Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key players in cellular trafficking and coordinate vital cellular processes, such as cytokinesis, pathogen defense, and ion transport regulation. With few exceptions, SNAREs are tail-anchored (TA) proteins, bearing a C-terminal hydrophobic domain that is essential for their membrane integration. Recently, the Guided Entry of Tail-anchored proteins (GET) pathway was described in mammalian and yeast cells that serve as a blueprint of TA protein insertion [Schuldiner M, et al. (2008) Cell 134(4):634-645; Stefanovic S, Hegde RS (2007) Cell 128(6):1147-1159]. This pathway consists of six proteins, with the cytosolic ATPase GET3 chaperoning the newly synthesized TA protein posttranslationally from the ribosome to the endoplasmic reticulum (ER) membrane. Structural and biochemical insights confirmed the potential of pathway components to facilitate membrane insertion, but the physiological significance in multicellular organisms remains to be resolved. Our phylogenetic analysis of 37 GET3 orthologs from 18 different species revealed the presence of two different GET3 clades. We identified and analyzed GET pathway components in Arabidopsis thaliana and found reduced root hair elongation in Atget lines, possibly as a result of reduced SNARE biogenesis. Overexpression of AtGET3a in a receptor knockout (KO) results in severe growth defects, suggesting presence of alternative insertion pathways while highlighting an intricate involvement for the GET pathway in cellular homeostasis of plants.GET pathway | TA proteins | SNAREs | ER membrane | root hairs

Highlights d Polarization and outgrowth of root hairs are temporally separate processes d Distinct RopGEFs are involved in ROP GTPase recruitment and growth regulation d GEF3 is required for ROP2 targeting to the emerging root hair initiation domain d GEF3 expression is sufficient to induce polar domain formation in epidermal cells

35Root hairs are tubular protrusions of the root epidermis that significantly enlarge the exploitable soil 36 volume in the rhizosphere. Trichoblasts, the cell type responsible for root hair formation, switch 37 from cell elongation to tip growth through polarization of the growth machinery to a pre-defined root 38 hair initiation domain (RHID) at the plasma membrane. The emergence of this polar domain 39 resembles the establishment of cell polarity in other eukaryotic systems [1][2][3]. Rho-type GTPases 40 of plants (ROPs) are among the first molecular determinants of the RHID [4, 5] and later play a 41 central role in polar growth [6]. Numerous studies have elucidated mechanisms that position the 42 RHID in the cell [7][8][9] or regulate ROP activity [10][11][12][13][14][15][16][17][18]. The molecular players that target ROPs to 43 the RHID and initiate outgrowth, however, have not been identified. We dissected the timing of the 44 growth machinery assembly in polarizing hair cells and found that positioning of molecular players 45 and outgrowth are temporally separate processes that are each controlled by specific ROP guanine 46 nucleotide exchange factor (GEFs). A functional analysis of trichoblast-specific GEFs revealed 47 GEF3 to be required for normal ROP polarization and thus efficient root hair emergence, while 48 GEF4 predominantly regulates subsequent tip growth. Ectopic expression of GEF3 induced the 49 formation of spatially confined, ROP-recruiting domains in other cell types, demonstrating the role 50 of GEF3 to serve as a membrane landmark during cell polarization. 51 3 RESULTS AND DISCUSSION 52 53 Temporal analysis of hair cell polarization reveals phased deployment of the tip-growth 54 machinery 55To dissect the process of breaking cellular symmetry and initiating polar growth in plants, we 56 analyzed the gradual assembly of the tip growth machinery in trichoblasts using specific markers 57 for cytoskeletal rearrangement, plasma membrane specialization, cell wall modification, vesicle 58 trafficking, and Rho-GTPase signaling. Using stable transgenic Arabidopsis lines expressing 59 fluorescently labeled versions of these protein markers, we determined the timing of their 60 polarization at the RHID during trichoblast differentiation. As the Arabidopsis root represents a time-61 axis of development from stem cells to mature cells, we numbered the developmental stages (-7 62 to +3) within a cell file, with the last cell before bulging labeled -1 and the first cell after the onset 63 of bulging labeled +1 ( Figure 1A). We used the integral plasma membrane marker GFP-LTI6B [19] 64 as a reference and calculated the polarity index of fluorescently labeled marker proteins to quantify 65 protein accumulation at the RHID (Figure 1B, C). Our survey unveiled a two-phase assembly of 66 the tip growth machinery: an initiation phase, during which the RHID is positioned and predefined, 67 followed by the tip growth phase. Consistent with previous reports [4, 5], mCitrine-labeled (mCit) 68GTPase ROP2 associ...