The Committee of Experiment Animals of China Agricultural University approved the protocols we used in our study for antibody production and tubulin purification. The rabbit was raised in standardized pathogen-free conditions in the Animal Care Facility at Beijing B&M Biotech Co., Ltd. Blood was drawn from the marginal ear vein under anesthesia to ameliorate suffering. For tubulin purification, porcin brain was taken from the slaughtered pigs, which were used as food material, from the No. 5 slaughtering company in Beijing.
The amino acid sequences of St901 (S. tuberosum cv. Desiree AY526087), TSB (S. lycopersicum AAM53961), MAP18 (Arabidopsis thaliana BAC41928), SB401 (S. berthaultii CAA65228), and SBgLR (S. tuberosum AAR29265) were blasted using the DNAMAN 6.0 software package (LynnonBiosoft). The imperfect repetitive V-V-E-K-K-N/E-E motifs were shaded in gray.
Purification of recombinant SBgLR protein and preparation of its antibody
The full-length coding sequence of SBgLR was cloned into pET30a vector (Novagen), and transformed into Escherichia coli
strain BL21 (DE3). Bacteria were cultured in LB liquid medium containing 500 mg/L kanamycin, to OD600
0.5, and then 50 mM isopropyl-β-d-thiogalactoside (IPTG) was added to induce the expression of the recombinant protein for 4 h. The bacteria were then centrifuged at 7 000 g
at 4°C for 10 min, and resuspended using lysate buffer (50 mM NaH2
, 300 mM NaCl, 20 mM imidazole, pH 8.0). After sonicating of the turbid liquid, the lysis solution was centrifuged at 30 000 g
at 4°C for 30 min. The suspension was added to the Ni-NTA agarose resin column (GE Healthcare, Sweden) and washed with lysate buffer containing 50, 100 and 150 mM imidazole. The recombinant protein was eluted with 250 mM imidazole elution buffer, and dialyzed against PEM buffer (0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4
, pH 6.9) at 4°C overnight. The purified protein was stored at −80°C for MT assay, or injected into a rabbit to elicit antiserum. The antiserum was purified using the Protein A resin column, and the cyanogen bromide resin column (Amersham, Pharmacia Biotech).
Purification and polymerization of tubulin
The tubulin was purified from porcine brain according to a previously described method 
. The tubulin was polymerized into MTs before use. After centrifuging the tubulin at 70 000 g
at 4°C for 30 min, 10 µl GTP (10 mM) and 90 µl tubulin was incubated at 37°C for 20 min, and then 2 µM, 20 µM and 200 µM taxol was added to the tubulin, and polymerized for 15 min at each concentration of taxol. The polymerized MTs were centrifuged at 13 523 g
at 25°C for 20 min and washed with PEMT (0.1 M PIPES, 1 mM EGTA, 1 mM MgSO4
, 20 mM taxol, pH 6.9) twice.
Tubulin and MT-binding assay
For the tubulin binding assay 2, 4, 8 and 16 µM of proteins (SBgLR, tubulin and BSA) in total volumes of 5 µl were spotted onto PVDF membranes (Millipore Corporation, USA) and air-dried. The membranes were then incubated with 3% BSA in TBST (50 mM Tris, 150 mM NaCl and 0.05% Tween20, pH 7.5) at 4°C overnight. The membranes were then incubated with 10 µM SBgLR, or 10 µM tubulin at room temperature for 2 h, and washed three times for 10 min; they were then incubated with anti-SBgLR or anti-β-tubulin antibody at the dilution of 1
1 000 and 1
500, respectively, at room temperature for a further 2 h. Following another washing with TBST, the membranes were incubated with the alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG (1
5000). The bounded proteins were detected using NBT/BCIP color development reagent (Promega, USA).
For MT co-sedimentation assays, recombinant SBgLR and BSA were centrifuged at 75 000 g for 30 min at 4°C before use. 0 to 12 µM recombinant SBgLR protein, 12 µM BSA, and 4 µM taxol-stabled MTs (tsMTs) was added to a 50 µl reaction volumes in PEMT buffer. Following a 30 min reaction at room temperature, samples were centrifuged at 75 000 g for 30 min at 25°C. The pellet was rinsed with PEMT, and resuspended using sample loading buffer (10 mM Tris-HCl pH 8.0, 10% glycerol, 5% β-mercaptoethanol, 2% SDS, 0.1% bromophenol). The samples were analyzed by 8% SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue R250. The amounts of the recombinant SBgLR bound to MTs were determined by gel scanning, and the binding ratio between the recombinant SBgLR and MTs was analyzed by the Image J 184.108.40.206 analysis system (National Institutes of Health).
MT-bundling assay and electron microscopy
For the MT-bundling assay, 0.5 µM taxol-stabled rhodamine-labeled MTs (tsrMTs) and 2 µM recombinant SBgLR were incubated at 25°C for 30 min, and then the mixture was fixed with 1% glutaraldehyde. The same amounts of tsrMTs without or with boiling denatured recombinant SBgLR were set as negative controls. Subsequently, a 1 µl sample was placed on the slide, and examined using a confocal microscope (SP5, Leica, Germany). Photographs were taken using a Leica AF Hardware configurator (Leica).
For the negative-staining assay, 0.5 µM tsMTs and 2 µM recombinant SBgLR were incubated at 25°C for 30 min; the same amounts of boiling denatured recombinant SBgLR were used as a negative control. Then the mixture was adsorbed on formvar-coated copper grids for 10 min, stained with 2% uranyl acetate for 3 min, and air-dried. The samples were examined with a Hitachi 7500 electron microscope (Japan). Photographs were taken using iTEM (OSIS, Germany).
For transmission electron microscopy (TEM), 0.5 µM tsMTs and 2 µM recombinant SBgLR were incubated at 25°C for 30 min, then centrifuged at 75 000 g at 25°C for 30 min. The pellet was fixed with fixation buffer (50 mM PBS, 1% glutaraldehyde, 4% paraformaldehyde and 5 mM EGTA, pH 6.8) at 4°C for 1 h, and post-fixed with 1% osmium tetroxide at 4°C for 30 min. The sample was dehydrated using an ethanol gradient, embedded in Spurr's resin, and sectioned into 90 nm thick slices. The sections were stained with 2% uranyl acetate and 2.66% lead citrate, and observed using a transmission electron microscope (7500, Hitachi) operated at 80 kV. Photographs were taken using iTEM (OSIS, Germany).
Co-localization of SBgLR and MTs
The 0.5 µM tsrMTs and 2 µM recombinant SBgLR were incubated at 25°C for 30 min. Next, the sample was incubated with antibody against SBgLR (1
500) at room temperature for another 1 h. After incubation, the sample was labeled with FITC-conjugated goat anti-rabbit IgG secondary antibody (1
100, Sigma-Aldrich, USA) at room temperature for 30 min. After being fixed with 1% glutaraldehyde, the sample was examined under a confocal microscope (SP5, Leica) at wavelengths of 488 nm and 561 nm, to visualize SBgLR and MTs, respectively. Incubation of boiling denatured recombinant SBgLR with tsrMTs was carried out as a negative control. Images were taken using the Leica AF hardware configurator (Leica).
Purified natural and 0.5% β-mercaptoethanol treated recombinant SBgLR protein was separated by 12% native polyacrylamide gel. The gel was stained with Coomassie brilliant blue R-250 (Amresco, USA). The molecular weight of the oligomers were estimated according to the equation log10
, molecular weight; x, relative migration rate; b and k, constant).
Plant transformation and molecular analysis
The vector p35S::SBgLR was constructed by cloning the full-length coding sequence of SBgLR
into pCambia2300. This was transformed into Agrobacterium tumefaciens
strain LBA4404. Tobacco transformation was carried out using the leaf disc method 
. The transgenic plants were confirmed by PCR amplification using SBgLR
(ORF) specific primer pair: Primer 1 5′-GAAAGTGGTGGCTGTGGAAA-3′
, and Primer 2 5′-AATTGGCTTGAT GGTCTCCT-3′
. For RT-PCR amplification, total RNA from T2
seedlings was extracted using TRIzol reagent (CWBIO, China), and the cDNA first strand synthesis was carried out according to the protocol of the Reverse Transcription System (A3500, Promega). RT-PCR amplification was performed using the same primer pair (Primer 1; Primer 2) used for PCR amplification.
For western blot analysis, 5 µg of proteins from different tissues were separated on 12% SDS-PAGE, and transferred to PVDF membrane (Millipore Corporation). The membrane was incubated with anti-SBgLR (1
5 000) or anti-actin primary antibody (1
500), and the alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (1
5000). The signals were detected with NBT/BCIP reagent (Promega).
Light microscopy (LM) and scanning electron microscopy (SEM)
For LM, cotyledons and hypocotyls were fixed in FAA (5% acetic acid, 5% formaldehyde solution, and 63% ethanol) at 4°C for 48 h. After dehydrating in an ethanol series of 50%, 70%, 80%, 90%, 95% and 100%, the samples were vitrified in a dimethylbenzene/ethanol series of 33%, 50%, 66% and 100%, and then embedded in paraffin. The embedded blocks were sectioned with a microtome (Leica) to a thickness of 10 µm. The sections were stained with 0.5% safranin and 0.5% fast green, and observed with a light microscope (BX51, Olympus, Japan).
For SEM, cotyledons, hypocotyls and pollen grains were observed using a scanning electron microscope (TM3000, Hitachi). For the measurements of hypocotyl epidermal cells, data was collected from at least 60 cells, and collected from two different hypocotyls. For the observation of cotyledon pavement cells, observations were consistently conducted on cells located in the mid-region of the cotyledon, at the same developmental stage. Data were obtained from the measurement of at least 170 cells, collected from four different cotyledons, and from three individual seedlings.
Immunofluorescence staining of MTs
Immunofluorescence staining assay was performed for the observation of MTs in roots. Roots were cut from 7-day-old seedlings and fixed in PEM containing 4% (w/v) paraformaldehyde at room temperature for 1 h. A mouse anti-β-tubulin monoclonal antibody (Sigma-Aldrich) at 1
800 dilution was applied as primary antibody, and TRITC-conjugated goat anti-mouse IgG antibody at 1
500 dilution (Sigma-Aldrich) was used as secondary antibody. Confocal microscopy observations and image acquisition were performed using a Leica SP5 confocal microscope.
Pollen I2-KI staining
Pollen grains were harvested from fresh opened anthers on a microscopic slide, and stained with I2-KI solution (0.5% I2 and 3% KI). They were observed with a light microscope (BX51, Olympus). For each line, pollen grains from at least four anthers and from two flowers were observed; pollens were observed under six fields of visions for statistical analysis.