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First published online September 15, 2009; 10.1104/pp.109.144030 Plant Physiology 151:1207-1220 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Large-Scale Analysis of Putative Soybean Regulatory Gene Expression Identifies a Myb Gene Involved in Soybean Nodule Development1,[W],[OA]Division of Plant Sciences, National Center for Soybean Biotechnology (M.L., K.T., A.H.-S., K.P., S.B., H.T.N., G.S.), and Digital Biology Laboratory, Computer Science Department (T.J., D.X.), C.S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211; and Donald Danforth Plant Science Center, St. Louis, Missouri 63132 (R.E.F., C.G.T.)
Nodulation is the result of a symbiosis between legumes and rhizobial bacteria in soil. This symbiosis is mutually beneficial, with the bacteria providing a source of nitrogen to the host while the plant supplies carbon to the symbiont. Nodule development is a complex process that is tightly regulated in the host plant cell through networks of gene expression. In order to examine this regulation in detail, a library of quantitative reverse transcription-polymerase chain reaction primer sets was developed for a large number of soybean (Glycine max) putative regulatory genes available in the current expressed sequence tag collection. This library contained primers specific to soybean transcription factor genes as well as genes involved in chromatin modification and translational regulation. Using this library, we analyzed the expression of this gene set during nodule development. A large number of genes were found to be differentially expressed, especially at the later stages of nodule development when active nitrogen fixation was occurring. Expression of these putative regulatory genes was also analyzed in response to the addition of nitrate as a nitrogen source. This comparative analysis identified genes that may be specifically involved in nitrogen assimilation, metabolism, and the maintenance of active nodules. To address this possibility, the expression of one such candidate was studied in more detail by expressing in soybean roots promoter β-glucuronidase and green fluorescent protein fusions. This gene, named Control of Nodule Development (CND), encoded a Myb transcription factor gene. When the CND gene was silenced, nodulation was reduced. These results, associated with a strong expression of the CND gene in the vascular tissues, suggest a role for CND in controlling soybean nodulation.
Nodulation involves the intimate relationship between soil bacteria (rhizobia) and legume plants, which results in the formation of a novel organ, the nodule, in which the bacteria reside and provide a steady source of nitrogen to the plant. Control of the initial host-symbiont interaction and subsequent nodule development is complex and appears to require a continuous exchange of chemical signals between the partners. For example, the compatible symbiont initially recognizes flavonoid molecules released by the host. This, in turn, leads to the production of a specific lipochitin nodulation signal (Nod factor) that is required for rhizobial infection and also triggers the development of the nodule primordium (Oldroyd and Downie, 2008  ).
Primarily through an examination of plant mutants defective in nodulation, the basic steps in the Nod factor signaling pathway have been elucidated (Oldroyd and Downie, 2008
The expression of some of these key TF genes is affected by rhizobial inoculation. For example, NSP2, ERN, and NIN expression was induced in response to root inoculation by the compatible symbiont (Kalo et al., 2005
Due to the low global expression level of many TF genes, DNA microarrays lack the sensitivity to accurately measure their expression (Czechowski et al., 2004 In this study, by mining public EST databases, 2,586 putative soybean (Glycine max) regulatory genes (i.e. TF genes as well as those involved in chromatin modification and translational regulation) were identified. PCR primers were designed and validated to create 1,149 specific primer sets, which can be used to specifically quantify gene expression by qRT-PCR. To demonstrate the usefulness of this resource, the expression of these genes was profiled during soybean nodule development by comparing nodulated and nodule-free roots. The results showed that 126 putative regulatory genes were differentially regulated during nodulation, including putative orthologs of MtHAP2.1 and MtERN. Most of these genes were regulated at the later stages when active nitrogen fixation is occurring. Further analysis distinguished those putative regulatory genes strictly regulated during nodulation, after nitrogen starvation, and/or those responsive to the addition of nitrate as a nitrogen source. Control of Nodule Development (CND), a Myb TF gene, was studied in more detail through the construction of GUS and GFP promoter fusions. Silencing of CND resulted in a decrease in nodule numbers. Our data clearly indicate that a specific, temporal pattern of putative regulatory gene expression correlates with nodule development.
Identification of 2,586 Putative Soybean Regulatory Genes in Public EST Databases
To begin compiling a library of putative regulatory genes in soybean, the sequences of predicted TF genes in Arabidopsis (2,290 and 2,250 predicted TF transcripts from the Database of Arabidopsis Transcription Factors [http://datf.cbi.pku.edu.cn/] and the Plant Transcription Factor Database [http://plntfdb.bio.uni-potsdam.de/v2.0/index.php?sp_id=ATH], respectively) were used for similarity searches to identify homologous soybean genes. Because the soybean genome sequences were not available at the time of the design of the primer sets, the identified sequences were compared against the National Center for Biotechnology Information G. max Unigene database (with BLAST e-value
To accurately quantify the expression levels of the identified putative regulatory genes, specific qRT-PCR primers were designed from each of the putative regulatory gene sequences using PRIMEGENS (Xu et al., 2002
The lack of specificity of the other primer sets is likely the result of the large number of soybean paralogs that arose from the whole genome duplications detected from analysis of the soybean genome (Schlueter et al., 2004
In addition to their specificity, primer efficiency is another important characteristic to validate suitable qRT-PCR primer sets. Theoretically, under ideal conditions, the number of amplicons should increase by two after each PCR cycle. Based on an earlier study (Libault et al., 2007 ), only a few primer sets are highly efficient across a set of tested conditions. In this study, the efficiency of each putative soybean regulatory gene primer set was measured using the LinRegPCR software (Ramakers et al., 2003). The average primer set efficiency was calculated when gene expression was detected in at least 20 out of the 24 different cDNA samples tested. Out of the 1,149 primer sets used, 1,043 were retained and exhibited an average primer efficiency of 1.97 ± 0.05 (Supplemental Table S3). None of the primer sets displayed an efficiency of less than 60%, and 953 primer sets (91.4% of the library) had an efficiency of greater than 90% (Supplemental Fig. 1C). These data provide confidence that the primer set library is of high specificity and can be used to accurately quantify putative regulatory gene mRNA expression levels.
The putative regulatory gene primer set library was first used to profile gene expression during nodule development. To identify regulated genes during nodulation, we compared gene expression in soybean roots inoculated with Bradyrhizobium japonicum and mock-inoculated roots at different stages of nodule development (4, 8, and 24 d after inoculation [DAI]). In our conditions, at 4 DAI, nodules were not visible but extensive root hair cell deformation could be seen, indicative of a strong plant response to B. japonicum. At 8 DAI, nodule primordia emerged, while nodules were fully developed at 24 DAI (i.e. leghemoglobin was present in the nodule [pink-red color], and nitrogen fixation occurred based on the absence of leaf chlorosis). Overall analysis of the expression pattern of soybean regulatory genes in mock-inoculated roots highlighted strong correlations between 4 and 8 DAI (Fig. 1B). A lower correlation between 4- to 8-DAI and 24-DAI mock-inoculated roots suggested that gene expression was modified during root development. This analysis suggests that the regulatory gene expression profiles were well established and stably maintained during the first stages of root development. During nodulation, the putative soybean regulatory gene expression profile was not affected during the first days following root inoculation by B. japonicum (i.e. the Euclidian distance between 4- and 8-DAI inoculated and mock-inoculated roots was low) but was strongly disrupted at 24 DAI (Fig. 1B). Such results suggested that a significant number of soybean regulatory genes were specifically expressed in mature nodules but not in developing nodules. On the other hand, based on our experience, mature nodules are plant organs extremely rich in mRNA. Consequently, comparisons between inoculated and mock-inoculated roots between 4 to 8 DAI and 24 DAI might also be explained by a strong dilution of the 4- to 8-DAI nodule mRNA by the overall root system mRNA, a dilution that was limited at 24 DAI when individual nodules can be harvested for analysis. These observations support the notion that a large number of putative regulatory genes were differentially expressed at 24 DAI. We identified for each time point the differentially expressed putative soybean regulatory genes. For example, 126 soybean regulatory genes were differentially expressed during soybean nodule development (P < 0.05 and fold change < 0.5 or > 2; Supplemental Table S4). Among them, 31% (39 genes) were up-regulated, while 69% (87 genes) were down-regulated (Fig. 2 ). As expected based on the calculation of the correlation between the different conditions tested, most of the putative regulatory genes were differentially expressed at 24 DAI (five, 19, and 111 genes were regulated at 4, 8, and 24 DAI, respectively; Fig. 2). Among the 126 genes, only eight genes were found to be differentially regulated at multiple time points of the time course: three CCAAT-Box, one AP2/EREBP, one Myb, one C2H2 Zinc Finger domain, one bHLH, and one RNA-dependent RNA polymerase gene. All eight of these genes were exclusively up-regulated. Such genes might have an important role in multiple stages of nodule development (e.g. infection of plant cells by bacteroids or control of nodule development).
Characterization of the Roles of Putative Regulatory Genes in Nitrogen Assimilation/Metabolism and Nodule Development A difficulty in profiling gene expression is choosing which tissues to compare. For example, during our analysis, it appears that 24-d mock-inoculated roots, in addition to lacking nodules, also exhibited symptoms of nitrogen starvation, such as chlorosis and leaf senescence. This stress condition might affect the expression pattern of the putative soybean regulatory genes, especially with respect to the comparisons between mock-inoculated and inoculated plants. Consequently, at 24 DAI, we made the assumption that some of the regulatory genes were regulated in response to nitrogen starvation in the mock-inoculated plants and did not have a specific role in nodule development. In order to obtain a clearer view of those genes that may be responding to the nitrogen provided by actively fixing bacteroids versus those that are strictly involved in the nodulation response, 24-DAI gene expression (inoculated versus mock inoculated) was compared in roots grown with and without nitrogen (KNO3 versus KCl; Supplemental Table S4).
Of the 111 genes regulated in 24-DAI nodulated roots, only 12 were also responsive to root nitrogen status (1.2% of the 1,034 genes analyzed; Supplemental Table S4). Among these 12 genes, 10 were repressed during both nodulation and KNO3 treatments. The remaining two genes were strongly up-regulated at 24 DAI but repressed in roots treated with KNO3. The remaining 99 genes (9.6%; Supplemental Table S4) were regulated specifically in nodulated roots, thereby supporting a role of these putative regulatory genes in the maintenance and activity of the nodule. The comparison of KNO3- and KCl-treated roots identified 38 genes responsive to nitrogen status but not involved in the nodulation process (3.7%; Supplemental Table S4). Recently, Ruffel et al. (2008)
We next compared the sequences of the 126 genes that we identified as differentially regulated in soybean nodule development with sequences of TFs from L. japonicus and M. truncatula with known roles in nodule development. Among them, Glyma02g35190 and Glyma10g10240 are homologous to M. truncatula HAP2.1 and its L. japonicus homologs CBF-A01 and CBF-A22 (Asamizu et al., 2008 In addition to the identification of the four putative soybean orthologs of MtHAP2.1, we also identified several soybean putative orthologs of MtERN (Glyma16g04410, Glyma16g27040, Glyma19g29000, Glyma02g08020, and Glyma08g12130). Based on this study, the expression of Glyma16g04410 was induced during early nodulation (Supplemental Table S4). An examination of the genomic regions of MtERN and Glyma16g04410 supports the notion that these genes are likely orthologous (Supplemental Fig. S3). A similar approach also identified Glyma19g29000 and Glyma08g12130 as putative MtERN orthologs. However, the genomic regions of Glyma16g27040 and Glyma02g08020 genes showed no microsynteny to the MtERN genome locus (Supplemental Fig. S3). Glyma16g27040, Glyma19g29000, Glyma02g08020, and Glyma08g12130 were not represented in the current qRT-PCR primer library. However, Glyma19g29000 and Glyma02g08020 were strongly expressed in soybean nodules, while Glyma16g27040 and Glyma08g12130 were not (Supplemental Table S5). Such observations reinforced the putative orthologous relationship between MtERN, Glyma16g04410, and Glyma19g29000. Note that even though the genome regions of Glyma08g12130 and MtERN showed significant microsynteny, Glyma08g12130 was not induced during nodulation. This result suggests that the regulation of Glyma08g12130 likely diverged from its progenitor following the duplication that gave rise to this paralog.
The identification of several putative soybean orthologs of MtHAP2.1 and MtERN is likely the result of the whole genome duplication events that occurred in soybean (Schlueter et al., 2004
Putative Soybean Regulatory Genes Have Temporally Defined Expression Patterns In order to examine in detail the response of a select number of putative regulatory genes during nodulation, the expression patterns of the eight genes found to be differentially expressed at multiple time points were enlarged by including additional time points (0, 16, and 32 DAI; for details, see Supplemental Table S6). In addition, the expression patterns of five additional genes (Glyma18g49360, Glyma19g34380, Glyma03g27250, Glyma03g31980, and Glyma06g08610) were also further analyzed. The detailed time-course analysis grouped these 13 putative regulatory genes into three classes based on their expression patterns (Fig. 4 ). Three genes were up-regulated only during the early stages of nodulation (Glyma16g04410, Glyma02g35190, and Glyma12g34510; Fig. 4, A–C), while six were up-regulated throughout all stages of nodulation (Glyma16g26290, Glyma10g10240, Glyma03g31980, Glyma06g08610, Glyma13g40240, and Glyma01g01210; Fig. 4, D–I). A third category of genes were regulated specifically during the later stages of nodulation (Glyma18g49360, Glyma17g07330, Glyma19g34380, and Glyma03g27250; Fig. 4, J–M). These three distinctive groups of genes clearly showed a temporally defined expression pattern that may reflect a specific developmental role for each gene. Based on these expression patterns, we hypothesized that the early induced putative regulatory genes might be involved in bacterial infection or in controlling the first steps of nodule development (e.g. cortical cell division). The late induced putative regulatory genes or those induced throughout nodule development might be involved in controlling the later steps of nodule development (e.g. cell elongation, bacteroid development, nitrogen metabolism, or control of nodule growth).
Investigation of Myb Gene Expression Patterns Using Promoter-Reporter Gene Fusions Our basic strategy is to identify TF genes important to soybean nodulation by examining their differential expression in response to inoculation. The identification of putative MtHAP2.1 and MtERN soybean orthologous genes (Glyma02g35190, Glyma16g04410, and Glyma10g10240) induced during nodulation provides some validation to this approach. Among the different genes regulated, Glyma03g31980 and Glyma17g07330, two Myb TF genes, were found to be regulated at several time points of the time course (Fig. 4, F and K; Supplemental Table S6).
Based on further characterization (see below), Glyma03g31980 was named CND. The expression profiles of CND and Glyma17g07330 during nodulation were similar. In a previous study, Koltai et al. (2001) To further examine the expression of CND during nodule development, promoter-GUS and -GFP fusions were constructed and introduced into soybean roots by Agrobacterium rhizogenes hairy root transformation. A 2-kb region 5' of the CND gene was identified from the Phytozome Web site and cloned upstream of the GFP and GUS reporter genes to construct transcriptional fusions. Early during nodule development (13 DAI), transformed roots showed CND promoter-GFP expression in emerging nodules and, at a lower level, in root cells (Fig. 5, A–D ). Although CND expression was apparent throughout the root, GFP signal was noticeably absent in emergent secondary root tips (Fig. 5, A and B). This result was also supported by the analysis of roots expressing the CND promoter-GUS construct, although in rare occasions secondary root tips did show some GUS staining (Fig. 5C, white arrow). CND expression was induced very early during nodule primordium formation, at the location of the infected zone (Fig. 5, C and H, black arrows). Later during nodule development (28 DAI and later), CND expression decreased in the infection zone of the nodule (Fig. 5, E, F, and I). Looking at transverse sections of GUS-stained transgenic roots and developing and mature nodules, CND was strongly expressed in the vascular tissues of all organs (Fig. 5, G–I). At a finer level, GUS staining was also observed in epidermal, endodermal, and cortical cells. These observations support a change in the expression pattern of CND during nodule development, especially in the infected zone of the nodule.
Characterization of the Role of CND during Nodule Development
RNAi silencing is a common strategy to characterize gene function when mutants are not available. Several studies focusing on legume nodulation have successfully used this strategy to investigate gene function. For example, silencing of MtDMI2 led to 75% of roots with no nodules compared with 0% to 20% of control roots with no nodulation (Limpens et al., 2003
Because the transcriptional response of a gene to a specific treatment does not necessarily imply an essential role in this process, we further investigated the role of CND in nodule formation. To specifically silence CND, a DNA fragment corresponding to the predicted 3' untranslated region (UTR) was cloned to create a hairpin structure (see "Materials and Methods"). As a control, pCGT5200 carrying an RNAi construct specific to the GUS gene was used. Three independent experiments were performed allowing the counting of nodule numbers on 44 CND-RNAi and 36 control RNAi-GUS roots. The silencing of CND consistently affected nodule development by significantly reducing nodule numbers by approximately 40% compared with the RNAi-GUS control roots (Student's t test, P
Based on the expression of CND in the infected zone of young nodules, we investigated more closely the infection of CND-RNAi nodules by B. japonicum. CND-RNAi and RNAi-GUS nodules were stained with SYTO-13, a nucleic acid-binding dye (Veereshlingam et al., 2004 ). By laser-scanning confocal microscopy, we did not observe a significant modification in the pattern of infected cells coincident to the silencing of the CND gene (Supplemental Fig. S6). Both the CND-RNAi nodules and the control nodules exhibited a clearly distinguishable infected zone. To confirm the silencing of CND, we quantified gene expression in RNAi-GUS control and CND-RNAi isolated nodules using qRT-PCR (i.e. for each of the three biological replicates of CND-RNAi and RNAi-GUS roots, small, medium, and large nodules were pooled and RNA was extracted). These experiments confirmed the silencing of CND at all stages of nodule development (Fig. 6C). We assume that the fact that nodule numbers were only reduced by 40% is likely due to the fact that 100% silencing of CND gene expression was not achieved. We also cannot rule out the possibility of functionally redundant TF genes that were not silenced by the CND-RNAi construct. Although primers were designed to specifically silence CND expression, the similarity of sequences between the members of the Myb TF family may have resulted in silencing of more than one gene. To test this hypothesis, the CND nucleotide sequence was BLAST searched against the available soybean genomic sequence (http://www.phytozome.net/soybean.php). The closest homolog was Glyma19g34740 (score = 379, e-value = 6.2e–103). Based on the strong identity between CND and Glyma19g34740 (83% and 87% identity of their nucleotide and amino acid sequences, respectively), we hypothesized that Glyma19g34740 might be homeologous to CND and that the function of these two Myb genes might be redundant. To better establish the relationship between these two genes, we mined soybean genome sequences to identify genes surrounding both Myb genes. Not surprisingly, the environment of these two genes in terms of gene content, order, and orientation was very similar, supporting their homeology (Supplemental Fig. S7). The strong nucleotide sequence identity between CND and Glyma19g34740 suggested that both genes might be silenced during our experiments. Accordingly, we also quantified the expression levels of Glyma19g34740 in the CND-RNAi and RNAi-GUS control nodules. The data showed that our construct specifically silenced the expression of the CND Myb gene (Fig. 6D). In addition, these analyses also demonstrated the strong expression of Glyma19g34740 in nodules, supporting a possible partial redundancy in the function of these two Myb genes. This result might be a second factor explaining the mild phenotype of the CND-RNAi plants.
The development and use of our soybean qRT-PCR library allowed quantification of the expression of 1,034 soybean putative regulatory genes. By focusing on nodule development, we validated the use of this approach by identifying putative soybean orthologs of MtHAP2.1 and MtERN as regulated during nodulation. In addition, using RNAi silencing, we identified a Myb TF gene, CND, involved in nodule formation. In addition to PHANTASTICA, our data suggest that probably several Myb genes including CND might be involved in nodule development.
Bacterial Culture
DH5
To silence the CND Myb gene, a 120-bp fragment specific to the 3' UTR of GmCND was amplified from soybean cDNA using the following primers: GmMyb-RNAifor (5'-TCGAGTAACAGTCGTAATGGACA-3') and GmMyb-RNAirev (5'-GACCAAGTCCTTCATTCAACG-3'). The amplified PCR fragment was cloned into the entry vector CGT11050 by TA cloning using two AhdI sites (engineered to produce 3' T overhangs). The RNAi entry vector was recombined with the destination vector CGT11017A (Supplemental Fig. S8) using the LR Clonase reaction (Gateway LR Clonase II enzyme mix; Invitrogen). The LR Clonase reaction combining the gene fragment in the entry vector and destination vector CGT11017A was used to produce GmCND-RNAi vector. The RNAi control vector used in these studies (CGT5200) was previously described by Govindarajulu et al. (2009) The Gateway system (Invitrogen) was used to clone GmMyb promoter upstream to the GFP and GUS cDNAs. By mining genomic sequences available on the Phytozome Web site (http://www.phytozome.net/soybean.php), we identified a 1,967-bp DNA fragment 5' to the first codon of the CND gene. By two independent PCRs, we created the AttB sites at the extremities of the promoter sequences. We used soybean cv Williams 82 genomic DNA as template and the following primers: GmMybpromoAttB-for (5'-AAAAAGCAGGCTCCTACATGAATATGTGTTCAAAATA-3') and GmMybpromoAttB-rev (5'-AGAAAGCTGGGTTTTGATGACTTAGACTACTCCTTC) first and the universal AttB primers attB1adaptor (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3') and attB2adaptor (5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3') second.
Using the Gateway BP Clonase II enzyme mix, the GmMyb promoter fragment was introduced first into the pDONR-Zeo vector (Invitrogen) and then into pYXT1 or pYXT2 destination vector using the Gateway LR Clonase II enzyme mix (Invitrogen). The pYXT1 and pYXT2 destination vectors carry the GUS and GFP reporter genes, respectively (Xiao et al., 2005 A. rhizogenes (strain K599) was transformed by electroporation with pCGT11017-3'UTR-GmMyb, GmMybpromoter-pYXT1, and GmMybpromoter-pYXT2 vectors.
Soybean seeds were surface sterilized according to Wan et al. (2005)
Soybean hairy root transformation was done essentially as described by Taylor et al. (2006)
CND-RNAi and control nodulated transgenic roots were isolated (these roots expressed the GFP reporter gene), and the nodules were isolated and then categorized based on the presence of leghemoglobin (i.e. a pink-red color in the infected zone of the nodule was used as a marker of nodule maturity; leghemoglobin gene expression increases proportionally with the age of the nodule [Marcker et al., 1984 For each category, nodule numbers were counted with a Leica MZFLIII stereomicroscope equipped with epifluorescence excitation and a GFP long-pass filter (500 nm long pass; no. 41018; Chroma Technology). Molecular Devices MetaMorph 7.5 was used to measure the area of each nodule (the areas of 73 large, 182 medium, 109 small mature, and 33 immature nodules from RNAi-GUS and 54 large, 178 medium, 88 small mature, and 32 immature nodules from CND-RNAi were measured). To observe B. japonicum infection in transgenic nodules, CND-RNAi and RNAi-GUS nodules were embedded in paraffin before sectioning. Five-micrometer sections were stained during 20 min with 10 µM SYTO13 in 1 M sodium phosphate buffer (pH 7.0). Stained sections were observed with a Zeiss LSM 510 META confocal microscope (488 nm excitation wavelength and 500- to 550-nm emission filter).
GmMyb promoter-GUS and -GFP transgenic roots were observed 13, 18, 29, and 40 DAI. GmMyb promoter-GUS roots were fixed and stained as described by Govindarajulu et al. (2008)
mRNAs isolated from different soybean tissues were reverse transcribed using standard protocols. After first- and second-strand cDNA synthesis, the cDNAs were end repaired prior to ligation of Solexa adaptors. The products were sequenced on an Illumina-Solexa platform.
Illumina Genome Analyzer II image data were base called and quality filtered using the default filtering parameters of the Illumina GA Pipeline GERALD stage. Alignments of passing 36-mer reads to all contigs of the Glyma1 8x Soybean Genome assembly (Soybean Genome Project, Department of Energy Joint Genome Institute) were performed using GSNAP, an alignment program derived from GMAP (Wu and Watanabe, 2005
To ensure maximum specificity of the designed primers, we used PRIMEGENS (http://digbio.missouri.edu/primegens/; Xu et al., 2002 To quantify specifically the expression of the Glyma19g34740 Myb gene, Glyma19g34740-for (5'-CCAAGACGCCAAACTAGAACA-3') and Glyma19g34740-rev (5'-TCGCTAGAACAATGAGGAGGA-3') primers were designed. Primer specificity was confirmed by dissociation curve profile after qRT-PCR amplification.
Total RNA was isolated using Trizol Reagent (Invitrogen) according to the manufacturer's instructions followed by chloroform extraction to improve their purity. Purified RNA was treated with TURBO DNase (Ambion) to remove any contaminating genomic DNA according to the manufacturer's instructions. In to the experiments, 100 µg of DNA-free total RNA was used for first-strand cDNA synthesis using Moloney murine leukemia virus (Promega) according to the manufacturer's instructions. The lack of genomic DNA contamination was verified by qRT-PCR using primers designed against soybean genomic DNA. Moreover, cDNA synthesis efficiency was analyzed by amplifying by qRT-PCR Cons6 cDNA with two different primer sets separated by 1 kb (Cons6-3' forward, 5'-AGATAGGGAAATGGTGCAGGT-3', and reverse, 5'-CTAATGGCAATTGCAGCTCTC-3'; Cons6-5' forward, 5'-AAAGGTGAAATTGCCTCTTCC-3', and reverse, 5'-CCCAAAGATCTGCCAAATGTA-3'). For each condition and replicate tested, less than one PCR cycle difference was measured between both primer sets, supporting the high quality of the cDNA synthesis.
qRT-PCR was performed as described by Libault et al. (2008)
The following materials are available in the online version of this article.
We thank Sandra Thibivilliers, Jeff Anderson, and Aaron J. Witte for critical reading of the manuscript. We also thank Yong-Li Xiao and Christopher Town for providing the pYXT1 and pYXT2 binary vectors, Christopher Taylor for providing CGT11050 and CGT11017 vectors, and Andrew Farmer and Gregory May for their help in the Illumina-Solexa sequencing analysis. Received June 30, 2009; accepted September 6, 2009; published September 15, 2009.
1 This work was supported by the National Science Foundation Plant Genome Research Program (grant no. DBI–0421620).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gary Stacey (staceyg{at}missouri.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.144030 * Corresponding author; e-mail staceyg{at}missouri.edu.
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TOP
ABSTRACT
RESULTS AND DISCUSSION
1e-12) to identify homologous soybean sequences. In addition, The Institute for Genome Research soybean Gene Indices (
Escherichia coli and K599 Agrobacterium rhizogenes were grown in Luria-Bertani medium supplemented with appropriate antibiotics at 37°C and 30°C, respectively. Bradyrhizobium japonicum USDA110 was grown at 30°C for 3 d in HM medium (Cole and Elkan, 1973
3.5 mm2 and <4.5 mm2], and large [
Ct). The expression level (E) of each gene was calculated according to the equation E = Peff(–
