI101-35S: AtFH2N282-TMD- eGFP-NOS | this paper | vector-promoter:protein construct | |
| Recombinant DNA reagent | pBI101-35S: OsFH8(N)-eGFP- NOS | this paper | vector-promoter:protein construct | |
| Recombinant DNA reagent | pBI101-35S: OsFH11(N)-eGFP- NOS | this paper | vector-promoter:protein construct | |
| Recombinant DNA reagent | pBI101-35S: OsFH15(N)-eGFP- NOS | this paper | vector-promoter:protein construct | |
| Recombinant DNA reagent | pBI101-35S: OsFH16(N)-eGFP- NOS | this paper | vector-promoter:protein construct | |
| Recombinant DNA reagent | pCambia1301-35S: AtFH2M-eGFP- NOS | this paper | vector-promoter:protein construct | |
| Recombinant protein | AtFH1-FH1FH2 | PMID: 15994911 | Q9SE97 | |
| Recombinant protein | 6His-AtFH2-ΔN | this paper | ||
| Recombinant protein | 6His-AtFH2-FH1FH2 | this paper | ||
| Recombinant protein | 6His-AtFH2-FH2 | this paper | ||
| Recombinant protein | 6His-AtFH2-FH2M | this paper | ||
| Recombinant protein | GST-AtFH2-FH2 | this paper | ||
| Recombinant protein | GST-AtFH2-FH2M | this paper | ||
| Chemical compound, drug | latrunculin B | Calbiochem | 428020 | |
| Software, algorithm | ImageJ | https://imagej.nih.gov/ij/ | version 1.51 | |
| Software, algorithm | IBM SPSS Statistics | other | version 25 |
Additional files
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Supplementary file 1
Primer form
- https://doi.org/10.7554/eLife.36316.057
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Transparent reporting form
- https://doi.org/10.7554/eLife.36316.058
