Characterization of cp3 reveals a new bri1 allele, bri1-120, and the importance of the LRR domain of BRI1 mediating BR signaling
© Shang et al; licensee BioMed Central Ltd. 2011
Received: 17 September 2010
Accepted: 11 January 2011
Published: 11 January 2011
Since the identification of BRI1 (BRASSINOSTEROID-INSENSITIVE1), a brassinosteroids (BRs) receptor, most of the critical roles of BR in plant development have been assessed using various bri1 mutant alleles. The characterization of individual bri1 mutants has shown that both the extracellular and cytoplasmic domains of BRI1 are important to its proper functioning. Particularly, in the extracellular domain, regions near the 70-amino acid island are known to be critical to BR binding. In comparison, the exact function of the leucine rich-repeats (LRR) region located before the 70-amino acid island domain in the extracellular cellular portion of BRI1 has not yet been described, due to a lack of specific mutant alleles.
Among the mutants showing altered growth patterns compared to wild type, we further characterized cp3, which displayed defective growth and reduced BR sensitivity. We sequenced the genomic DNA spanning BRI1 in the cp3 and found that cp3 has a point mutation in the region encoding the 13th LRR of BRI1, resulting in a change from serine to phenylalanine (S399F). We renamed it bri1-120. We also showed that overexpression of the wild type BRI1 protein rescued the phenotype of bri1-120. Using a GFP-tagged bri1-120 construct, we detected the bri1-120 protein in the plasma membrane, and showed that the phenotypic defects in the rosette leaves of bri1-301, a kinase-inactive weak allele of BRI1, can be restored by the overexpression of the bri1-120 proteins in bri1-301. We also produced bri1-301 mutants that were wild type in appearance by performing a genetic cross between bri1-301 and bri1-120 plants.
We identified a new bri1 allele, bri1-120, whose mutation site has not yet been found or characterized. Our results indicated that the extracellular LRR regions before the 70-amino acid island domain of BRI1 are important for the appropriate cellular functioning of BRI1. Also, we confirmed that a successful interallelic complementation occurs between the extracellular domain mutant allele and the cytoplasmic kinase-inactive mutant allele of BRI1 in vivo.
Numerous plant developmental processes, such as germination, cell elongation, photomorphogenic responses, and male fertility are regulated by the plant-specific steroidal hormones, brassinosteroids (BR). BR-biosynthetic or BR-perceiving mutants have exhibited defective growth patterns in various tissues that persist throughout their entire life span, indicating the critical role of BR in plant development [1, 2]. Although studies researching the BR signaling process began much more recently than any of the other plant hormones, the identification of BRASSINOSTEROID-INSENSITIVE1 (BRI1), a receptor of BR , and several other important components involved in BR signaling have provided much insight into many important components in plant development . Plasma membrane-localized BRI1 and its co-receptor BRI1-ASSOCIATED KINASE1 (BAK1) are receptor-like serine/threonine kinases containing leucine-rich repeats (LRR-RLKs) [5, 6]. N-terminal LRRs are found in the extracellular portion of the plasma membrane. BRI1 constitutively forms a homodimer in the plasma membrane. In the absence of BR, the activity of the BRI1 homodimer is inhibited by the BRI1 kinase inhibitor 1 (BKI1) by binding BKI1 to the C-terminal portion of BRI1. In the presence of BR, BKI1 is released by the direct binding of BR to the 70-amino acid island region in the extracellular domain of BRI1 . Then, BRI1 recruits BAK1, forming heterodimerized-receptor complexes in the plasma membrane [8, 9], leading to the activation of the BES1 and BZR1 transcription factors that regulate the expression of the BR-associated genes [2, 10, 11].
BRI1 is considered to be a master regulator that plays a critical role in the direct binding of BR and subsequent BR signaling processes , while BAK1 has been found to be a partner not only for BRI1 but also for other LRR-RLKs, such as FLS2 and EFRs, which are involved in the plant innate immunity responses [13, 14]. To date, genetic screening looking for BR-insensitive mutants has resulted in the identification of only two genes, BRI1 and BIN2 [3, 15]. Since the first report of BRI1 in 1997 , more than 30 different mutant alleles have been identified in several different Arabidopsis ecotypes, including Col-0, Ws-2, and En-2 during last two decades. Large numbers of mutant alleles that have mutations in various positions of a specific gene provide information regarding how that gene acts, because the mutation sites themselves are indicators of their importance to the functioning of the gene. In that sense, studying multiple mutant alleles of BRI1 will be likely to reveal important information regarding its function. Detailed analyses of the characteristics of each mutation have shown that both the extracellular and cytoplasmic domains of BRI1 are required for full BRI1 functioning, because the mutation sites of all of the bri1 mutant alleles are dispersed in both an extracellular domain and a cytoplasmic kinase domain [4, 16].
The extracellular domain of BRI1 consists of LRRs and a 70-amino acid island containing unique sequences that show little homology to any other protein. Since BRI1 was discovered, it has been considered to have 25 LRRs with a 70-amino acid island flanking the 21st and 22nd LRR. However, Vert et al, (2005)  suggested that BRI1 contains 24 LRRs, postulating that the 21st LRR is actually an atypical formation. It appears evident that the region near the 70-amino acid island allows for the extracellular binding of BR. It is interesting to note that most of the mutation sites in the extracellular domain of BRI1 are clustered in the 70-amino acid island domain and in the 4 LRRs situated before the transmembrane domain. There are very few examples of mutant alleles containing defects in the LRR regions that occur before the 70-amino acid island. This may be partially because the mutations in these LRR regions of BRI1 were neglected due to the lack of any discernible phenotypic alterations. Or, at the opposite extreme, they may lethally affect plant development, resulting in no viable mutants for further analyses. Here, we report a new mutant allele of BRI1, bri1-120. A point mutation in the region encoding the 13th LRR of BRI1 in bri1-120 caused the defective growth and reduced BR sensitivity of the plant. Using this mutant allele, we demonstrated successful interallelic complementation using a kinase-inactive mutant allele, bri1-301 and performed a detailed analysis of BR sensitivity.
Phenotypic analyses of the weak bri1-looking semi-dwarf mutant, cp3
Identification of the weak bri1 mutant allele, bri1-120
To verify this notion, we generated a transgenic cp3 plant overexpressing BRI1 by introducing a BRI1 promoter-driven BRI1:BRI1-GFP construct. The growth of the BRI1-overexpressing cp3 plants was more similar to that of the wild type as compared to the non-transformed cp3 plants (Figure 3B). We confirmed that the BRI1-GFP transgene was highly expressed in the transgenic cp3 plants by RT-PCR analyses using primers that amplified transgene specifically (Figure 3C). The cp3 plants overexpressing BRI1-GFP showed nearly normal overall growth patterns with elongated leaves and petiole length as well as total height, similar to those observed with Ler (Figure 3D). In addition, the cp3 transgenic plants overexpressing BRI1 showed restored BL sensitivity, exhibiting a BL-induced transcriptional inhibition of CPD expression (Figure 3E). These results suggest that the growth retardation of the cp3 mutant accompanied by the dark green coloring is caused by a mutation in the extracellular domain of BRI1. Therefore, we renamed the cp3 mutant bri1-120, referring to the order of naming for bri1 mutant alleles 
BRI1(S399F) protein is localized in plasma membrane and the overexpression of BRI1(S399F) in bri1-301 resulted in the leaf elongation of bri1-301 and co-suppression of the endogenous bri1-301
We introduced nucleotide C instead of T at the 1196th position of BR1 by site-directed mutagenesis to generate the bri1-120 mutated BRI1, using the BRI1-GFP construct as a template. The resulting construct (BRI1:bri1-120-GFP) was transformed into the wild type Col-0, bri1-301 plants to produce a mutated BRI1(S399F). After the wild type plant was transformed with BRI1:bri1-120-GFP, we first observed the intracellular localization of the BRI1(S399F) protein using a confocal microscope by detecting the GFP that was fused with BRI1(S399F) in the plasma membrane of the cells (Figure 4A), which indicated that bri1-120 possesses the plasma membrane-localized BL receptor, although BRI1(S399F) may not be fully functional protein. In comparison, the mutated BRI1 proteins, BRI1(C69Y) in bri1-5 and BRI1(S662F) in bri1-9, in which both mutations are in the extracellular domain of BRI1, are known to be localized to the endoplasmic reticulum (ER) .
Bri1-301 and bri1-120complemented each other to form a functional BRI1 receptor
Different BL sensitivity was observed in the bri1-301 transformed with BRI1:bri1-120-GFP and the bri1-301 crossed with bri1-120
BRI1-120revealed the importance of the LRR region in the extracellular domain of BRI1
The degree of phenotypic alteration caused by each bri1 allele depends on the specific affected mutation sites . Mutants that have amino acid changes in the cytoplasmic kinase domain usually show very strong mutant phenotypes, which can be attributed to loss of BRI1 kinase activity. Bri1-301 is an exceptional case. Although bri1-301 was shown to be a kinase-inactive protein, the mutant plant exhibits only mild phenotypic changes. Bri1-301 contains two nucleotide changes (GG to AC) in the cytoplasmic kinase domain of BRI1, resulting in a change from Gly989 to Ile . However, Gly989 is not a conserved amino acid, and its position is slightly out of the critical region of the kinase domain. So, it is possible that Gly989 is important for maintaining the proper conformation of the BRI1 protein to retain its kinase activity, but, not for controlling the kinase activity itself.
In comparison, most of the mutations in the extracellular domain of BRI1 produced relatively mild mutant phenotypes. A more thorough examination of the extracellular domain of BRI1 revealed that the 70-amino acid island domain and the subsequent four LRRs before the transmembrane domain are frequent mutation sites, indicating their functional importance to the BRI1 protein. In addition, the first cysteine pair before the beginning of the LRRs is thought to be critical for BRI1 as seen in the mutant bri1-5 (C69Y). So far bri1-4 is the only mutant in which the mutation occurred in the LRR regions preceding the 70-amino acid island domain . However, a 10-bp deletion in the 3rd LRR of BRI1 in bri1-4 introduced a premature stop in translation and did not provide any clues regarding the functional importance of the LRR domains of BRI1.
In this study, we analyzed the BR-related phenotypes of cp3 grown from the CS48 seeds obtained from ABRC to have more natural mutants with similar morphologies to known bri1 mutants, although the phenotypic strength of bri1-120 is relatively weak compared to other bri1 mutants, such as bri1-5 or bri1-9. Cp3 has the COMPACTA3 (cp3) mutation, and cp3 mutants show altered phytochrome A signaling . However, the mutated gene has not been characterized yet. From the direct sequencing of the genomic DNA region containing BRI1, we found that this plant contains a mutation in BRI1 called bri1-120. Bri1-120 contains phenylalanine instead of serine at the 399th position in the 13th LRR due to a nucleotide change (T to C) at the 1196th position (Figure 3A). When we overexpressed wild type BRI1 in bri1-120, mutant phenotypes of bri1-120 were rescued not only morphologically but also in terms of their sensitivities to BR (Figure 3). Overexpression of the bri1-120 protein in wild type plants produced transgenic plants with bri1 mutant phenotypes (Figure 4 and Supplementary Figure 2). We believe that bri1-120 is the first example of a natural mutant allele with a point mutation in the LRR region of the extracellular domain of BRI1. These results suggest that the LRR region before the 70-amino acid island domain is also important in maintaining a fully functional BRI1.
Tandem array of repeating LRR are known to provide protein-protein interaction motif . The plant-specific LRR motif out of seven subfamilies contains 23-25 amino acids that form an extended β-strand connected with an α-helix by a loop . Especially, first 11 amino acid residues (LxxLxLxxNxL) in LRR are highly conserved and corresponds the region forming β-strand and loop [21, 23]. Leucine residues can be compatible with isoleucine (I), valine (V), and phenylalanine (F), which form the hydrophobic core . Asparagine (N) in the 9th position is important for half-turn in LRR unit, and serine or threonine are the preferred amino acid in the 8th position, just before the asparagine . We found that the first part of amino acid sequence in the 13th LRR of BRI1 (LLTLDLSSNNF from 392nd to 402nd amino acid in BRI1) is well matched with the known consensus sequence. Compared with that, the serine residue at the 399th position of BRI1 in front of the asparagines is changed to phenylalanine in bri1-120 mutant. Regarding that serine or threonine is able to form an additional hydrogen bond with other part of proteins, it is highly possible that hydrophobic phenylalanine instead of serine residue in bri1-120 causes conformational change of LRR motif in the BRI1. Among other genes encoding the LRR-RLKs, CLAVATA1 (CLV1) which involves in meristem differentiation has been reported to have three missense mutant alleles within LRRs: cla1-10 in LRR4, clv1-4 in LRR5, and clv1-8 in LRR9. These mutations were likely to be harmful for the dimerization of CLV1 with other receptors . The HAR receptor that regulates the nodulation in legumes possesses 21 LRRs. Mutation in the LRR7 in har1-4, which alters β-strand structure, led to the reduced ligand binding . Therefore, it is possible that conformational changes due to a mutation in the 13th LRRs of BRI1 affect receptor dimerization or reduce ligand binding capacity. Recently, several mutants generated by the TILLING method were reported to have amino acid changes in the LRR region of the extracellular domain of BRI1  (http://tilling.fhcrc.org), and they are awaiting further analysis to reveal the functional significance of the LRR domain of BRI1.
Interallelic complemented bri1-301 showed different BL sensitivity as compared to the bri1-301 overexpressing a BRI1:bri1-120-GFP
There have been many reports that the compact and downward-curling rosette leaves that are considered to be weak bri1 mutant phenotypes can be restored by the overexpression of the genes encoding the positive regulators of BR signaling, such as BAK1 [5, 6], BSK1  and BES1 , and BRI1 itself . Bri1-9, bri1-5 and bri1-301 are frequently used in these types of studies. Here, we showed that the phenotypic defects in the rosette leaves of bri1-301 can be restored in two ways. First, we overexpressed BRI1:bri1-120-GFP, causing the bri1-120 mutation in bri1-301, and we showed that the transgenic bri1-301 displayed an elongated leaf and petiole growth pattern similar to that of the wild type (Figure 4B and 4C). Secondly, we generated plants by crossing bri1-120 with bri1-301. Receptors that require the assembly of homodimers in order to become active signaling complexes were interallelically complemented [30, 31]. However, to date, it has not been elucidated that whether the bri1 alleles that have the extracellular domain mutation are able to complement kinase-inactive bri1 alleles. By showing that more than half of the F2 plants had perfectly wild type-looking overall rosette morphologies, we demonstrated a successful interallelic complementation with two different bri1 alleles (Figure 5). The possibility that the genetic recombination between one homologous chromosome with a bri1-120 mutation and the other homologous chromosome with a bri1-301 mutation occurs during the self fertilization of a F1 progeny after the initial cross, resulting in a homologous chromosome without either mutation, cannot be completely ruled out. However, that event seems to occur very rarely, because both mutations are less than 2 Kb apart.
Interestingly, during our analysis, we found significant differences in growth patterns and the BR sensitivities between the bri1-301 plants rescued by the genetic cross with bri1-120 and the bri1-301 plants rescued by the transformation of a BRI1:bri1-120-GFP construct. The overall rosette phenotype of the rescued bri1-301 plants generated by any one of the methods was similar to that of the wild type plants. However, the bri1-301 plants overexpressing BRI1(S399F) due to the transformation of BRI1:bri1-120-GFP showed reduced root and hypocotyl growth in normal growth conditions compared to the wild type plants. Moreover, the BR sensitivities of these plants were similar to the BR sensitivity of bri1-120 based on the inhibition of root growth and CPD expression in response to BL. On the other hand, both root and hypocotyl growth and BR sensitivity almost completely reverted to wild type levels in the plants heterozygous for each mutated allele due to the cross of bri1-301 and bri1-120 (Figure 6). It is possible that although the bri1-301 phenotypes could be rescued by both a transgenic approach, transformation of BRI1:bri1-120-GFP gene, and a genetic cross with bri1-120, different growth pattern in detail and the BR sensitivity between both lines were resulted from the more accumulation of the BRI1-120-GFP proteins in transgenic bri1-301, because expression level of transgene was diverse in each transgenic plant. We also cannot rule out the possibility that the increased amount of BRI1-120-GFP proteins in transgenic bri1-301 affected only rosette development with unknown mechanisms yet. Taken together, these results suggest that observing the shape of the rescued rosette, including the elongated leaves and petioles, is not likely to be a precise way to determine BR sensitivity. A recent publication supported this view. Albrecht et al. (2008)  reported that the overexpression of AtSERK4 in bri1-301 led to the appearance of the rescued compact rosette leaves but did not promote hypocotyl growth. Additionally, we previously showed similar phenomena when BAK1 was overexpressed in bri1-301 . Conventionally, several indicators, such as the conversion of the rosette leaf phenotypes from compact, curled and dark-green elongated, the inhibition and promotion of the root and hypocotyl growth, respectively, the transcriptional inhibition of CPD expression, and the BL-induced accumulation of dephosphorylated BES1, have been used to denote normal BR sensitivity. We believe that each experimental method represents a different degree of BR sensitivity. In that sense, the rescued rosette phenotype does not reflect heightened BL sensitivity as compared to any other method. However, the changes observed in the outward appearance of the weak bri1 mutant phenotype can still be regarded as useful indicators the genetic suppressor screening of bri1 mutants to find additional regulators involved in BR signaling. A BRI1 co-receptor BAK1 , BRS1 (a secreted carboxpeptidase) , BRL1 (BRI1-like1) , BSU1 (a serine/threonine protein phosphatase) , and BEN1 (a dihydroflavonol 4-reductase-like protein) , and recently published TCP1 (a transcriptional modulator of DWARF4, BR biosynthetic gene)  are examples of bri1 suppressors identified in the activation-tagged bri1-5. In addition, the proteins involved in ER quality control were revealed allele-specifically in the genetic suppressor screening of EMS-mutagenized bri1-9 [39–41]. Bri1-301 was also used for the suppressor screening in the activation tagged pools, resulting in the identification of several ATBS genes, including one encoding a bHLH transcription factor that regulates BR signaling (ATBS1)  and YUCCA, which is involved in tryptophan-dependent auxin biosynthesis (ATBS3 to ATBS6) . These results imply that the suppressor screening of bri1 mutant alleles with rosette leaf phenotypes can allow for the mining of genes related to diverse cellular functions in addition to BR signaling. We believe that bri1-120 is a suitable mutant allele for this purpose. We are currently performing genetic screening to search for modulators of bri1-120, to expand the understanding of the functions of this gene.
In summary we demonstrated that the mutant previously referred to as cp3 that shows retarded growth and reduced BR sensitivity is allelic to bri1, and we renamed it bri1-120. The analysis of a point mutation in the 13th LRR that resides before the 70-amino acid island portion of the extracellular domain of BRI1 has indicated that this specific LRR region is critical for proper BRI1 functioning. Using bri1-120 and bri1-301, we revealed that interallelic complementation is able to occur between the extracellular domain mutant allele and the cytoplasmic kinase-inactive mutant allele of BRI1 in vivo.
Plant growth condition
We used Arabidopsis thaliana Landsberg (Ler) as the wild type for the comparison with phenotypic changes of bri1-120 (seeds from CS48) and used Arabidopsis thaliana Columbia (Col-0) as the wild type for the comparison with phenotypes of the transgenic bri1-301 plants. All transgenic plants used here were made by floral dipping into suspensions of Agrobacterium tumerfaciens (GV3101) containing appropriate binary plasmid constructs. Seed sterilization was performed by washing the seeds with 75% ethanol containing 0.05% Tween-20 for 15 minutes, and then washing them twice with 95% ethanol. Sterilized seeds were plated in 1/2 MS (Duchefa) containing 0.8% phytoagar. After stratification at 4°C for 2 days, plates were transferred to a growth room set at 22°C under long-day conditions (16 hours L/8 hours D). To observe the plant phenotypes, the seeds were sown directly onto soil (Sunshine #5) top-layered with fine particles of vermiculite.
Construction of plasmids
The plasmid containing the bri1-120 mutation in BRI1 to express the mutated BRI1 protein, BRI1(S399F), was made by in vitro site-directed mutagenesis using a QuickChange Site-Directed Mutagenesis Kit (Stratagene) with pPZP212-BRI1:BRI1-GFP as a template. The sequences of the primers used were a 5-cgttagatctcagcttcaacaatttctccgg-3' (forward) and 5'-ccggagaaattgttgaagctgagatctaacg-3' (reverse). All of the resulting plasmids were fully sequenced to confirm the presence of the intended changes and the absence of other alterations. After confirmation, the plasmid, BRI1:bri1-120-GFP, was transformed into wild type and bri1-301 plants by Agrobacterium tumefaciens-mediated floral dipping.
Confocal microscopic analysis of the subcellular localization of BRI1(S399F)
The localization pattern of BRI1(S399F) was analyzed by examining the root tips of 5-day-old BRI1:bri1-120-GFP transgenic seedlings using a Zeiss LSM510 Meta confocal microscope with excitation set at 488 nm and a 500-530-nm band-path filter was used to detect the GFP.
Root growth inhibition assay
To determine the BR sensitivity of the plants, the sterilized seeds of interest were placed in a line on 1/2 MS containing 0.8% phytoagar plates supplemented with or without brassinolide (BL) at the indicated concentrations. The seeds of the different plants of interest were seeded in the same plate to minimize ambient differences. Three sets of plates were plated vertically and grown for 10 days at 22°C under long-light conditions (16 hours L/8 hours D) for root elongation. Root lengths were measured for 20-30 seedlings in each line. To determine the hormone sensitivity of bri1-120, we added 20 μM of IAA, GA, kinetin, and ACC and 50 μM of JA to 1/2 MS MS plates and processed them the same way. All of the chemicals were purchased from Duchefa Biochemie except IAA (Sigma Aldrich) and BL (Synthchem. Inc.) All experiments were repeated twice.
We grew the sterilized seeds of interest on the 1/2 MS (Duchefa) containing 0.8% phytoagar plates supplemented with or without brassinolide (BL) for 10 days and extracted total RNA from each seedling. For the northern hybridization, the total RNA was run on a formaldehyde-containing 1% agarose gel, blotted onto a nylon membrane (GE Healthcare) and hybridized with the 32P-labeled CPD probe (32α-P-dCTP, 10 mCi/mol, IZOTOP) at 42°C in a hybridization solution (1M NaCl, 1% SDS, 1% dextran sulfate (Sigma Aldrich), and 50% formamide). For the RT-PCR analysis, the RNA was treated with RNase-free RQ1 DNases (Promega), and the first-strand cDNA was synthesized using the SuperscriptIII-MMLV reverse transcriptase (Invitrogen) and oligo d(T15) primer. The same aliquot of first-strand cDNA was used as a template in the second polymerase chain reaction, in which the CPD transcript was amplified for 23 cycles with the primers CPD-RTF: 5'-gccttcaccgcttttctcctcctc-3' and CPD-RTR: 5'-atttgacggcgagagtcatgatcg-3'.
Confirmation of BRI1 expression by RT-PCR analysis
RNAs were purified from the seedlings grown for two weeks on 1/2 MS plate, and treated with RNase-free RQ1 DNase (Promega). First-strand cDNA synthesis was performed using the SuperscriptIII-MMLV reverse transcriptase (Invitrogen) according to manufacturer's protocol. Second step of polymerase chain reactions were performed with the same aliquot of first-strand cDNA as a template. Polymerase chain reaction was as followings: pre-denaturation at 94°C for 4 min., denaturation at 94°C for 30sec., primer-annealing at 52°C for 30 sec., elongation at 72°C for 30 sec. for 22 cycles, and post-elongation at 72°C for 7 min. The primer sequences for detection of endogenous BRI1 expression are 15F7: 5'-tgcgatggatacgcatttaa-3' (forward) and BRI1 3'UTR: 5'-tcggactgacccttagatg-3' (reverse). The primer sequences for detection of transgene-derived BRI1 expression are GFPSEQF: 5'-acaacatcgaagacggcggcgtg-3' (forward) and KH002: 5'-cagtaggattgtggtgtgtgcgc-3' (reverse). The expression of each gene was normalized to β-Tubulin with primers of TUBF 5'-atgcgtgagattcttcacatcc-3' (forward) and TUBR 5'-tgggtactcttcacggatcttag-3' (reverse).
Genotyping of bri1-120 and bri1-301mutations
For the bri1-301 genotyping, the genomic DNA region adjescent to the bri1-301 mutation was amplified in a polymerase chain reaction (PCR) with the primer set 5'-ggaaaccattgggaagatca-3' (forward) and 5'-gctgtttcacccatccaa-3' (reverse) and then digested with DPNII. One of the restriction sites for DPNII in the PCR-amplified fragment is lost in bri1-301, so DNA fragments with different sizes can be distinguished in the 1% agarose gels after electrophoresis. For the bri1-120 genotyping, we PCR-amplified the genomic DNA with specifically designed dCAPS primers 5'- ccgcttcgttgctaacgttagatctaagct-3' (forward) and 5'-ccagttaagattggtacagttacttaaacc-3' (reverse), to generate a HindIII site only in bri1-120. HindIII-digested PCR products were run on a 3% agarose electrophoresis gel. Wild type Col, Ler, bri1-120, bri1-301, and the F1 plants crossed with bri1-120 and bri1-301 were always included in the experiments as controls.
Detection of BRI1 proteins by western blot analysis
Total protein crude extracts were prepared from 3-4 leaves of 3-week-old soil-grown plants with the extraction buffer (50 mM HEPES (pH 7.4), 10 mM EDTA, 0.1% Triton X-100, and a protease inhibitor cocktail (1 tablet/50 mL, Roche)). Equal amounts of total protein were separated by 7.5% SDS-PAGE and blotted onto a PVDF membrane (Bio-Rad) with the BIO-RAD Mini PROTEAN and Criterion systems, respectively. A western blot analysis was carried out with anti-BRI1 antibodies and peroxidase-conjugated secondary antibodies (Goat anti-rabbit IgG, Pierce). Protein bands were visualized with an ECL plus western blotting detection system (GE Healthcare).
This work were supported by the Korean Science and Engineering Foundation (grant # R01-2007-000-20074-0 to K.H.N.), by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant # 2010-0022823 to K.H.N.) and by the National Institute of Health Grant (GM060519 to J.L).
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