ChromoTek | Date: 2013-10-28
Chemical preparation for scientific and industrial purposes. Pharmaceutical reagents for medical scientific use.
ChromoTek | Date: 2013-10-28
Chemical preparations namely, diagnostic reagents for scientific use by academic, pharmaceutical, and industrial scientists for molecular biology research. Pharmaceutical preparations, namely, biological reagents comprising antibody compounds for medical use, used by academic and industrial scientists for molecular biology research. Scientific services, namely, scientific research and development of products for use in the fields of chemicals and pharmaceuticals by academic and industrial scientists and for scientific laboratory use; scientific research and development for others.
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment. Arabidopsis thaliana accession Columbia (Col-0) was used as the wild type. Seeds of T-DNA insertion lines were obtained from ABRC and NASC, and T-DNA insertions were confirmed by genomic PCR (Extended Data Table 1). The insert sites were determined by sequencing of the PCR products, as described in Extended Data Fig. 1c. Plant growth conditions and transformation methods were described previously6. C. rubella seeds were obtained from ABRC (accession CS22697; ref. 29), and C. rubella plants in the rosette stage were subjected to vernalising cold treatment (8-h photoperiod at 4 °C for about 1 month) for flowering induction. To investigate candidate RLKs responsible for AtLURE1 signalling, RLK genes encoding proteins with extracellular domains and displaying notable and specific expression in the pollen tube were selected as follows. Whether the more than 80 genes expressed in dry pollen or pollen tubes13 were expressed predominantly in the mature pollen was determined using the Arabidopsis eFP Browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi)30. Twenty-three pollen-dominant genes and their related genes were selected: PRK1–8 (see Extended Data Table 1), AT2G18470 (PROLINE-RICH EXTENSIN-LIKE RECEPTOR KINASE 4, PERK4), AT4G34440 (PERK5), AT3G18810 (PERK6), At1g49270 (PERK7), AT1G10620 (PERK11), AT1G23540 (PERK12), AT4G29450, AT3G13065 (STRUBBELIG-RECEPTOR FAMILY 4, SRF4), AT1G78980 (SRF5), AT4G18640 (MORPHOGENESIS OF ROOT HAIR 1, MRH1), AT5G45840, AT1G29750 (RECEPTOR-LIKE KINASE IN FLOWERS 1, RKF1), AT3G23750 (BAK1-ASSOCIATING RECEPTOR-LIKE KINASE 1, BARK1; or TMK4), AT1G19090 (CYSTEINE-RICH RLK 1, CRK1) and AT4G28670. A further five RLK genes of a subclass of the CrRLK1L family (AT3G04690 (ANXUR1), AT5G28680 (ANXUR2), AT4G39110, AT2G21480 and AT5G61350) were also pollen-dominant but were not examined in this study. T-DNA insertions in the coding or promoter regions of these selected 23 genes were identified by genomic PCR and sequencing of the PCR products. Semi-in-vivo pollen tubes from one or more lines for each gene were assessed by an attraction assay using the AtLURE1.2 peptide, as described below. Recombinant His-tagged AtLURE1.2 peptide was expressed in Escherichia coli, purified and refolded, as described previously6. The refolded His–AtLURE1.2 peptide was suggested to be a conformational isomer by reverse-phase high-pressure liquid chromatography (HPLC) using a Phenomenex Jupiter C18 column and a Jasco analytical instrument equipped with a UV-2077 plus detector and PU-2080 plus pumps. A construct for His–AtLURE1.2(GGGG) was generated from pET-28a-AtLURE1.2 by site-directed mutagenesis using the primers 5′-GTATGgGAgGGGGTggGTATATTC-3′ and 5′-cACCCCcTCcCATACAAGCTC-3′ (lowercase bases denote mutated bases from the original AtLURE1.2). No aggregation due to inappropriate folding was observed during refolding or concentration of the His–AtLURE1.2(GGGG) peptide. Alexa488-labelled His–AtLURE1.2 was produced using the refolded His–AtLURE1.2 peptide and the Alexa Fluor 488 Protein Labelling Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. For the semi-in-vivo attraction assay, pollen tubes were grown through cut styles of A. thaliana on solid pollen germination medium poured into a mould made with 2-mm thick silicone rubber and cover glasses31. About 4–5 h after hand-pollination, the topside cover glass was removed and the medium was covered with hydrated silicone oil (KF-96-100CS; Shin-Etsu). The assay for T hemizygous C. rubella plants was performed similarly using A. thaliana or C. rubella pistils as pollen acceptors. Attraction of pollen tubes towards the peptide was evaluated using gelatine beads (5% (w/v) gelatine (Nacalai) in the pollen medium without agar) containing 5 μM His-tagged AtLURE1.2 peptide under an inverted microscope (IX71, Olympus) equipped with a micro-manipulator (Narishige), as described previously6. The percentages of attracted pollen tubes are shown for the total number of pollen tubes in at least two assays. In the assay using hemizygous plants, the presence of the transgene in the pollen tube containing the transgene was confirmed by fluorescence observations after assessment of pollen tube responsiveness as a simple blind test. For the AtLURE1-responsive wavy assay, the purified AtLURE1.2 peptide was added to solid pollen germination medium, which was melted at 70 °C and then cooled to a certain degree. The mixture was mixed by vortexing and poured into the mould. Pollen tubes of each genotype were grown through cut styles, as described earlier. Plasmids encoding green and red fluorescent proteins, pcDNA3-Clover and pcDNA3-mRuby2 (gifts from M. Lin, Addgene plasmids 40259 and 40260)32, respectively, were used as templates to prepare binary vectors as follows. The original Clover was converted to A206K mutant form to prevent potential dimerization, and a restriction site KpnI in the nucleotide sequence was eliminated by a silent mutation, designated as monomeric Clover (mClover). Modified binary vectors pPZP211, pPZP221 (ref. 33) and pMDC99 (ref. 34) derivatives, pPZP211G (ref. 35), pPZP221G, and pMDC99G, were used for cloning of the mClover and mRuby2. pPZP221G was produced by the same procedure as that used for pPZP211G (ref. 35), and pMDC99G was produced by removal of ccdB by EcoRI digestion and self-ligation31 and by inserting multiple cloning sites, green fluorescent protein (GFP), and the NosT cassette of pPZP211G via HindIII and EcoRI sites. To add linkers to both the amino-terminal and carboxy-terminal of mClover and mRuby2, three rounds of PCR were performed with DNA templates for mClover and mRuby2, respectively, using three sets of primers: (5′-aggtggaggtggaATGGTGAGCAAGGGCGA-3′ and 5′-tccacctccacctgaCTTGTACAGCTCGTCCA-3′; 5′-tctggaggtggaggttcAGGTGGAGGTGGA-3′ and 5′-cggggtacccactagtttaattaagaattcTCCACCTCCACCTG-3′; 5′-aggcgcgccTCTGGAGGTGGAG-3′ and 5′-cggggtacccactagtttaattaagaattcTCCACCTCCACCTG-3′) (lowercase bases denote additional nucleotides for template DNAs). The PCR fragments were digested with AscI and KpnI and ligated into pPZP211G, pPZP221G and pMDC99G by replacing the GFP sequence, resulting in pPZP211Clo, pPZP221Clo, pPZP211Ru, pPZP221Ru, pMDC99Clo and pMDC99Ru vectors. For the expression of full-length PRK6, kinase domain-deleted PRK6 (K-del), cytosolic domain-deleted PRK6 (cyto-del-2) and PRK6 orthologue of C. rubella (CrPRK6) as mRuby2-fusion protein under the control of their own promoter, genomic sequences of PRK6 or CrPRK6 containing promoter and coding regions were amplified and were cloned into the pPZP221Ru using SalI and AscI sites, resulting in pPZP221-pPRK6::PRK6-mRuby2, -pPRK6::PRK6 (K-del)-mRuby2, -pPRK6::PRK6 (cyto-del-2)-mRuby2, and -pCrPRK6::CrPRK6-mRuby2 vectors. These constructs were introduced into prk6-1, prk3-1 prk6-1, prk3-1 prk6-1 prk8-2 and prk1-2 prk3-1 prk6-1 plants by the floral dip method. For the heterologous expression of PRK6 in C. rubella, the pPZP221-pPRK6::PRK6-mRuby2 vector was used for C. rubella transformation by the floral dip method after flowering induction. Genomic sequences of PRK6 or PRK3 containing promoter and coding regions were also cloned into pMDC99Clo using SalI and AscI sites, and these constructs were introduced into prk3-1 prk6-1. Primers used for these constructs are listed in Supplementary Table 1. For all transgenic lines expressing PRK proteins, T transformants were screened by moderate or weak fluorescence intensity in approximately half of the pollen grains, implying single insertion. Note, when pollen grains showing mid to strong fluorescence intensity were used for the semi-in-vivo pollen tube growth assay, few or no fluorescent pollen tubes emerged from the cut end, probably owing to the growth defect caused by excess PRK expression. T homozygous plants obtained from several selected T lines were used for the semi-in-vivo AtLURE1-responsive wavy assay. To prepare constructs for the BiFC assay in the leaf epidermal cells of Nicotiana benthamiana, cauliflower mosaic virus 35S promoter was introduced to the binary vector pPZP211G (ref. 35) using HindIII and PstI sites. Then, the GFP sequence was replaced by nucleotide sequences encoding each of amino acids 1–174 and 175–239 of enhanced yellow fluorescent protein (nYFP and cYFP, respectively) with the same linkers as the mClover and mRuby2 constructs, described above, resulting in pPZP211-p35SnY and pPZP211-p35ScY vectors. Genomic PRK2 and PRK6 were amplified and connected upstream of the cYFP sequence of pPZP211-p35ScY. The genomic sequences of PRK6, PRK3, LIP1 and LIP2 were connected upstream of the nYFP sequence of pPZP211-p35SnY. Genomic ROPGEF8, ROPGEF9, ROPGEF12, ROPGEF13 and ROPGEF12ΔC (encoding amino acids 1–443 of ROPGEF12 (ref. 8)) were amplified and connected downstream of the nYFP sequence in pPZP211-p35SnY. Primers used for these constructs are listed in Supplementary Table 1. Transient expression in N. benthamiana leaves was performed by agro-infiltration according to a method described previously20. In brief, Agrobacterium tumefaciens strains GV3101 (pMP90) containing each expression vector were cultured overnight in LB media. Equal amounts of Agrobacterium cultures for nYFP and cYFP constructs and the p19 silencing suppressor were mixed to a final A of 1.0 and collected and resuspended in infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl and 150 μM acetosyringone). The mixed suspensions were incubated at room temperature for ~3 h and infiltrated into leaves of N. benthamiana grown at 25 °C. Two to three days after infiltration, the leaves were cut into pieces for confocal microscope observation. To analyse pollen tube growth and guidance in the pistil, Col-0 pistils emasculated 1 day before were abundantly hand-pollinated with two or three fully dehiscent anthers from each genotype. Two types of aniline blue staining were performed 12 or 24 h after pollination as follows. For measurement of pollen tube growth inside the transmitting tract, aniline blue staining was performed, as described previously36. Pollinated pistils were dissected to remove a pair of ovary walls and then fixed in a 9:1 mixture of ethanol and acetic acid for more than 2 h. They were washed with 70% ethanol for ~30 min, treated with 1 N NaOH overnight, and stained with aniline blue solution (0.1% (w/v) aniline blue, 0.1 M K PO ) for more than several hours. The pistils were observed under ultraviolet illumination using an upright microscope (DP71, Olympus). Multiple images for each pistil were combined using Adobe Photoshop CS4 (Adobe Systems), and lengths from the top of the stigma to the tip of the longest pollen tube were measured for maximum pollen tube length using the MacBiophotonics ImageJ software (http://www.macbiophotonics.ca/). To evaluate pollen tube guidance after emergence on the septum surface of the pistil, dissected pistils were stained directly with modified aniline blue solution (5:8:7 (v/v) mixture of 2% aniline blue, 1 M glycerol, pH 9.5, and water), as described previously37, and observed under ultraviolet illumination using an upright microscope (DP71, Olympus). Quantitative analysis was performed by evaluating pollen tube growth on 10 upper ovules of both sides (total, 20 ovules per pistil) to eliminate bias in ovule number in a pistil. Confocal images were acquired using an inverted microscope (IX81, Olympus) equipped with a spinning disk confocal scanner (CSU-X1, Yokogawa Electric Corporation), 488 nm and 561 nm LD lasers (Sapphire, Coherent), and an EM-CCD camera (Evolve 512, Photometrics). For A. thaliana pollen tubes, a 60× silicone immersion objective lens (UPLSAPO60XS, Olympus) and a 1.6× intermediate magnification changer were used. For time-lapse imaging of PRK6–mRuby2 during pollen tube attraction towards a gelatine bead containing 5 μM Alexa488-labelled His-AtLURE1.2, sequential images using 488 nm and 561 nm lasers were acquired every 5 s. For the BiFC assay in N. benthamiana leaves, a 20× objective lens (UPLFLN20X, Olympus) was used. The confocal microscope system was controlled and time-lapse images were processed by MetaMorph (Universal Imaging). Images were edited with MacBiophotonics ImageJ. To prepare transient expression vectors in N. benthamiana leaf cells, the cauliflower mosaic virus 35S promoter was introduced into the binary vector pPZP211Clo via HindIII and PstI sites, resulting in the pPZP211-p35SClo vector. The 3× Flag tag sequence was introduced into pPZP211-p35S using the AscI and SacI sites, resulting in the pPZP211-p35SFlag vector. For co-immunoprecipitation of PRK-mClover and ROPGEF12-3 × Flag proteins, genomic sequences of full-length PRK3, full-length PRK6, PRK6 (K-del), PRK6 (cyto-del-1), and ROPGEF12 were inserted into the pPZP211-p35SClo or pPZP211-p35SFlag vectors. One of the PRK-mClover or mClover proteins plus the p19 silencing suppressor and ROPGEF12-3 × Flag were co-expressed in N. benthamiana leaves as described for the BiFC assay. The leaves were ground in mortars with liquid nitrogen and suspended in 3–3.5 × (w/v) extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, protease inhibitor cocktail (cOmplete EDTA-free, Roche)). The extracts were centrifuged twice at 10,000g for 10 min at 4 °C to remove precipitates. The supernatants, with the exception of the mClover sample, were ultracentrifuged at 100,000g for 30 min at 4 °C, and the pellets were solubilized in extraction buffer containing 0.5% Triton X-100. The solubilised membrane fraction samples and mClover sample plus 0.5% Triton X-100 were incubated with GFP-trap agarose beads (ChromoTek, gta-20) with rotation for 2 h at 4 °C. The beads were washed with buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) four times. Then, the bound proteins were eluted with SDS sample buffer by heating at 70 °C for 5 min. The protein samples were separated on SDS–PAGE and subjected to immunoblot analysis. The immunoblot analysis was conducted on PVDF membranes (Immobilon-P, Millipore) using primary antibodies (anti-GFP (ab290, Abcam), or monoclonal anti-DYKDDDDK tag (Wako) for Flag tag) and secondary antibodies (goat anti-rabbit IgG peroxidase-labelled antibody or goat anti-mouse IgG peroxidase-labelled antibody (KPL)). Signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore), detected with Light-Capture (ATTO).
No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment. The Arabidopsis thaliana wild-type (Col-0), T-DNA insertion mutants mdis1-1 (GABI_463E06), mdis1-2 (GABI_090F03), mdis2 (SALK_004879) and Capsella rubella were obtained from ABRC stock centre. mik1 (SALK_095005) and mik2 (SALK_061769) were obtained from J. Zhou. The E. salsugineum seeds were obtained from Q. Xie. Plants were grown at 22 °C under long-day conditions (16-h light/8-h dark cycles). For C. rubella and E. salsugineum, the sterilized seeds were vernalized on the MS media at 4 °C for 30 days and then grown at 22 °C under long-day conditions. Pollen tubes were germinated on the germination media (1 mM CaCl , 1 mM Ca(NO ) , 1 mM MgSO , 0.01% H BO , 18% sucrose and 0.5% agarose) and cultured for 5 h at 22 °C. The germination ratio was scored under light microscopy. Mean value was calculated from three independent experiments and for each experiment, more than 300 pollen were scored. For in vivo tube growth, pollen from the wild-type and mutants were pollinated on the emasculated pistil with mature stigma as reported20. The pistils were collected at 3, 6 and 8 h after pollination and fixed for aniline blue staining. The pollen tubes in the pistil were photographed with Leica M205 microscope. The length of pollen tubes was measured with Image J software (http://rsb.info.nih.gov/ij/). Flowers at 12c stage were emasculated and left to grow for 12–24 h to achieve pistil maturation. Then about 20 pollen grains from wild-type or mutant plants, respectively, were dispersed on the stigma papillar cells with a tiny brush. After 24 h, pistils were excised and fixed in Carnoy’s fixative (75% ethanol and 25% acetic acid) as reported21, 22. The pistils were washed in 50 mM PBS buffer (NaHPO /NaH PO , pH 7.0) three times and immersed in 1 M NaOH overnight for softening. Then after three washes with PBS, the pistil was stained with 0.1% aniline blue (pH 8.0 in 0.1 M K PO ) for 6 h. The stained pistils were observed under Axio Skop2 microscope (Zeiss) equipped with an ultraviolet filter set. Ovules with micropylar guidance defect and the ratio of fertilized ovules to the number of pollen tubes in the style were calculated and the mean values from three independent experiments were compared with that of the wild type. For the dominant-negative constructions, the kinase domains were inactivated by replacing the conserved lysine residue in the intracellular ATP-binding domain with glutamic acid to generate dominant-negative constructs. For the atypical kinase, the intracellular domain was chimaerically replaced with that of BRASSINOSTEROID INSENSITIVE1 (BRI1)23 receptor kinase with an inactive kinase domain (K to E substitution). For GFP and GUS reporter expression, genomic sequences containing 2 kb native promoters and the genomic coding sequence for MDIS1 and MDIS2 were subcloned into the pCAMBIA1300-GFP binary vector. For complementation of mik mutants, full-length coding sequence driven by LAT52 promoter was cloned into pCAMBIA1300. Similarly, full-length LURE1.2 fused with a C-terminal Flag tag driven by the 35S promoter was cloned into the pCAMBIA1300. For complementation assay, the genomic fused GFP constructs were transformed into the mutant using Agrobacterium-mediated floral dip method24. To break down the reproductive isolation barrier, the full-length MDIS1 coding sequence under the LAT52 promoter was introduced into C. rubella by floral dip method. LURE1.2 and PDF2.1 lacking the putative N-terminal signal peptides (71 and 55 amino acids, respectively) were fused N-terminally with a His-tag using pET28a vector (Novagen). Similarly, the ectodomains of MDIS1, MDIS2, MIK1, MIK2 and PRK3 lacking the predicted signal peptides were fused with an N-terminal GST tag using a pGEX4T-2 vector. The fused proteins were expressed in Escherichia coli strain Rossetta DE3 (Stratagene). Cells were grown to an A value of 0.6 at 37 °C and then induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 6 h at 22 °C. The cells were lysed by sonication on ice in lysis buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, Complete Protease Inhibitor Cocktail (Roche) and 1 mg ml−1 lysozyme (Wako). After centrifugation at 12,000 g for 20 min at 4 °C, the supernatants and pellets were collected separately; the pellet was washed three times with the lysis buffer. For LURE1, the insoluble His–LURE1.2 peptides in the inclusion bodies were solved in 1 M urea supplemented with 6 M guanidine-HCl (in Tris-HCl buffer, pH 8.0) for 1 h on ice. Then the peptides were diluted at 1:10 and refolded for 3 days at 4 °C using glutathione (reduced form: oxidized form = 10:1, MERCK) and l-arginine ethyl ester dihydrochloride (Sigma-Aldrich). The folded peptides were dialysed with 3-kDa centrifugal filter (Millipore) and eluted with 50 mM Tris-HCl (pH 8.0) and then used for pull-down, co-IP, protoplasts treatment, pollen tube guidance assays and antibody generation. For purification of GST-tagged ectodomain of MDIS1, MDIS2, MIK1, MIK2 and PRK3 proteins, cells from 2 l culture were collected and lysed respectively as described above. The supernatants were used for affinity purification by glutathione agarose beads (GE, 17-0756-01) to avoid extra folding process, although more fused proteins were in the pellets than the supernatant. For GST pull-down assay, the purified proteins were mixed and incubated for 3 h and then subjected to pull-down assay with glutathione agarose beads for 3 h at 4 °C. The beads were collected by centrifugation and then washed five times with buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Triton X-100 and 0.1% SDS. Finally, the proteins bound on the beads were boiled with 1× SDS sample buffer in 95–100 °C water bath and then subjected to SDS–PAGE and immunoblot with anti-GST (GE Healthcare, 27-4577-01) and anti-His (Santa Cruz) antibody. For mobility shift detection of phosphorylated proteins, phosphatase inhibitor phrostop (Roche) was added during purification and incubation. Moreover, 50 μM Phos-tag (AAL-107) and 50 μM MnCl was added to the gel according to the manufacturer’s procedure. After electrophoresis, the gel was treated with 10 mM EDTA, pH 8.0, for 10 min to remove the Mn2+ before immunoblot assay. Seedlings of LURE1.2-Flag transgenic plants were ground to fine powder in liquid nitrogen and solubilized with extraction buffer (0.05 M HEPES-KOH, pH 7.5, 150 mM KCl, 1 mM EDTA, 0.1% Triton X-100 with freshly added proteinase inhibitor cocktail (Roche)). The extracts were centrifuged at 10,000g for 10 min, and the supernatant was incubated with pre-washed anti-Flag M2 magnetic beads (Sigma-Aldrich, M8823) for 3 h at 4 °C, and then the beads was washed six times with the extraction buffer. The immunoprecipitates were eluted with 3 × Flag peptides. For co-IP in protoplasts, the transformed protoplasts expressing MDIS1–HA, MIK–HA and BAK1–HA were incubated with the purified LURE1.2–Flag or the 200 nM folded His–LURE1.2 purified from E. coli for 10 min and lysed for co-IP with pre-washed anti-HA agarose beads (Sigma-Aldrich, A2095). The precipitates were diluted with SDS sample buffer, separated on a 10% SDS–PAGE gel and subjected to immunoblot with the corresponding antibodies (anti-Flag, Sigma-Aldrich, F1804; anti-HA, Santa Cruz, sc-7392; anti-His, Santa Cruz, sc-803). Arabidopsis protoplast transformation was performed as reported previously25. For the His–LURE1-protoplast binding assay, the protoplasts incubated with 10 μm LURE1.2 for 5 min, washed three times with the culture buffer and then lysed for SDS–PAGE and immunoblot. For the enhanced interaction between MDIS1 and MIK proteins by LURE1.2, the protoplasts co-transformed with MDIS1–HA and MIK1–Flag were divided into two equal volumes. One was incubated with 0.5 nM LURE1.2 and another with equal volume of 50 mM Tris-HCl (pH 8.0) as mock control for 10 min and subjected to anti-HA immunoprecipitation. For the phosphorylation test, the transformed protoplasts were divided equally into two and incubated for 10 min with 200 nM LURE1.2 or 50 mM Tris-HCl (pH 8.0), respectively. For competition assay, protoplasts expressing MDIS1–HA, MIK1–HA and MIK2–HA were each divided equally into four centrifuge tubes and incubated with purified LURE1.2–Flag. Then active His–LURE1.2 of different concentrations was added to the protoplasts and incubated for 10 min and subsequently co-immunoprecipated with anti-HA conjugated agarose beads. For co-IP in planta, the flowers opened in the morning were collected in the afternoon at the estimated time when the pollen tubes are approaching the ovules. Total proteins were subjected to co-IP with anti-GFP conjugated agarose (ChromoTek, gta-200) or anti-LURE1.2 and protein-A-conjugated magnetic beads (Bio-Rad, 161–4013). The immunoprecipitates was subjected to SDS–PAGE and immunoblot with the corresponding antibodies (anti-GFP-HRP, Miltenyi Biotec, 130-091-833). All the co-IP experiments were repeated at least three times. For A. thaliana, the same germination media as that for in vitro germination was used. For C. rubella, a modified media (4 mM CaCl , 4 mM Ca (NO ) , 0.01% H BO , 10% sucrose and 0.5% agarose) was used. Semi-in-vitro germination and ovule-pollen attraction assay were performed as reported in A. thaliana3. Pollen tubes entered the micropyle were scored as successful breakdown of the reproductive isolation and the pollen tubes bypass outside the micropyle within 20 μm were scored as failing to enter the micropyle. For the attraction assay, gelatin (Nacalai) beads containing 40 μM LURE1.2 were made and placed beside the pollen tube tip using a micro-manipulator (Narishige) equipped with an inverted microscope (Zeiss AxioVert. A1) as described previously26. Behaviour of pollen tubes was monitored and recorded with a CCD camera. Pollen tubes growing to the beads with >30° direction change were regarded as effective pollen tube attraction. Total RNA was extracted from pollen, in vitro germinated pollen tubes (3 h after pollination) and seedlings with TRIzol reagent (Invitrogen) and then treated with DNase I (RNase-free DNase kit, Qiagen) to remove DNA. SuperScript III Reverse Transcriptase (Invitrogen) was used for the reverse transcription reactions. qPCR was performed with Power SYBR Green PCR Master Mix on the Bio-RAD C1000 Thermal Cycler using Tubulin 2 as the internal control for quantitative normalization. The specificity of the primers was examined by running the PCR products on 2.5% agarose gels and sequencing. The affinity of the purified GST, GST–MDIS1ECD, MDIS2ECD, MIK1 ECD, MIK2 ECD, ERECTAECD and PXY ECD to His–LURE1.2 was measured using the Monolith NT.115 (Nanotemper Technologies). The GST-fusion proteins were fluorescently labelled according to the manufacturer’s procedure. The solution buffer was exchanged to labelling buffer and the protein concentration was adjusted to 2 μM. Then fluorescent dye NT-647-NHS was added and mixed and incubated for 30 min at 25 °C in the dark. Finally, the labelled proteins were dialysed with column B (Nanotemper L001) and eluted with 50 mM Tris-HCl (pH 8.0) supplemented with 0.02% Tween 20. For each assay, the labelled protein (about 1 μM) was incubated with the same volume unlabelled His–LURE1.2 of 12 different serial concentrations in 50 mM Tris-HCl (pH 8.0) supplemented with 0.02% Tween 20 at room temperature for 10 min. The samples were then loaded into silica capillaries (Polymicro Technologies) and measured at 25 °C by using 20%–40% LED power and 20% MST power. Each assay was repeated three times. Data analyses were performed using Nanotemper analysis software and OriginPro 9.0 software. The constructs containing MDIS1-NE (MDIS1 fused with the N-terminal YFP), MIK1-CE and MIK2-CE (MIK1 and MIK2 fused with the C-terminal YFP, respectively) were generated as described previously8. The Agrobacterium tumefaciens EHA105 strains carrying MDIS1-NE and MIK-CE were equally mixed with and without EHA105 strain carrying LURE1.2–Flag and transformed into half of the same tobacco leaf. The transformed leaves were photographed 2 days later with a confocal laser scanning microscope (Zeiss Meta 510). Images were acquired using the same optical setting and average total pixel intensity values were calculated by sampling images of different leaves using the ImageJ software as reported27. Mean values of three experiments, each with five transformed leaves, were compared using Student’s t-test for biological significance. The E. coli cells expressing the fusion proteins were lysed and centrifuged at 4 °C. The affinity-purified fusion proteins from the supernatants were subjected to mass spectrometry. His–MDIS1KD was incubated with GST–MIK1KD in vitro in kinase assay buffer (25 mM Tris-HCl, pH 8.0, 10 mM MgCl and 100 mM ATP) for 1 h at 30 °C. The proteins were separated by 10% SDS–PAGE and the gel was stained with Coomassie blue G250. The corresponding proteins band were cut into slices and subjected to alkylation/tryptic digestion followed by LC–MS/MS as reported previously28. For disulfide bonds determination, GST–MDIS1ECD, GST–MIK1ECD and GST–MIK2ECD were affinity purified from the supernatants of the bacterial lysis and eluted with 50 mM Tris-HCl, pH 8.0. Then disulfide bonds were determined by mass spectrometry as previously reported29. Alignment of protein sequences were aligned using ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Phylogenetic tree of the alignment were drawn with MEGA5 (http://www.megasoftware.net/) using the neighbour-joining method with bootstrapping based on 1,000 replicates. The leucine-rich repeat domains were predicted with LRRfinder (http://www.lrrfinder.com/) and HHPREP program. The transmembrane domains were predicted with TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The signal peptides were predicted with SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/). The coding sequences of MDIS1 or MIK1 and MIK2, respectively, were cloned into the pBT3-SUC bait or pPR3-N prey according to the manufacture’s procedure (DualsystemBiotech). Yeast strain NMY51 was co-transformed with the bait and prey constructs and grown on the selective medium lacking Trp, Leu, His and adenine. Total RNA was extracted from pollen, leaf, flower and total plant of C. rubella and E. salsugineum with TRIzol reagent (Invitrogen) and then treated with DNase I (RNase-free DNase kit, Qiagen) to remove any contaminating DNA. SuperScript III Reverse Transcriptase (Invitrogen) was used in reverse transcription reactions. ACTIN11 was used as the control for quantitative normalization. The specificity of the primers was confirmed by sequencing of the band after electrophoresis. The accession numbers for the amplified genes are as follows: CrMDIS1 (XM_006280043), EsMDIS1 (XM_006398206), CrMIK1 (XM_006285722), EsMIK1 (XM_006412864), CrMIK2 (XM_006286915), EsMIK2 (XM_006397188), CrACTIN11 (XM_006297859) and EsACTIN11 (XM_006407307). The histochemical GUS activity assay was performed in the solution containing 2 mM X-Gluc (Sigma) in 50 mM PBS (pH 7.0) and 0.5 mM potassium/ferrocyanide. GUS solution was added to the samples and incubated at 37 °C overnight. Digital images were taken with a Zeiss Axio Skop2 plus microscope. For GFP observation, images were taken with Zeiss confocal laser scanning microscope with a setting of 488 nm excitation (Carl Zeiss, Meta 510 confocal microscope). The semi-in-vitro germinated MDIS1–GFP pollen tubes were treated with 500 nM LURE1.2 and photographed by CLSM 780 (Zeiss) after different times. The anti-MIK1 and anti-MIK2 antibodies were raised in mouse with the purified His-tagged extracellular domains lacking the predicted N-terminal signal peptide. Anti-LURE1.2 antibody was raised in mouse with the folded active His–LURE1.2 fusion protein. For MIK1 and MIK2, the specificity of antibodies was tested with the fusion proteins expressed in protoplasts and the total proteins of pollen from the wild-type and corresponding mutant plants. For LURE1.2, the antibody specificity was tested with the total protein from the leaves of LURE1.2–Flag-overexpressing plants. For immunostaining, the semi-in-vitro germinated pollen tubes were fixed in 3.7% paraformaldehyde (3.7% formaldehyde, 1 mM CaCl , 1 mM MgSO , 50 mM HEPES, 5% sucrose, pH 7.4) for 30 min, washed with PME buffer (50 mM PIPES, 1 mM MgCl , 5 mM EGTA, pH 6.8) three times and then subjected to 1% Driselase and 1% cellulase for 10 min. The sample was sequentially washed with PBS buffer (pH 7.4) three times, NP40 buffer (0.5% Nonidet P-40, 1% BSA, in PBS, pH 7.4) and PBS buffer once. Antibodies diluted 1:500 (with PBS containing 3% BSA) were incubated with the sample overnight at 4 °C and then washed with PBS three times. The samples were incubated for 1 h at 4 °C with FITC-labelled goat anti-mouse secondary antibody (KBL, 202-1806) and washed with PBS three times. Anti-fade mounting medium (Invitrogen, P36934) was used for signal detection by confocal laser scanning microscopy (Zeiss Meta 510) with 488 nm excitation.
Brown C.J.,A Star Inc. |
Quah S.T.,A Star Inc. |
Jong J.,A Star Inc. |
Goh A.M.,A Star Inc. |
And 11 more authors.
ACS Chemical Biology | Year: 2013
By using a phage display derived peptide as an initial template, compounds have been developed that are highly specific against Mdm2/Mdm4. These compounds exhibit greater potency in p53 activation and protein-protein interaction assays than a compound derived from the p53 wild-type sequence. Unlike Nutlin, a small molecule inhibitor of Mdm2/Mdm4, the phage derived compounds can arrest cells resistant to p53 induced apoptosis over a wide concentration range without cellular toxicity, suggesting they are highly suitable for cyclotherapy. © 2012 American Chemical Society. Source