Supporting Online Material for

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1 Supporting Online Material for Local Positive Feedback Regulation Determines Cell Shape in Root Hair Cells Seiji Takeda, Catherine Gapper, Hidetaka Kaya, Elizabeth Bell, Kazuyuki Kuchitsu, Liam Dolan* *To whom correspondence should be addressed. This PDF file includes: Materials and Methods Figs. S1 to S4 Tables S1 and S2 References Published 29 February 2008, Science 319, 1241 (2008) DOI: /science

2 Supporting online material (SOM) Materials and Methods Plant growth conditions and strains Arabidopsis seeds were sterilized in 5% sodium hypochlorite, washed in water and sown on Murashige and Skoog (Duchefa, Haarlem, The Netherlands) medium (ph 5.8) containing 1% sucrose and 0.8% Phytagel. New rhd2 mutants used in this study were GABI-Kat lines, 841E09 (rhd2-5) and 203A02 (rhd2-6) in which the T-DNA is inserted in the fifth exon and ninth intron, respectively (Fig. S1A). Both mutants have severe short root hair phenotype (Fig. S1C). Fluorescent protein fusion constructs GFP was amplified using GFPattB1F and GFPwosattB2R for GFP without stop codon, or GFPattB1F and GFPstopattB2R with stop codon, and introduced to pdonr207 by BP reaction (Gateway technology, Invitrogen) to generate pentrgfpwos and pentrgfpstop, respectively. For the construction of GFP:genomic fusion genes, the Gateway multisite technique (Invitrogen) was used. RHD2 promoter or promoter+gene fragments were amplified by PCR and cloned into pdonrp4p1r by BP reaction to generate pentrp4p1r clones. 3 region or gene+3 region were amplified and cloned into pdonrp2rp3 to generate pentrp2p3r clones. To generate N-terminal fusion, the promoter in pentrp4p1r, pentrgfpwos, and the gene+3 region in pentrp2rp3 were mixed with pgwbmultisite, which had been modified by M. Tomlinson (John Innes Centre, UK) from pgwb1 (a gift from T. Nakagawa, Shimane University, Japan) and used for LR reaction. For C-terminal fusion, promoter+gene in pentrp4p1r, pentrgfpstop and 3 region in pentrp2rp3 were used in the same way. Point mutated RHD2 genomic fragment was generated

3 by a PCR-based site direct mutagenesis method. The gene structure is shown in Fig. S1B. Primer sequences are listed in Table S2. Plant transformation and screening of T1 plants Agrobacterium tumefaciens strain LBA4404.pBBR1MCS virgn54d was used for plant transformation by the floral dipping method as described (S1). T1 plants were screened on MS and agar plates containing 50 µg/ml hygromycin and 50 µg/ml kanamycin. Imaging and microscopy 4-day grown plants were mounted in liquid MS media (LM: 1 x MS and 3% sucrose, ph 5.8) for imaging. GFP was imaged with Leica TCS SP system attached to Leica DMRE confocal microscope using 488 nm of an Ar/Kr laser or a CCD Hamamatsu TSU ORCA-ER digital camera attached to Nikon Eclipse E600 was used with Metamorph software. For FM4-64 treatment, 4-day old seedlings were incubated in 25 µm FM4-64 solution in LM made from 16.5 mm stock solution in DMSO. For BFA treatment 4-day seedlings were incubated in 25 µm BFA in LM made from 100 mm stock solution in DMSO for 30 min. Images were processed on ImageJ ( and Adobe photoshop CS (Adobe). Recombinant Protein Expression A cdna fragment of RHD2 corresponding to the amino acids was amplified by PCR and cloned into pgex-kg using NcoI and XhoI sites and GST added at the N-terminal end. Amino acid substitution was introduced by PCR. To express theses proteins, the plasmids were transformed into the E. coli strain Rosetta DE3 plyss (Novagen). After growth IPTG was added to the culture to a final concentration of 0.1 mm followed by a further incubation at 30 C for 3 hours. Cell pellets were resuspended in 1/10 culture volume of homogenisation buffer (50 mm Tris ph 8, 250 mm

4 NaCl, 0.5% Tween-20, 0.1% β-mercaptoethanol, Complete Protease Inhibitor cocktail (Roche Diagnostics). Cell lysis was performed by sonication on ice. The cell debris was removed by centrifugation at 4 C for 15 minutes at 10,000 g. 3 ml of glutathione agarose (Sigma, prepared as per manufacturers instructions) was added to the supernatant and this mixture was incubated with gentle agitation at 4 C for a 1 hour. The glutathione agarose was recovered by centrifugation at 4 C for 15 minutes at 3,000 rpm. The resin was applied to an empty gravity column and washed with 2 bed volumes of wash buffer (50 mm Tris ph 8, 250 mm NaCl, 0.5% Tween-20). The bound proteins were then eluted with elution buffer (50 mm Tris ph 8, 250 mm NaCl, 20mM Glutathione). Concentration was determined by Bradford Assay. Concentration was adjusted to 1 mg/ml and proteins were stored in 20% glycerol at -20 C. Protein extraction Plant roots were harvested from 14 day old Col-0 seedlings. Roots were excised from just below the hypocotyl and immediately frozen in liquid nitrogen. The frozen plant material was ground under liquid nitrogen using a mortar and pestle. 10 ml of extraction buffer (50mM HEPES ph 7.5, 5 mm EDTA, 1% w/v PVP, 1 mm β-mercaptoethanol, Roche complete protease inhibitor cocktail) was added and vortexed briefly. Samples were then centrifuged for 30 minutes at 4 C at 20,000 g. The supernatant was sterile filtered using a 22 µm filter. Protein concentration was determined by Bradford Assay. Proteins contained in the filtered supernatant were either used directly or rebuffered with an appropriate starting buffer using PD-10 columns (BioRad) and analyzed by SDS-PAGE. In vitro kinase assay For in vitro kinase assays, 5 µl of total plant protein extract or eluted proteins were incubated with 18 µl kinase assay buffer (50 mm HEPES/KOH ph 7.5, 20 mm MgCl 2, 1 mm EGTA (for Ca 2+ free

5 assays) or 10 µm CaCl 2 (for Ca 2+ assays). To this 5 µl of GST-tagged fusion proteins were added. To start the kinase reaction 2 µl of ATP mix (0.1 µl γ-32-p-atp, 0.5 µl 2mM ATP, 1.4 µl water) was added and the reaction allowed to proceed for 30 minutes at room temperature. The reaction was stopped by adding 5 µl loading buffer and incubating at 95 C for 3 minutes. The samples were then separated by SDS-PAGE. Following stain and destain, the gel was vacuum dried and exposed to a PhosphoImager screen to detect radioactive signal. ROS production assay in heterologous expression system Full length of RHD2 cdna was amplified by RT-PCR and cloned into pdonr207 by Gateway technology (Invitrogen). RHD2 carrying point mutation in phosphorylation residues and EF-hand motifs were produced by PCR. The RHD2 fragments were subcloned into pcdna3.1 with FLAG tag at its amino-terminal. Transient transfection of HEK293T cells, immune blotting and ROS measurements were carried out as described before (S2). Production of ROS was determined via chemiluminescence and expressed as relative luminescence units per second (RLU/s, means ± S. E., N=3).

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10 Supplemental tables Table S1. Number of measured roots and root hairs Number of roots Number of root hairs Col rhd GFP:RHD GFP:RHD GFP:RHD E250A E250A E250A Table S2. Primer sequences used for this work Primer name Sequence (5 to 3 ) GFPattB1F GFPwosattB2R GFPstopattB2R GGGG ACA AGT TTG TAC AAA AAA GCA GGC TCA ATG AGT AAA GGA GAA GAA CTT TTC GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA TTT GTA TAG TTC ATC CAT GCC GGGG AC CAC TTT GTA CAA GAA AGC TGG GTG TCA TTT GTA TAG TTC ATC CAT GCC RHD2PattB4F RHD2PattB1R RHD2GattB1R RHD2GattB2F RHD2UattB2F RHD2UattB3R GGGG ACA ACT TTG TAT AGA AAA GTT GTT AAG CTT CCT CCT GTT TCG AAT GGGG AC TGC TTT TTT GTA CAA ACT TGC TTT TTA ACA CAC TCT ACC TGA GGGG AC TGC TTT TTT GTA CAA ACT TGC GAA ATT CTC TTT GTG GAA GGA GGGG ACA GCT TTC TTG TAC AAA GTG GAA ATG TCT AGA GTG AGT TTT GAA GGGG ACA GCT TTC TTG TAC AAA GTG GAA AGG ACA CAC AGA GTT AAA AGG GGGG AC AAC TTT GTA TAA TAA AGT TGC AGA TCT ATA AGA TCA TTT CCA Supplemental references S1. S. J. Clough, A. F. Bent, Plant J 16, 735 (1998). S2. Y. Ogasawara et al., J. Biol. Chem /jbc.M (2008).