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. 2013 Oct;163(2):713-20.
doi: 10.1104/pp.113.224501. Epub 2013 Aug 6.

ROOT HAIR DEFECTIVE3 Family of Dynamin-Like GTPases Mediates Homotypic Endoplasmic Reticulum Fusion and Is Essential for Arabidopsis Development

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Free PMC article

ROOT HAIR DEFECTIVE3 Family of Dynamin-Like GTPases Mediates Homotypic Endoplasmic Reticulum Fusion and Is Essential for Arabidopsis Development

Miao Zhang et al. Plant Physiol. .
Free PMC article

Abstract

In all eukaryotic cells, the endoplasmic reticulum (ER) forms a tubular network whose generation requires the fusion of ER membranes. In Arabidopsis (Arabidopsis thaliana), the membrane-bound GTPase ROOT HAIR DEFECTIVE3 (RHD3) is a potential candidate to mediate ER fusion. In addition, Arabidopsis has two tissue-specific isoforms of RHD3, namely RHD3-like (RL) proteins, and their function is not clear. Here, we show that a null allele of RHD3, rhd3-8, causes growth defects and shortened root hairs. A point mutant, rhd3-1, exhibits a more severe growth phenotype than the null mutant, likely because it exerts a dominant-negative effect on the RL proteins. Genetic analysis reveals that the double deletion of RHD3 and RL1 is lethal and that the rhd3 rl2 plants produce no viable pollen, suggesting that the RL proteins are redundant to RHD3. RHD3 family proteins can replace Sey1p, the homolog of RHD3 in yeast (Saccharomyces cerevisiae), in the maintenance of ER morphology, and they are able to fuse membranes both in vivo and in vitro. Our results suggest that RHD3 proteins mediate ER fusion and are essential for plant development and that the formation of the tubular ER network is of general physiological significance.

Figures

Figure 1.
Figure 1.
Characterization of rhd3-8, a null mutant of RHD3. A, Schematic diagram of the genomic locus of RHD3. Exons and introns are shown in boxes and lines, respectively. Mutation sites for rhd3-8 and rhd3-1 are indicated. B, The transcription levels of RHD3 in wild-type (Columbia [Col-0]), rhd3-8, and rhd3-1 plants were analyzed by reverse transcription and real-time PCR. EF1α was used as an internal control. The data represent means ± sd from three replicates. C, The protein levels of RHD3 in wild-type (Col-0), rhd3-8, and rhd3-1 plants were determined by immunoblotting (IB) with anti-RHD3 antibodies (top panel). Coomassie blue staining of the RuBisCO subunit (RbcS) served as a loading control (bottom panel). D, Mature plants of Col-0, rhd3-8, and rhd3-1 shown in top view (top panel) and side view (bottom panel). E, Root and root hairs of wild-type (Col-0), rhd3-8, and rhd3-1 plants. The corresponding 5-d-old seedlings are shown in insets. Bars = 0.5 mm. F, Wild-type (Col-0) and rhd3-8 root cells in a Q4 line as an ER marker were visualized by fluorescence confocal microscopy. Bars = 2 μm. G, Wild-type (Col-0) and rhd3-8 leaf epidermal cells were transfected with the ER marker ss-GFP-HDEL and were visualized by fluorescence confocal microscopy. The right panels show enlarged views of the highlighted areas. Bars = 10 μm.
Figure 2.
Figure 2.
Genetic interactions between RHD3 and RLs. A, rhd3 was crossed with either rl1 or rl2. F3 seedlings were genotyped and counted. B, Mature plants of rhd3−/−, rhd3−/− rl2+/−, and rhd3−/− rl2−/−. C, The flowers of the wild type (Col-0), rhd3−/− rl2+/−, and rhd3−/− rl2−/− were imaged. The bottom panels show enlarged views. Note that rhd3−/− rl2−/− has no visible pollen grains. Bars = 1 mm. D, The anthers of the wild type (Col-0) and rhd3−/− rl2−/− were collected at stage 12 (left panels) and 13 (middle panels) and were imaged. Pollen viability was analyzed by Alexander staining (right panels). Note that the viable pollen grains are purple and the inactive ones are green. Bars = 100 μm.
Figure 3.
Figure 3.
RHD3 proteins can replace Sey1p in yeast. A, Wild-type (wt) RHD3 proteins or the indicated mutants were expressed under the control of the endogenous SEY1 promoter in S. cerevisiae cells lacking Sey1p and Yop1p (sey1Δ yop1Δ cells). For expression levels, see Supplemental Figure S7. The ER was visualized by expressing Sec63-GFP, focusing the microscope on either the periphery or the center of the cells. sey1Δ yop1Δ cells expressing Sey1p or empty vector were also analyzed for comparison. Bar = 1 μm. B, The percentage of cells with abnormal ER was determined from 80 to 200 cells per mutant.
Figure 4.
Figure 4.
ER-ER fusion mediated by RHD3 proteins in yeast. A, sey1Δ cells of opposite mating types expressing either ss-RFP-HDEL or cytosolic GFP were mixed, placed on an agarose pad, and imaged at 1-min intervals. Selected images from the time-lapse video are shown. Time 0 min is the first image taken after cell fusion, as indicated by GFP in both cells. Bar = 2 μm. B, As in A, but with sey1Δ cells expressing wild-type RHD3. C, The average time between cell fusion and ER fusion during mating was determined from eight to 10 cells per sample. Values shown are means ± sd.
Figure 5.
Figure 5.
RHD3 proteins mediate membrane fusion in vitro. A, RHD3, RL2, or the indicated mutants were purified and reconstituted into proteoliposomes. Flotation in a sucrose gradient (right) shows efficient reconstitution of the proteins (T, top fraction; B, bottom fraction). B, Donor (D; with NBD- and rhodamine-labeled lipids at quenching concentrations) and acceptor (A; unlabeled) proteoliposomes containing RHD3, RL2, or the indicated mutants were analyzed by SDS-PAGE and Coomassie blue staining. C, GTPase activity of full-length wild-type RHD3, RL2, or the indicated mutants was measured by phosphate release. The data represent means ± sd from six replicates. Kcat, Catalytic constant. D, Full-length RHD3 was reconstituted at equal concentrations into donor and acceptor vesicles. Fusion was monitored by the dequenching of the NBD-labeled lipids present in the donor vesicles and was initiated by the addition of GTP. Control experiments were performed in the absence of Mg2+ or the presence of GDP or GTPγS instead of GTP. E, As in D, but with full-length RL2. F, Fusion with RHD3 or RL2 was compared with that of the indicated GTP-binding mutants. WT, Wild type.

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