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

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.In all eukaryotic cells, the endoplasmic reticulum (ER) comprises a continuous membrane system of sheets and tubules (Baumann and Walz, 2001; Shibata et al., 2006). ER tubules frequently connect through homotypic membrane fusion to form a reticular network (Lee and Chen, 1988; Prinz et al., 2000; Du et al., 2004). ER fusion in metazoans is mediated by the atlastins (ATLs), a class of dynamin-like, membrane-bound GTPases (Hu et al., 2009; Orso et al., 2009). ATL possesses a cytoplasmic N-terminal GTPase domain, followed by a helical domain, two closely spaced transmembrane domains, and a C-terminal cytosolic tail. ATL proteins localize mostly to ER tubules and they interact with the tubule-shaping proteins, reticulons and DP1 (Hu et al., 2009). A role for the ATLs in ER fusion is suggested by the fact that depletion of ATLs leads to long, nonbranched ER tubules in cultured cells (Hu et al., 2009) and to ER fragmentation in Drosophila melanogaster (Orso et al., 2009), possibly due to insufficient fusion between the tubules. Nonbranched ER tubules are also observed upon the expression of dominant-negative ATL mutants (Hu et al., 2009). In addition, antibodies to ATL inhibit ER network formation in Xenopus laevis egg extracts (Hu et al., 2009). Moreover, proteoliposomes containing purified D. melanogasterATL undergo GTP-dependent fusion in vitro (Orso et al., 2009; Bian et al., 2011). The physiological significance of ER fusion is supported by the observation that mutations in human ATL1, the dominant isoform in the brain, cause hereditary spastic paraplegia (Zhao et al., 2001), a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity and weakness of the lower limbs (Salinas et al., 2008).Many organisms lack ATL homologs. In yeast (Saccharomyces cerevisiae), another dynamin-like GTPase, Sey1p, has been found to share the same signature motifs and membrane topology as ATL (Hu et al., 2009). Recent work suggests that Sey1p mediates ER membrane fusion both in vivo and in vitro (Anwar et al., 2012). Cells lacking Sey1p grow normally (Hu et al., 2009), but additional mutation of an ER SNARE Ufe1p, which probably represents an alternative ER fusion mechanism in yeast, causes severe growth defects (Anwar et al., 2012). In Arabidopsis (Arabidopsis thaliana), the potential functional ortholog of ATL appears to be ROOT HAIR DEFECTIVE3 (RHD3; Hu et al., 2009), which was initially discovered by a genetic screen of root hair-defective mutants (Schiefelbein and Somerville, 1990). It is sequence related to Sey1p over the entire length (Wang et al., 1997; Brands and Ho, 2002). Mutations of RHD3 cause short and wavy root hairs (Schiefelbein and Somerville, 1990; Wang et al., 1997; Stefano et al., 2012) and defects in cell expansion (Wang et al., 2002).Despite the sequence homology between Sey1p and RHD3, it was reported that Sey1p could not replace RHD3 in plants and vice versa (Chen et al., 2011). Therefore, it is not clear whether RHD3 can mediate ER fusion. Another complication in plants is that the Arabidopsis RHD3 family also contains two RHD3-like (RL) proteins (Hu et al., 2003): RL1 is expressed only in pollen, whereas RL2 is expressed ubiquitously, but both are present at very low levels. Deletion of either RL protein causes no detectable defects in root hair development or overall growth (Chen et al., 2011). Whether RL proteins support the role of RHD3 in a tissue-specific manner remains to be investigated.Here, we have analyzed the function of RHD3 and RL proteins in Arabidopsis. We show that RHD3 and the two RL proteins play redundant roles but function during different stages of Arabidopsis development. In addition, we show that RHD3 proteins can functionally replace Sey1p in yeast and mediate ER membrane fusion.