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First published online September 3, 2004; 10.1104/pp.104.041665 Plant Physiology 136:2747-2761 (2004) © 2004 American Society of Plant Biologists
Two Transcription Factors Are Negative Regulators of Gibberellin Response in the HvSPY-Signaling Pathway in Barley Aleurone[w]Commonwealth Scientific and Industrial Research Organisation Plant Industry, Canberra, Australian Capitol Territory 2601, Australia
SPINDLY (SPY) protein from barley (Hordeum vulgare L. cv Himalaya; HvSPY) negatively regulated GA responses in aleurone, and genetic analyses of Arabidopsis thaliana predict that SPY functions in a derepressible GA-signaling pathway. Many, if not all, GA-dependent responses require SPY protein, and to improve our understanding of how the SPY signaling pathway operates, a yeast two-hybrid screen was used to identify both upstream and downstream components that might regulate the activity of the HvSPY protein. A number of proteins from diverse classes were identified using HvSPY as bait and barley cDNA libraries as prey. Two of the HvSPY-interacting (HSI) proteins were transcription factors belonging to the myb and NAC gene families, HSImyb and HSINAC. Interaction occurred via the tetratricopeptide repeat domain of HvSPY and specificity was shown both in vivo and in vitro. Messenger RNAs for these proteins were expressed differentially in many parts of the barley plant but at very low levels. Both HSImyb and HSINAC inhibited the GA3 up-regulation of  -amylase expression in aleurone, both were activators of transcription in yeast, and the green fluorescent protein-HSI fusion proteins were localized in the nucleus. These results are consistent with the model that HSI transcription factors act downstream of HvSPY as negative regulators and that they in turn could activate other negative regulators, forming the HvSPY negative regulator-signaling pathway for GA response.
GAs are essential for a number of processes, including gene expression in cereal aleurones, seed germination, elongation growth, and flowering. Significant advances have been made in understanding how GAs exert these effects, especially in the area of GA biosynthesis. Enzymes of the biosynthetic pathway have been studied by the identification of mutants and by the cloning and the characterization of the genes and enzymes involved, including aspects of regulation (Hedden and Kamiya, 1997  ; Olszewski et al., 2002). By contrast, the area of GA signaling remains less well characterized. In the aleurone, the receptor is predicted to be located on the plasma membrane (Hooley et al., 1991; Gilroy and Jones, 1994.), and models for signaling pathways have been proposed based on functional and genetic analyses with a number of protein components (Richards et al., 2001; Olszewski et al., 2002). These factors have been identified, cloned, and characterized in both monocotyledonous and dicotyledonous species. These factors could be classified into positive and negative regulators of GA signaling; the functionality of a factor was assigned based on its effects on the final GA-induced responses. The positive regulators include rice (Oryza sativa) D1 (-subunit of the trimeric G protein complex; Ashikari et al., 1999; Fujisawa et al., 1999), F-box proteins in rice (GID2) and in Arabidopsis (Arabidopsis thaliana; SLEEPY1 or SLY1; McGinnis et al., 2003; Sasaki et al., 2003), and barley (Hordeum vulgare) GAmyb (Gubler et al., 1995).
The negative regulators are Arabidopsis SPINDLY (SPY; Jacobsen and Olszewski, 1993
The first GA-signaling gene cloned was Arabidopsis SPY (Jacobsen et al., 1996
The cloning of animal UDP-N-acetyl-D-glucosamine:protein
The TPR domain is important for Arabidopsis SPY protein function, since a number of spy alleles contained mutations only in the TPR domain (Tseng et al., 2001 Protein factors in the GA-signaling pathway have been identified by genetic screens and subsequent cloning in Arabidopsis and in other species using the Arabidopsis sequences. Further genetic screens identified mainly alleles of known mutants, suggesting that the identification of additional factors may not be accomplished by genetic screens that look for a limited number of obvious GA response phenotypes resulting from mutations. In order to overcome the limitations of genetic screens, an alternative approach was applied in this study, which used the yeast two-hybrid (Y2H) screen to identify proteins that would interact with the HvSPY protein. The GA-signaling pathway would require protein components to interact for the signal to be transduced from the receptor to downstream components. The presence of the TPR domain in the HvSPY protein and the pleiotropic effects of spy mutations would suggest that there may be many HvSPY-interacting (HSI) proteins for a number of SPY pathways. Some of these proteins would function in the GA-signaling pathway, as upstream components which may regulate HvSPY activity or as downstream components which may receive signals from HvSPY. This article reports the construction and screening of Y2H libraries in barley, the identification of HvSPY interacting proteins, and the functional and molecular characterization of two transcription factors. This study shows that these transcription factors are novel negative regulators of GA response in barley aleurone.
Identification of HvSPY-Interacting Proteins by Y2H Screen To clone proteins that specifically interact with HvSPY, a Y2H screen was carried out using the whole HvSPY protein as bait and barley two-hybrid cDNA libraries as prey (see "Materials and Methods"). In total, 8.3 million colony-forming units were screened to identify 41 clones corresponding to 15 cDNAs (Supplemental Tables I and II, available at www.plantphysiol.org).
Five cDNA clones (clones 354, 597, 1132, 1342, and 1356) encoded the same overlapping sequence, with clone 1132 having the longest sequence. The database search using the deduced amino acid sequence showed that it belonged to the R2R3 class of myb transcription factors (Fig. 1A). The sequence, HSImyb for HvSPY-interacting myb, encodes a 388-amino acid polypeptide with the R2R3 myb domain located toward the amino end of the protein. The database search identified many plant R2R3 myb sequences with the highest sequence identity for a homologous rice gene at 97.2% using the myb domain alone; however, no closely related sequence could be identified using the C-half of the protein sequence except for the rice homolog. The absence of monocot expressed sequence tags (ESTs) suggests a low abundance of expression for this class of myb genes (see below). When the Arabidopsis genome database was searched with the amino acid sequences from the myb domain and non-myb domain separately, AtMyb83 was identified with the highest identity score for the myb domain. Significantly, it identified a motif at the carboxyl end that is conserved in myb genes between cereals (barley and rice) and dicotyledonous plants (Arabidopsis and soybean [Glycine max]), which is likely to reflect functional conservation (Fig. 1A).
Clone 531 encodes a member of the NAC gene family of transcription factors (Fig. 1A). The N-terminal half of the open reading frame encodes the highly conserved NAC domain. There is no in-frame stop codon before the proposed start codon in the fusion clone, thus we cannot be certain that this clone contains the complete open reading frame. However, comparison with a number of sequences in the NAC gene family suggests that clone 531 contains a complete open reading frame, since the number of amino acid residues before the subdomain A of the NAC domain is relatively short for NACs, which is less than 20 amino acid residues. It is even possible that the translation start may be from the second ATG, since there are 43 amino acid residues before the subdomain A for the proposed open reading frame. Based on these comparisons, it is proposed that clone 531 encodes a polypeptide of 391 amino acid residues in length (Fig. 1A) and that the clone be named as HSINAC (HvSPY-interacting NAC). The amino-half is the well-defined NAC domain with 166 amino acid residues, and it consists of five (A–E) subdomains (Aida et al., 1997 ; Kikuchi et al., 2000). The domain is highly conserved among a number of genes across genera, but it is found only in plants (Riechmann et al., 2000). BLAST searches have identified many sequences with the NAC domain from both dicotyledonous and monocotyledonous plant species such as Arabidopsis and rice. When the search was carried out using the sequence unique to HSINAC (see below), no gene was identified, but 12 cereal ESTs were found. It thus appears that this part of the sequence may be unique to monocots. The identification of relatively few ESTs suggests that the transcript may also be expressed at very low levels, and this was later confirmed (see below). To estimate gene copy numbers of HSImyb and HSINAC, genomic Southern-blot experiments were carried out. When a filter containing Himalaya genomic DNA digested with five restriction enzymes was hybridized with the non-MYB fragment of HSImyb, a single band in each of the digests was detected (Fig. 1B). With the non-NAC sequence from HSINAC used as a probe, single hybridizing bands were detected in all lanes except for EcoRI (Fig. 1C). The presence of two bands for the EcoRI-digested DNA is in agreement with the presence of an internal EcoRI site in the non-NAC region of HSINAC. There are also two HindIII sites in the non-NAC region, however, two smaller fragments are most likely to have run off the gel and the largest 5' fragment is the hybridizing band. Therefore, these results suggest that both HSImyb and HSINAC are unique genes in the barley genome.
To investigate the specificity of the interaction between HvSPY and the HSI proteins, and to examine which domain of the HvSPY protein was responsible for the interaction, additional interaction analyses were carried out with the Y2H system. The relative strengths of interactions were quantified using the lacZ reporter gene system with o-nitrophenyl
HvSPY Interacts with HSImyb and HSINAC in Vitro To demonstrate that HvSPY interacts with HSImyb and HSINAC in another system, their interactions were tested in vitro using recombinant proteins. Recombinant HvSPY, HSImyb, and HSINAC were expressed in Escherichia coli with 6x His tag, and the expression conditions were optimized to produce small but soluble amounts of HvSPY, HSImyb, and HSINAC. HvSPY was purified from soluble fractions of E. coli protein extract using the His tag. The interaction was tested by incubating the purified HvSPY and the soluble E. coli proteins containing the recombinant HSImyb or HSINAC. The protein complex was immunoprecipitated with a polyclonal antibody raised against the HvSPY TPR domain. The immunoprecipitated complex was then examined by SDS-PAGE, followed by staining to show the protein profile, as well as by anti-His western-blot analysis for the recombinant proteins (Fig. 3). For both HSImyb and HSINAC, the stained gel showed that there were very few proteins in the immunoprecipitated complex, except for the major band of rabbit IgG, which was isolated by the Protein A. Most of the soluble E. coli proteins were in the supernatant fraction. Anti-His western-blot analysis showed that HvSPY coprecipitated with HSImyb (Fig. 3A) and HSINAC (Fig. 3B) and there was little HvSPY in the supernatant. The excess HSImyb and HSINAC were also present in the supernatant. The many smaller bands in the HSINAC western blot were shorter peptides of the HSINAC. These results show that in addition to the interactions in vivo as hybrid proteins in yeast cells, both HSImyb and HSINAC interacted with HvSPY in vitro.
The Two Transcription Factors Are Activators of Transcription In order to investigate whether the two transcription factors functioned as activators or repressors of transcription, HSImyb and HSINAC proteins were tested in the yeast one-hybrid transcriptional activation assay. These proteins were isolated as fusion proteins with the GAL4 activation domain (AD), so the activation of the reporter genes in Y2H could have been by the GAL4 AD or an AD of the transcription factors. When HSI proteins were fused to the GAL4 DNA binding domain (BD), they would be placed proximal to the transcription start site of the reporter genes. If the HSI proteins had transcriptional activation activity, the activity could be detected by the activities of the reporter genes HIS3 and lacZ (Fig. 4A).
When the yeast cells were transformed with the vector containing the GAL4 DNA BD alone, the cells failed to grow in the absence of His and there was no blue color in the lacZ assay (Fig. 4B). By contrast, when HSImyb was fused to the GAL4 DNA BD, cells containing the plasmid grew without His and also developed a blue color from the  -galactosidase encoded by lacZ. The GAL4 DNA BD-HSINAC fusion protein was also capable of activating reporter genes HIS3 and lacZ. Thus, both transcription factors, HSImyb ad HSINAC, were transcription activators in yeast. By contrast, the GAL4 DNA BD-HvSPY fusion protein did not activate the reporter genes as previously shown when HvSPY was tested for its ability to autoactivate. One of the HSImyb clones (clone 597) was isolated, even though it was out of frame with the GAL4 AD protein. The original clone must have been able to activate the reporter genes causing its isolation, and this provides additional evidence that HSImyb is an activator of transcription. Barley SLN1 is a negative regulator of GA signaling and is a member of the DELLA class of proteins that includes Arabidopsis GAI and RGA. SLN1 was tested for its role in transcriptional regulation in the yeast system. Similarly to HSImyb and HSINAC, SLN1 was also able to activate both reporter genes, HIS3 and lacZ, showing that it too was a transcriptional activator.
To investigate where HSIs are expressed in barley, northern-blot experiments were carried out using those cDNA fragments containing sequences unique in detecting HSImyb and HSINAC (Fig. 1A). However, no signal could be detected in total RNA samples from different parts of the plant (data not shown), presumably due to a low abundance of mRNAs. Alternatively, semiquantitative duplex reverse transcription (RT)-PCR analyses were carried out with a pair of gene-specific primers and an internal standard, EF1
HSImyb and HSINAC Are Negative Regulators of GA Response in Barley Aleurone
The bait protein, HvSPY, is a negative regulator of GA signaling. The HSI proteins could function as negative or positive regulators, or perhaps not be involved in GA signaling at all. To investigate these possibilities, the HSI proteins were functionally tested in the barley aleurone transient expression system. In these assays, the effects of HSI proteins on the GA3-induction of high-pI
When no effector protein was expressed (UbiCass), the promoter activity increased 5.6-fold in response to GA3 (Fig. 6B), showing the GA responsiveness of the system. When HSImyb protein was overexpressed under the control of a maize ubiquitin promoter, this increase was completely abolished and the activity was lower than the control background value. Similarly, the overexpression of HSINAC also blocked the GA3-induced promoter activity, but not as effectively as the overexpression of HSImyb. These results show that both HSImyb and HSINAC can inhibit the GA response in aleurone.
The inhibitory effect may have been specific to the GA response or may have been a general effect on any gene. To differentiate between these two possibilities, further functional assays were carried out using promoters from two genes, an abscisic acid (ABA)-induced gene, barley dehydrin (Dhn), and a constitutively expressed gene, rice actin (Act). Effects on the promoters were measured using the reporter gene GUS (Fig. 6A). The promoter activity of the Dhn gene increased more than 30-fold in response to ABA (Fig. 6C). When HSImyb was expressed, the activity increased significantly by 50%, while it decreased slightly from the overexpression of HSINAC, but this decrease was not significant at 99% confidence level. The promoter activity of the Act gene increased by 35% in response to GA3, and the increase was significant (Fig. 6D). These results show that the Act gene expression is not completely constitutive. The promoter activities did not increase in additional control experiments when HSImyb and HSINAC were overexpressed in the absence of hormones (data not shown). Together, these results show that the inhibitory function of HSImyb and HSINAC protein is specific to the GA up-regulated
Transcription activators are expected to be localized in the nucleus in order to function. To investigate if these HSI transcription activators were localized in the nucleus, and also to determine whether their protein abundance or their localization changed in response to hormones, the expression of green fluorescent protein (GFP)-HSI fusion proteins was examined in barley aleurone protoplasts that had been transfected with GFP-HSI constructs under the control of a maize ubiquitin promoter (Fig. 7A). The expression of GFP alone and of the GFP-HvSPY fusion protein were also examined. These GFP-fusion HSI proteins were functionally active in the barley aleurone layer transient assays, which showed results similar to those obtained with nonfusion proteins. The promoter activity of high-pI
Barley aleurone protoplasts contain three major organelles, which are the nuclei, protein storage vacuoles containing smaller protein storage bodies, and the cytoplasm in between (Fig. 7B). Protoplasts were examined for GFP expression 18 h after the transfection. In the emission range between 500 and 600 nm, strong signals were detectable in the nucleus when GFP-HSINAC was overexpressed. There were also smaller spots of fluorescence in the protein storage vacuoles (Fig. 7E). The background autofluorescence was detected in the emission range between 600 and 770 nm (Fig. 7F), and small fluorescent spots were present in the protein storage vacuoles. These two images were overlaid to identify the GFP-specific signal. The signal from the nucleus was the only GFP fluorescence and the signal from the protein storage vacuoles was the autofluorescence signal (Fig. 7G). The analysis of many transfected protoplasts showed that GFP-HSINAC was expressed exclusively in the nucleus.
When GFP-HSImyb was overexpressed in the protoplasts, the protein was also localized in the nucleus, but not exclusively (Fig. 7, H–J). GFP fluorescence was also present in the cytoplasm. When a large number of protoplasts expressing GFP-HSImyb were examined in replicated transfection experiments, there was a correlation between the localization and the stages of protoplast aging (Bush et al., 1986 GFP-HvSPY was present in the nucleus and in the cytoplasm at very low levels (Fig. 7C). No changes were detected in cellular localization or abundance of expression by hormone treatments (data not shown). When GFP alone was expressed, it was expressed to very high levels in the nucleus and in the cytoplasm at all times (Fig. 7D). To investigate whether the localization and/or the abundance of HSI proteins are regulated by GA3 and/or ABA, the expression of GFP-HSI fusion protein was examined following various hormone treatments. For GFP-HSINAC, when the transfected protoplasts were incubated without any hormone (C), 3.06% ± 0.52% of the cells showed detectable GFP fluorescence in the nucleus (Fig. 8). When the cells were incubated in 1 µM GA3, 2.13% ± 0.21% of the cells showed detectable GFP, a significant decrease compared to the control cells. The decrease in the number of cells with detectable GFP fluorescence was quite rapid, as cells incubated without hormone for 15 h followed by 3 h in GA3 (C-GA) also had significantly fewer cells expressing GFP, 2.16% ± 0.28%. When treated with ABA, the proportion of cells with the GFP fluorescence was 3.18% ± 0.28%, and this was similar to the control cells. The GFP expression was exclusively in the nucleus in all treatments. By contrast, the proportion of cells expressing GFP-HSImyb did not change significantly in response to hormone treatments. The results with GFP-HSImyb also show that the decrease in the expression of HSINAC in GA3-treated cells is not a general proteolysis response. Were this the case, we would have seen a decreased expression of several GFP-fusion proteins.
SPY and HvSPY proteins are negative regulators of GA signaling based on genetic evidence and functional analyses in Arabidopsis and in barley (Jacobsen and Olszewski, 1993 ; Robertson et al., 1998; Richards et al., 2001; Olszewski et al., 2002). Arabidopsis spy mutants show GA-independent seed germination and growth, faster transition to flower, and partial male sterility (Jacobsen and Olszewski, 1993). Unlike other GA-signaling components such as GAI and RGA, SPY is the only factor to influence all known GA-dependent processes as observed in spy mutants. In addition, spy mutants also show phenotypes that may not be GA related, suggesting that SPY proteins may be involved in regulating response pathways other than the GA pathway (Swain et al., 2002). Consistent with this, ABA up-regulated dehydrin promoter activity was increased by an overexpression of HvSPY in barley aleurone; however, the action of HvSPY was independent of the ABA pathway (Robertson, 2003). Mutations at the SPY locus have pleiotropic effects in Arabidopsis, and these observations are consistent with the idea that the protein is essential for many aspects of normal plant development. The diversity of affected responses suggests that SPY protein is a hub for many signaling pathways, some involving GA, others not. Such a signaling hub would be receiving and transmitting signals between many upstream and downstream factors as a part of SPY function. In agreement with the role of SPY in signaling, many HSI factors were identified in the Y2H. These HSI proteins are expected to define components upstream and downstream of HvSPY, which could be receiving signals from diverse input ligands and conveying the signal downstream. Most of the identified factors, including the two transcription factors described in this article, were previously unknown. Therefore, the use of the Y2H screen has proved a useful method, complementary to mutant screens, in isolating novel components.
SPY proteins are members of a protein family with TPR motifs that are repeats of 34 semiconserved amino acid residues (Goebl and Yanagida, 1991
GA-signaling components include enzymes (SPY, SLY/GID), heterotrimeric G proteins (G
Several myb transcription factors have been shown to be involved in regulating GA response in cereal aleurone. The myb factor described in this article, HSImyb, is a R2R3 myb, and functional assays have shown that it acts as a negative regulator of GA response. The well-known positive regulator of aleurone GA response is GAmyb with a R2R3 DNA BD (Gubler et al., 1995
The fact that these transcription factors were identified by protein-protein interaction would strongly suggest that they are downstream factors of HvSPY. If they were upstream factors regulating HvSPY activity, the interaction between the transcription factors and HvSPY would have been through the promoter of HvSPY gene. The sequence similarity of HvSPY and animal OGTs indicates that HvSPY is likely to be a plant OGT, and it is known that transcription factors are substrates for GlcNAc modification. Recent studies have shown that GlcNAc modification regulated the activities of transcription factors. For example, GlcNAc modification of transcription factors has been found both to decrease transcriptional activities of mER-
Transcription factors regulate gene expression through modulating transcription. They can be repressors or activators of transcription. R2R3 myb proteins are shown to be both transcriptional activators and repressors (Stracke et al., 2001
How is it that the two transcription factors both function as negative regulators of GA response? It is possible that different downstream negative regulators may be the targets of these transcription factors, thus they would define steps of divergence in signaling. This possibility is consistent with our observation of no change in
In addition to HvSPY and HSI transcription factors, two other negative regulators of
Transcription factors are active only when they are in the nucleus, and these proteins are expected to contain the nuclear localization signal (NLS) enabling their transport into the nucleus, following translation in the cytoplasm. With HSImyb, NLS is predicted to be KR.{45}KKRL, by the PredictNLS program (Cokol et al., 2000
Using the same approach, GFP-HvSPY was localized both in the nucleus and the cytoplasm, in agreement with the animal OGTs (Kreppel et al., 1997 In conclusion, the Y2H screen identified a number of candidate proteins for inclusion in the HvSPY-signaling pathway. The identification of many novel components confirms the advantage of the Y2H approach, which would complement the genetic-screening approach, in advancing our understanding of GA signaling through the identification of its components. This article also describes two HSI transcription factors regulating GA response in aleurone. The results predict signaling pathways composed of negative regulators and that this type of pathway may be an important characteristic of the regulation of GA responses.
Y2H cDNA Library Construction Total RNA was extracted from the first leaf blade of 5- to 7-d-old seedlings of barley (Hordeum vulgare L. cv Himalaya) that were germinated in petri dishes. The blade was frozen and stored in liquid N2. Poly(A)-RNA was isolated using Poly(A) Quik (Stratagene, La Jolla, CA) according to the manufacturer's instructions. cDNA was synthesized from 5 µg poly(A)-RNA using a cDNA synthesis kit (Stratagene) according to the manufacturer's instructions, except that double-stranded cDNA was size fractionated on a gel and cDNAs larger than 0.6 or 0.8 kb were purified from the gel to make two cDNA libraries, V1 and V2, respectively.
Lambda ACT2 vector was obtained from Dr. S. Elledge (Baylor College of Medicine, Houston) and propagated in Escherichia coli LE392. Lambda DNA was isolated using Qiagen tip 100 columns (Qiagen, Hilden, Germany), digested with EcoRI and XhoI, and treated with calf intestine phosphatase. Following the ligation of lambda arms and the cDNA, the libraries were packaged using MaxPlax packaging extract (Epicentre Technologies, Madison, WI) according to the manufacturer's instructions. Primary barley Y2H libraries in
About 5 x 107 pfu of each of the two Y2H libraries were mass excised from the
Prior to screening for HSI proteins, the bait constructs and GAL4 DNA BD vector plasmid (pAS2-1) in the Matchmaker 2 system (CLONTECH, Palo Alto, CA) were transformed into Y187 and Y190 yeast hosts according to the TRAFO procedure (Agatep et al., 1998
The yeast cells Y190 carrying HvSPY (whole) as a bait [pAS::HvSPY (whole)/Y190] were transformed with the cDNA library in pACT2 by the TRAFO procedure (Agatep et al., 1998
HIS3 positive colonies were tested for lacZ reporter gene activity by colony-lift filter Preliminary sequence analysis showed that the majority of the inserts encoded Rubisco small subunit (RSU) isozymes. These sequences were in frame but had a nonspecific interaction for the lacZ reporter gene (data not shown). Therefore, it was necessary to identify clones encoding RSU among the HIS3 and lacZ positive colonies and this was carried out by duplex PCR reactions using yeast cell lysates as a template, which contained only the prey plasmid. Briefly, yeast cells carrying the prey plasmid were grown on SD –Leu plates and were lysed in water by freeze/thaw in liquid N2. An aliquot of the lysate was directly used in plasmid PCR reactions to amplify inserts and RSU, if present, using vector primers and an RSU-specific forward primer. When two bands were produced, with the smaller one around 400 to 800 bp, these clones were designated as RSU and discarded. The remaining clones that produced single bands, now designated as HvSPY-HSI clones, were studied further.
The prey cDNA plasmids were isolated from yeast cells, transformed into E. coli XL1-blue MRF' and plasmids were isolated from E. coli for sequence analysis. Those clones with long open reading frames, which were in frame with the GAL4 AD protein, were tested for interaction specificity. HSI clones in Y190 were mated with Y187 cells containing different bait plasmids, which would encode negative control proteins [pAS2-1/Y187 and pLAM5'1 (laminC)/Y187], the positive control protein [pAS::HvSPY (whole)/Y187] and test proteins [pAS::HvSPY (TPR)/Y187 and pAS::HvSPY (C-half)/Y187]. The mated samples were plated on SD –Leu, -Trp plates to select for diploid cells carrying both the prey (cDNA) and bait plasmids. At least three diploid cells from each mating were tested for HIS3 and lacZ reporter gene activities. Those clones with specific interaction were selected and grouped into sequence family groups when they had overlapping sequences. A quantitative
The vector plasmid pAS2-1 and plasmid constructs carrying GAL4 DNA BD-HSI protein fusions (pAS2-1::HSImyb and pAS2-1::HSINAC) and GAL4DNABD-HvSPY (pAS2-1::HvSPY) were transformed into yeast Y190 cells (TRAFO) and the transformants were tested on SD –Trp, -His, +3-AT (40 mM) plates for the HIS3 reporter gene, and by lacZ filter assays (YPH) using colonies growing on SD –Trp plates.
The transient assays were carried out essentially as described (Robertson et al., 1998
Barley aleurone protoplasts were prepared from Himalaya barley (1998 harvest at Pullman, Washington University) as described (Robertson et al., 1995
Barley genomic Southern experiments were carried out as described (Robertson et al., 1998
Barley plant parts were harvested from immature seedlings and mature Himalaya plants. They were stored frozen in liquid N2 and total RNA was isolated as described (Robertson et al., 1998
To produce recombinant proteins, bacterial cells carrying His-tagged HvSPY, HSImyb, and HSINAC (HIS-HvSPY/BL21-RIL, HIS-HSImyb/BL21-RP, and HIS-HSINAC/BL21-RP) were grown at 37°C to mid-log phase. The cultures were then incubated at 20°C and the expression was induced with 0.6 mM isopropylthio- For immunoprecipitation, the purified HIS-HvSPY was incubated with soluble protein extracts from E. coli containing the recombinant HIS-HSImyb or HIS-HSINAC in TBS-NP40 (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1% NP-40) at 4°C for 1 h. HvSPY and associated proteins were precipitated with an antibody raised against the TPR domain of HvSPY and then with protein-A Sepharose (Pharmacia, Uppsala) for 1 h each at 4°C. The precipitates were washed three times with TBS-NP40. Both the precipitates and the supernatant were analyzed by SDS-PAGE and stain or western blot using anti-HIS MAb (Qiagen).
Bait Constructs
GATEWAY Vectors For recombinant protein productions, the region flanked by aatR1 and aatR2 sites in pDEST17 was amplified with primers NarR1 5'-ATTGGCGCCCACCTCGAATCAACAAGT-3' and KpnR2 5'-AAGGTACCTGTTAGCCTCGAATC-3' and Pfx DNA polymerase (Invitrogen). The fragment with ccdB and Cmr genes with attR1 and attR2 sites was cloned into the EheI and KpnI sites of pProEx-1 (Invitrogen) to produce pDEST-ProEx. To modify the effector construct vector UbiCassNotI for the transient assays, the NotI site of pDEST26 was eliminated by digesting the plasmid with NotI, the overhang was filled in using Klenow DNA polymerase (Perkin-Elmer, Foster City, CA), and the resulting blunt ends were ligated using T4 ligase to produce pDEST26 (no Not). The region flanked by attR1 and attR2 sites in pDEST26 (no Not) was amplified using vector primers 5'-ATGGTACCGCGGACCATGGCGTAC-3' and 5'-ATATGAGCTCTCAACCACTTTGTACAAGAA-3', and Pfx DNA polymerase (Invitrogen). The fragment with ccdB and Cmr genes with attR1 and attR2 sites was cloned into the KpnI and SacI sites of Ubi1CassNotI (Z. Li, Plant Industry, Commonwealth Scientific and Industrial Research Organisation), to produce pDEST-Ubi1CassNotI.
For the protein localization study in aleurone protoplasts, a vector was constructed to clone GFP fusion proteins under the control of a constitutive promoter, maize ubiquitin. An enhanced GFP sequence (Chui et al., 1996
pENTR Constructs
Expression Constructs For one-hybrid analysis, HvSPY, HSImyb, and HSINAC open reading frames in pENTR constructs were cloned into pDEST-AS2-1 by LR clonase (Invitrogen) to produce pAS2-1::HvSPY, pAS2-1::HSImyb, and pAS2-1::HSINAC, respectively. Plasmid DNAs were prepared by Qiagen mini-spin columns for restriction enzyme analysis and transformation into yeast. For transient assay effector constructs, open reading frames of HSImyb and HSINAC in pENTR constructs were cloned into pDEST-Ubi1Cass by LR clonase (Invitrogen) to produce Ubi::HSImyb and Ubi::HSINAC, respectively. Effector plasmid DNAs for transfection were prepared by Qiagen maxi column. For protein localization studies, open reading frames of HvSPY, HSImyb, and HSINAC in pENTR constructs were cloned into pDEST-UbiGFPCassNotI by LR clonase (Invitrogen) to produce Ubi::GFP-HvSPY, Ubi::GFP-HSImyb, and Ubi::GFP-HSINAC, respectively. The resulting expression constructs were prepared using Qiagen mini-spin columns for restriction enzyme analysis. Plasmids used for transfection into aleurone layers and protoplasts were prepared by Qiagen maxi columns. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY672068 and AY672069 for HSImyb and HSINAC, respectively.
We thank Dr. S. Elledge (Baylor College of Medicine) for  ACT2 vector, Dr. Gietz for yeast Y190 and advice on yeast transformation, Drs. F. Gubler and Z. Li for SLN cDNA and the UbiCassNotI, Jan van de Velde for results in Figure 6, and Judy Radik and Robyn East for technical assistance. Received February 29, 2004; returned for revision June 20, 2004; accepted June 28, 2004.
[w] The online version of this article contains Web-only data.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041665. * E-mail masumi.robertson{at}csiro.au; fax 61 (2) 6246–5000.
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