www.sciencemag.org/cgi/content/full/319/5867/1241/DC1
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. E-mail: [email protected]
Published 29 February 2008, Science 319, 1241 (2008)
DOI: 10.1126/science.1152505
This PDF file includes:
Materials and Methods
Figs. S1 to S4
Tables S1 and S2
References
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
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
(http://rsb.info.nih.gov/ij/index.html) and Adobe photoshop CS (Adobe).
Recombinant Protein Expression
A cDNA fragment of RHD2 corresponding to the amino acids 316 – 351 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
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 MgCl2, 1 mM EGTA (for Ca2+ free
assays) or 10 µM CaCl2 (for Ca2+ 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).
Supplemental tables
Table S1. Number of measured roots and root hairs
Number of roots Number of root hairs
11
290
Col-0
rhd2
9
222
GFP:RHD2-1
GFP:RHD2-2
GFP:RHD2-3
20
13
21
517
304
466
E250A-1
E250A-2
E250A-3
18
15
19
423
297
392
Table S2. Primer sequences used for this work
Primer name
Sequence (5’ to 3’)
GFPattB1F
GGGG ACA AGT TTG TAC AAA AAA GCA GGC TCA ATG AGT AAA GGA GAA GAA CTT TTC
GFPwosattB2R
GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA TTT GTA TAG TTC ATC CAT GCC
GFPstopattB2R
GGGG AC CAC TTT GTA CAA GAA AGC TGG GTG TCA TTT GTA TAG TTC ATC CAT GCC
RHD2PattB4F
GGGG ACA ACT TTG TAT AGA AAA GTT GTT AAG CTT CCT CCT GTT TCG AAT
RHD2PattB1R
GGGG AC TGC TTT TTT GTA CAA ACT TGC TTT TTA ACA CAC TCT ACC TGA
RHD2GattB1R
GGGG AC TGC TTT TTT GTA CAA ACT TGC GAA ATT CTC TTT GTG GAA GGA
RHD2GattB2F
GGGG ACA GCT TTC TTG TAC AAA GTG GAA ATG TCT AGA GTG AGT TTT GAA
RHD2UattB2F
GGGG ACA GCT TTC TTG TAC AAA GTG GAA AGG ACA CAC AGA GTT AAA AGG
RHD2UattB3R
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. 10.1074/jbc.M708106200 (2008).