Supplemental Figure 1. The wxr3 mutant exhibits decreased expression of CYCB1;1, SCR and SHR compared with the control.A and B, Expression of ProCYCB1;1:GUS in the control line (A) and the wxr3 mutant (B). C and D, Expression of ProSCR:GFP in the control line (C) and the wxr3 mutant (D). E and F, Expression of ProSHR:SHR-GFP in the control line (E) and the wxr3 mutant (F). Bar is 1mm. All seedlings are 7 days old.

Supplemental Figure 2. Phenotypic characterization of the wxr1, wxr3 mutant and wxr1 wxr3 double mutant. Seedlings in A are 10 days old. Bar is 1cm.

PIN2 PIN1 PIN2

WT wxr3

60’ BFA

60'BFA50 30'washout 60'washout 90'washout 60'BFA50 30'washout 60'washout 90'washoutDR5 DR5 DR5 DR5 wxr3-3 wxr3-3 wxr3-3 wxr3-3

00.10.20.30.40.50.60.70.80.9

1Cells with BFA bodies

Supplemental Fig. 3. The wxr3 mutation does not affect response to BFA. Roots of wild type and mutant were treated with 50µM BFA for 60 min and washed for a subsequent 90min. Samples were immunostained for PIN1/PIN2 (top). The fraction of cells containing PIN1-staining BFA bodies is shown below.

Supplemental Fig. 4. Positional cloning of the wxr3 mutation. A, The wxr3 mutation is mapped onto F16L2 BAC. B, DNA sequencing shows that a G to A mutation is found at the 3’ end of the second exon of At3g45890. The arrows show the location of primers used for RT-PCR in (C). C, The wxr3 mutation causes an RNA splicing error that retains the second intron in the mature mRNA identified by RT-PCR and following sequencing. D and E, Complementation assay is performed by transformation of ProWXR3:WXR3-GUS construct into the wxr3 mutant (DR5rev:GFP background). The transgenic lines display recovered primary root elongation (D) and DR5rev:GFP response after one day 2,4-D treatment (E). Seedlings are 4 days old in D and 5 days old in E. Three independent transgenic lines are shown. F, The structure of RUS1/WXR3 protein. The green box represents a unique N-terminal extension of RUS1/WXR3 protein, the purple box is a glycine rich region, and the blue box is DUF647 domain of RUS1/WXR3 protein.

Supplemental Fig. 5. Expression pattern and subcellular localization of RUS1/WXR3 protein.A to I, Expression pattern of RUS1/WXR3 in the plant as determined using ProWXR3:WXR3-GUS lines. J to L, RUS1/WXR3 is located in the plastid. J, Subcellular localization of WXR3-GFP. K, Subcellular localization of Pt-mCherry. L, Overlay of J and K. Bar is 50µm .

Supplemental Figure 6. Inducible overexpression of WXR3 enhances root hair initiation and elongation.A and B, The phenotype of WT (A) and pMDC7:WXR3-GFP roots (B) treated with DMSO for 2 days. C and D, WT root (C) and root hair (D) after 4 µM estradiol treatment for 2 days. E and F, pMDC7:WXR3-GFP root (E) and root hair (F) after 4uM estradiol treatment for 2 days. G, Root hair density of WT and pMDC7:WXR3-GFP seedlings after estradiol treatment. Error bars represent SEM. All seedlings are 7 days old.