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First published online April 20, 2007; 10.1105/tpc.106.048264 The Plant Cell 19:1221-1234 (2007) © 2007 American Society of Plant Biologists An ERF Transcription Factor in Medicago truncatula That Is Essential for Nod Factor Signal Transduction[W]
a Department of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom 3 To whom correspondence should be addressed. E-mail giles.oldroyd{at}bbsrc.ac.uk; fax 44-1603-450045.
Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor–induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.
Legume/rhizobial symbiosis plays an important role in the nitrogen cycle through the fixation of atmospheric dinitrogen. Recognition of rhizobial bacteria by the appropriate legume host induces the formation of a unique structure, the nodule, on the plant root. Microaerobic conditions are maintained within the nodule to allow activity of the bacterial enzyme nitrogenase that is responsible for nitrogen fixation.
The early signaling and transcriptional events leading to nodule formation have been well studied. Rhizobia recognize flavonoids released from the host legume roots and respond with the production of lipochito-oligosaccharide signaling molecules termed Nod factors (Long, 1996
Root hair deformation leads to the entrapment of rhizobial bacteria within an infection focus inside the curled root hair. Infection threads elongate from this infection focus, and this allows bacterial invasion into the developing nodule primordium. The infection thread is an invagination of the cell wall and plasma membrane, forming a tube-like structure that extends from the infection focus toward the cortex (Gage, 2002
Although biochemical proof is yet to be obtained, it is widely believed that Nod factors are perceived by receptor-like kinases that contain sugar binding LysM domains. Such receptor-like kinases exist as gene families with at least three members, NFP, LYK3, and LYK4 (Ben Amor et al., 2003
Downstream of the hypothetical Nod factor receptors in M. truncatula are a receptor-like kinase with Leu-rich repeat motifs, DMI2 (Catoira et al., 2000
It is likely that the Nod factor–induced calcium spiking signal is transduced via CCaMK to activate gene expression. This is supported by pharmacological evidence indicating that the inhibition of calcium signaling blocks Nod factor–induced gene expression (Charron et al., 2004
Transcriptome analysis in M. truncatula of the Nod factor signaling mutants nfp, dmi1, dmi2, dmi3, nsp1, and nsp2 revealed that all six genes are absolutely required for rhizobial and Nod factor–induced gene transcription, suggesting that these six genes may function in a linear genetic pathway leading to early gene induction (Mitra et al., 2004a Here, we report the identification of ERN, an AP2-like transcription factor in the ERF subfamily, which is necessary for nodulation and functions in early Nod factor signaling. Like the GRAS proteins previously identified, this gene functions downstream of CCaMK. ERF transcription factors have well-defined DNA binding activity; thus, ERN is an excellent candidate for a transcription factor that could work with the GRAS proteins to activate the expression of nodulation genes.
Identification of a Gene Necessary for Infection Thread Development We undertook a genetic screen in a population of fast neutron mutagenized M. truncatula plants to identify components necessary for nodulation (Starker et al., 2006  ). We identified a number of mutants that were unable to form nodules, classified as Nod–. For one of these Nod– mutants, bit1-1 (Table 1
), the F2 of a backcrossed population segregated in a 3:1 ratio (37 Nod+:12 Nod–;  2 = 0.007), indicating a single recessive mutation. When inoculated with mycorrhizal fungi, we saw normal colonization in this mutant, indicating that the mutated gene was not necessary for mycorrhizal infection (M. Harrison, personal communication).
Upon further characterization, we found that bit1-1 could form small bumps when in the presence of M. truncatula's rhizobial partner Sinorhizobium meliloti for in excess of 2 months (Figure 1F ), indicating the formation of underdeveloped nodule primordia. At the equivalent time point, large and numerous nodules had formed on the wild-type plants (Figure 1C). Infection with S. meliloti carrying the hemA-LacZ fusion allowed us to assess the level of bacterial infection in this mutant. We found that the formation of infection foci in the bit1-1 mutant was similar to that of the wild type (bit1-1, 28 ± 6; wild type, 32 ± 8 infection events per root), but in most cases, there was no further development of infection structures beyond the infection focus in bit1-1 (Figure 1D). However, a limited number of infection threads did develop in this mutant, but these generally terminated within the root hair cell (Figures 1G and 1H). In contrast with the wild type, which had small infection foci and unbranched infection treads (Figure 1A), the infection foci of bit1-1 were often distended (Figure 1D), and infection threads commonly showed complex branched structures within the root hair cell (Figures 1G and 1H); hence, we chose to call this mutant allele branching infection threads1-1 (bit1-1). To determine whether infection threads could progress successfully in this mutant, we assessed the infection events associated with the small bumps forming on bit1-1 after long periods of time in the presence of S. meliloti. When we stained these bumps, it was possible to visualize the ramification of infection threads into the cortex (Figure 1E), but again these infection threads terminated prematurely with little expansion into the nodule primordium as seen in wild-type plants (Figure 1B).
The bit1-1 mutant phenotype is similar to mutants previously described with defects in infection thread development and shows particular similarity to lin (Kuppusamy et al., 2004 ). Allelism tests with lin revealed that these two mutants represent two separate complementation groups (Table 1). From these data, we concluded that we had identified a gene that is necessary for initiation and maintenance of infection thread growth.
In parallel to analysis of fast neutron populations, phenotypic screens for Nod– mutants were conducted in ethyl methanesulfonate–mutagenized populations (Penmetsa and Cook, 2000 Despite the general similarity of infection arrest phenotypes, bit1-2 is distinguished by abnormal root development (Figures 1I and 2 ). Root hairs develop in alternating regions of dense and sparse hairs, giving the root a "poodle's tail" appearance (Figure 2A); hence, the previous reference to this allele as poodle. Here, we refer to this allele as bit1-2. Similarly to bit1-1 infection threads initiated in bit1-2, however, they were never observed to exit the epidermal root hair cells, and as in bit1-1, the formation of infection foci was comparable to the wild type (bit1-2, 94 ± 18; wild type, 116 ± 20 infection events per root) (Figure 2B). Growth conditions for quantification of infection events for bit1-1 and bit1-2 differ (see Methods), and this explains the discrepancies in the levels of infection observed. Semithin longitudinal sections through wild-type and bit1-2 roots revealed that the patches of dense root hair development were accompanied by a disorganized subtending root cortex (Figures 2C and 2E). Such patches were characterized by reduced longitudinal cell expansion, increased radial cell expansion, the presence of occasional intracellular spaces between cortical cells, and adjacent clustering of root hair cells. Wild-type cells of the inner cortex have rectangular length and width dimensions (162 ± 117 and 23 ± 13 µm, respectively), whereas the homozygous bit1-2 plants have regions where inner cortical cell dimensions approximate to that of a square (72 ± 19 µm length x 69 ± 18 µm width). In contrast with bit1-1, which had a wild-type root system in the absence of S. meliloti, the roots of bit1-2 had a much reduced root growth rate (Figure 1I). Scoring for nodule structures and bacterial infection 21 d after inoculation revealed fully developed functional nodules in the wild type (Figure 2F) in contrast with bit1-2, which showed arrested infections and no noticeable inner cortical cell division (Figure 2D). Cosegregation of the Nod– and poodle-like phenotypes in all 485 F2 progeny from backcrossed plants suggests that these phenotypes derive from the same single gene mutation.
BIT1 Is Necessary for Nod Factor Signal Transduction The CCaMK encoded by DMI3 is essential for Nod factor signal transduction (Levy et al., 2004 ; Mitra et al., 2004b). For the kinase to function, and thereby relay the Nod factor–derived signal, an autoinhibitory domain must be inactivated through the binding of calmodulin (Patil et al., 1995). Gleason et al. (2006) have shown that by removing the autoinhibitory domain, the kinase is constitutively active. When plants are transformed with this construct, spontaneous nodules form in the absence of rhizobia (Gleason et al., 2006; Figure 3A
). As such, this construct can be used for testing the epistatic relationship of genes within the Nod factor signaling cascade.
When 98 bit1-1 and 61 bit1-2 plants were transformed with autoactivated CCaMK (DMI31-311), spontaneous nodules failed to form (Figure 3B). By contrast, spontaneous nodules did form on seven out of 17 dmi3-1 plants transformed with DMI31-311. To ensure that the bit1 roots were transformed with this CCaMK construct, 10 transformed root systems were pooled and the presence of the cotransformed kanamycin gene was shown to be present (Figure 3B, inset). This result indicates that DMI3 is dependent on the downstream action of BIT1 for the production of spontaneous nodules, showing that BIT1 functions downstream of CCaMK in the activation of nodule organogenesis. These results suggest that BIT1 plays a role not only in infection thread growth but also in nodule formation.
The necessity for BIT1 function in CCaMK induction of spontaneous nodulation was surprising because the mutant phenotype indicated a role for BIT1 in the initiation and maintenance of infection threads. However, the results of the autoactivated CCaMK experiments suggested that BIT1 may function in the Nod factor signal transduction pathway. To further test this hypothesis, we analyzed the ability of the Nod factor to activate the Nod factor reporter ENOD11:ß-glucuronidase (GUS) in the bit1-1 mutant background. Within 6 h of Nod factor application, wild-type plants showed strong induction of ENOD11:GUS (Figure 3C), while bit1-1 mutants showed no or severely diminished ENOD11:GUS induction (Figure 3D). Although widely reported (Journet et al., 2001
To further explore the role of BIT1 in the induction of early nodulation gene expression, we assessed rhizobial-induced genes that have previously been shown to require Nod factor signaling components. Mitra et al. (2004a)
Transcript-Based Gene Cloning Identified Downregulated Genes in bit1-1
All three of these transcripts occur within the sequenced BAC mte1.32m6 located on LG7, suggesting that a single deletion might be causal of the reduced transcript levels. We found that BIT1 mapped to LG7 and cosegregated with marker 002E05, which resides within the same BAC contig as mte1.32m6, suggesting that the deletion we had identified may contain BIT1. PCR analysis with primers specific for one of the candidate transcripts (an arabinogalactan-peptide gene) confirmed the presence of a DNA deletion that cosegregated with the bit1-1 mutant phenotype in a backcrossed population (Figure 4C ). The extent of the deletion was estimated by progressive amplification of genomic DNA fragments flanking the AGP transcript. Using this method, a region of  40 kb is estimated to be deleted in bit1-1. The centromeric deletion boundary was only tentatively identified, since the PCR product was contained within a multicopy retrotransposon, thus confounding the interpretation of the PCR amplification. The PCR results confirm that at least five genes are affected by this deletion: an arabinogalactan peptide, an unknown gene, an  -tubulin, an ERF transcription factor, and a karyopherin. Neither the unknown gene nor the karyopherin was detected by the transcript-based cloning because the unknown gene was not on the Affymetirx GeneChip and the expression of the karyopherin was very low in the wild type, such that its absence in bit1-1 could not be detected.
Positional Cloning Identifies bit1-2 In parallel to deletion analysis, we undertook the positional cloning of the bit1-2 allele. bit1-2 was located on contig 995 (http://medicago.org/genome) between markers TC87156_1 and TC80512_1 on LG7 and cosegregated with marker 29P9F that was generated from the end sequence of BAC 29P9 (Figure 4A). This genetic region was contained within a single BAC clone (Figure 4A), and the sequencing of this region revealed four genes between markers TC87156_1 and TC80512_1: an -tubulin, an ERF transcription factor, a karyopherin, and an exostosin-like protein. Sequencing these four genes in the bit1-2 mutant revealed a single mutation within the ERF transcription factor and wild-type alleles of the other three candidate genes. The mutation causes an amino acid change of Gly to Asp at a residue that is completely conserved within the DNA binding domain of all 147 Arabidopsis AP2-like genes. The affected ERF in bit1-2 is the same gene as the ERF transcription factor contained within the deleted region of bit1-1 (Figure 4B). Importantly, in mapping bit1-2, both the nonsymbiotic and symbiotic phenotypes were used, and all recombinants were mutant for both symbiotic and nonsymbiotic phenotypes. As all genes between the cosegregating markers were sequenced and only the mutation in the ERF was found, we can attribute both phenotypes to this gene.
bit1-1 Is Complemented by Agrobacterium rhizogenes–Mediated Transformation Using the ERF Transcription Factor
ERN Is a Member of a Previously Uncharacterized Group of ERF Transcription Factors Among the AP2 transcription factors of Arabidopsis thaliana, ERN shows closest similarity to RAP2.11, a member of the ERF subfamily of AP2 transcription factors, with 41.3% overall identity and 96.6% identity within the AP2 DNA binding domain (Figure 6A ). Close homologs were identified in L. japonicus, Pisum sativum, Phaseolus vulgaris, Glycine max, and Populus alba (poplar). The L. japonicus homolog is contained within a genomic region that is highly syntenic to that of ERN in M. truncatula (Figure 4B). Within the AP2 DNA binding domain, there is 100% amino acid sequence identity between the legume proteins and near complete conservation among all plants (Figure 6A). Two additional domains, CMV3 and CMV4 (conserved motif of Group V), have been bioinformatically identified in this class of ERF transcription factors in Arabidopsis and rice (Oryza sativa) (Nakano et al., 2006 ). These domains show moderate conservation between the legume proteins and other plant homologs of ERN (Figure 6A). However, there are significant differences in both composition and size of these domains in the legume and poplar proteins compared with Arabidopsis and rice. Yet within the legumes and poplar there is considerable similarity. In addition, a fourth domain of homology is apparent in the legume and poplar proteins between the CMV3 and CMV4 domains (Figure 6A). Arabidopsis RAP2.11 has previously been classed in Group V of the ERF group of AP2 transcription factors together with a number of other Arabidopsis ERF transcription factors, including the SHN1 gene (Aharoni et al., 2004; Nakano et al., 2006). Using the AP2 domain, we assessed the phylogenetic relationship of the Group V class of ERFs to the closest homologs of ERN in legumes and nonlegumes (Figure 6B). ERN clusters with RAP2.11 along with the poplar and rice homologs, but this grouping appears to be distinct from the other Group V members in Arabidopsis.
ERN Is Rapidly Induced in Response to S. meliloti Inoculation It was previously shown that TC102418, encoding ERN, is upregulated in response to S. meliloti inoculation based on data from Affymetrix arrays (Starker et al., 2006 ). The 1.5-fold induction 1 d after rhizobial inoculation is maintained for at least 21 d after inoculation (Figure 7A
; see Supplemental Table 3 online). Semiquantitative RT-PCR confirmed that ERN is rapidly induced (Figure 7B). However, in the RT-PCR work, induction was observed at 6 h after inoculation with S. meliloti and this induction is maintained. Despite slight differences between the Affymetrix and RT-PCR data, both implicate the induction of ERN by S. meliloti. This pattern is also observed in the roots of bit1-2 plants, although the onset of expression induction appears to be delayed (Figure 7B). ERN is expressed in M. truncatula roots prior to S. meliloti infection but is not expressed in the shoot.
To assess whether ERN induction by rhizobia required components of the Nod factor signaling pathway, we looked at the rhizobial induction of ERN on Affymetrix gene profiles in the Nod factor signaling mutants. ERN was not induced in nfp, dmi1, dmi2, dmi3, nsp1, or nsp2 plants 24 h after S. meliloti inoculation but was induced in hcl at an equivalent level to the wild type (Figure 7C). This shows that the S. meliloti induction of ERN is dependent on the Nod factor signaling components but is independent of HCL.
In the last few years, several components of the Nod factor signaling pathway have been defined from the model legumes M. truncatula and L. japonicus (Oldroyd and Downie, 2004 , 2006). Nod factor perception at the plasma membrane involves multiple receptor-like kinases that are necessary to activate oscillatory calcium changes associated with the nucleus (Ben Amor et al., 2003; Limpens et al., 2003; Radutoiu et al., 2003). Perception of the calcium signal is likely to be facilitated through CCaMK, the activation of which is sufficient to induce nodule organogenesis (Levy et al., 2004; Mitra et al., 2004b; Gleason et al., 2006; Tirichine et al., 2006). Downstream of CCaMK are two GRAS family transcriptional regulators (Kaló et al., 2005; Smit et al., 2005). Such GRAS proteins have been proposed to act as transcriptional activators, though the mechanism remains elusive (Bolle, 2004). Here, we show that ERN, a protein with homology to ERF transcription factors and with a highly conserved AP2 DNA binding domain, is necessary for Nod factor signaling. We show that ERN functions in the Nod factor signaling pathway downstream of CCaMK and, based on the mutant phenotype, probably downstream of the GRAS family proteins. ERN is a strong candidate to provide specific binding to the nodulin promoters for Nod factor–dependent activation of gene expression.
ERN is a member of Group V of the ERF subfamily of AP2 transcription factors (Nakano et al., 2006 As ERN encodes a transcription factor, genes downregulated in the mutant may give clues as to the function of ERN. Of the 46 genes known to be up- or downregulated by twofold in response to rhizobial inoculation, 29 of these genes were not induced or repressed in the bit1-1 mutant. While predefined nodulins and other transcription factors were among these genes (see Supplemental Table 1 online), it is difficult to draw conclusions regarding the regulation of nodulation by ERN from these regulated genes.
Three lines of evidence strongly support the statement that ERN functions in Nod factor signal transduction. First, nodulin reporter gene studies show that Nod factor–dependent ENOD11 expression is absent or greatly reduced in bit1-1 compared with the wild type. Second, the spontaneous nodulation induced by a modified CCaMK requires ERN activity. Finally, Nod factor–induced genes show altered expression in bit1-1 plants. However, unlike nfp, dmi1, dmi2, dmi3, nsp1, and nsp2, the rhizobial induction of these genes in not completely blocked in bit1-1. A function for ERN in Nod factor signaling would implicate this protein in regulating processes activated by the bacterial signal, namely, nodulin gene expression, infection thread development, and induction of the nodule meristem. Consistent with this hypothesis, we see defects in all of these processes in the bit1 mutants. ERF transcription factors are known to function in hormone signaling, most notably that of ethylene (Ohme-Takagi and Shinshi, 1995
Despite the requirement for ERN in Nod factor signaling, both bit1 mutants have phenotypes less severe than plants mutated in the previously defined components of the Nod factor signaling pathway. Specifically, in contrast with earlier mutants, rhizobial-induced gene expression still occurs in bit1-1 despite a reduced number of up- or downregulated genes showing significant expression changes in bit1-1 compared with wild-type plants. Additionally, bit1-1 and bit1-2 are infected by S. meliloti though show only limited infection thread development. Because bit1-1 is a complete deletion of ERN, the capacity of bit1-1 plants to partially transduce the Nod factor signal suggests that ERN is not as essential for Nod factor signaling as the other transcriptional regulators in the pathway, NSP1 and NSP2. It is perhaps important to note from the Affymetrix analysis that it is apparent that the expression of DMI2, DMI3, and NSP2 are unaffected in the bit1-1 mutant (data not shown). The limited Nod factor signaling activity remaining in the absence of ERN is sufficient to activate a subset of early responses but not adequate to complete the rhizobial infection process. This is consistent with previous observations showing greater stringency for Nod factor perception during infection stages compared with the induction of earlier responses, such as gene expression (Ardourel et al., 1994
The bit1-2 phenotype shows distinct differences to bit1-1. In particular, bit1-2 has altered root cortical and epidermal cell development in the absence of rhizobia or the Nod factor. It is possible that the inability of the roots of bit1-2 plants to nodulate stems not just from abnormal Nod factor signaling but an inability for either the clustered root hairs or aberrant cortical cells to support, respectively, root hair–mediated infection or rhizobia-induced cortical cell division. However, the absence of any discernible aberrant root hair or cortical cell phenotype in the null allele bit1-1 suggests that the bit1-2 mutant protein (BIT1-2) has a novel function that interferes with normal root development. The fact that bit1-2 results from a single amino acid change in the AP2 DNA binding motif is consistent with the possibility that BIT1-2 has altered DNA binding properties, perhaps binding nontarget promoter sequences or altered affinity to cognate promoter sequences. Regardless of functional changes to BIT1-2, we note that ERN and other components of the Nod factor signaling pathway are expressed in nonsymbiotic tissue (e.g., all of the DMI genes are expressed prior to Nod factor application), consistent with the idea that presymbiotic cells contain receptors and transduction pathways ready to initiate Nod factor signaling. Mutations that change the function of such pathway components may display phenotypes in the absence of rhizobial signals, such as has been demonstrated for particular C-terminal truncations of DMI3 (Gleason et al., 2006
The AP2 domain of Arabidopsis ERF1 has been resolved and shown to bind to DNA (Allen et al., 1998
Plant Growth and Bacterial Strains Medicago truncatula cv Jemalong A17 was used as the wild type. Backcrosses of bit1-1 and bit1-2 were to A17 and mapping crosses to A20. ENOD11 promoter GUS fusions were introduced into bit1-1 plants by crossing with a wild-type ENOD11:GUS line and selection through a series of generations to produce plants homozygous for both bit1-1 and ENOD11:GUS. For plate assays, seeds were scarified with sandpaper and sterilized in bleach for 3 min. Seeds were imbibed for 2 h in water and geminated inverted on damp filter paper for 2 d at 4°C, followed by 1 d at room temperature. Seedlings were then transferred to BNM medium supplemented with 0.1 µM aminoethoxyvinylglycine. Plates were infected with Sinorhizobium meliloti by flooding and subsequent draining of the plate with 1 mL of overnight culture, spun down and resuspended in BNM. For infection thread analyses and visualization, S. meliloti 1021 carrying pXLGD4, expressing LacZ under a hemA promoter, was used to inoculate plants. Agrobacterium rhizogenes ARqua was used as the transformation vector in all hairy root transformations.
Phenotypic Characterization For infection analysis of bit1-1, plants were grown on BNM supplemented with 0.1 µM aminoethoxyvinylglycine. By contrast, bit1-2 infection was assessed in aeroponic culture. In both treatments, plants were inoculated with S. meliloti 1021 carrying pXLGD4 and stained 3 d after inoculation. The number of infection events was assessed.
Affymetirx GeneChip construction, RNA preparation, and chip hybridizations were performed on bit1-1 plants at 1 or 4 d after inoculation with either S. meliloti 1021 or BNM as described by Mitra et al. (2004a)
For A. rhizogenes–mediated hairy root transformation of the DMI31-311 construct (Gleason et al., 2006
Gene Isolation Using an F2 mapping population of 1302 individuals from a cross between bit1-2 and M. truncatula A20 plants, the nod– phenotype and the cosegregating bit1-2 root phenotype were located between cleaved-amplified polymorphic sequence markers 58T and DK427R at a genetic distance of 0.7 and 0.3 centimorgans, respectively, on linkage group 7. Marker DK427R was selected for chromosome walking and used to identify the sequenced BAC clone Mth2_15C20 (GenBank accession number AC126009). Sequence information from this clone was used to generate a new genetic marker, 15c20SSRat_1, and identify BAC clone Mth2_16I24 that had been survey sequenced. Sequence information of Mth2_16I24 allowed the generation of a new genetic marker TC80512_1 by which BAC clone Mth2_49o13 was identified and then survey sequenced. Based on this, the marker TC87156_1 was generated, and the forward end sequence of BAC Mth2_29P9 (GenBank accession number CG929807) was identified from which 29P9F-specific primer pairs were generated. The sequenced region between the two bit1-2 recombinant markers was submitted to GenBank (accession number DQ984942). Primers are as follows: 58T forward primer, 5'-AAGGGCTTTTAATTTCTGTCTG-3'; 58T reverse primer, 5'-CCAAACCAATCTAAAATAATAAAC-3'; DK427R forward primer, 5'-CCAAACAAGGAAAAGTGTTGGTGTCA-3'; DK427R reverse primer, 5'-ATGAGAAACTTTTGAAATTTAGGATACGATAG-3'; 15C20SSRat_1 forward primer, 5'-AATAGGGACATTAAGATG-3'; 15C20SSRat_1 reverse primer, 5'-TTGTGAAATAGTCGTGAT-3'; TC80512_1 forward primer, 5'-CATAACAAAAACCCAAAGTGAGA-3'; TC80512_1 reverse primer, 5'-AGTTATAATATGTCGGTGCCAATC-3'; TC87156_1 forward primer, 5'-TGGAGCTATATGTTCTTTTGGTGT-3'; TC87156_1 reverse primer, 5'-GTGGTGGCCTGGGTTTGA-3'; 29P9F 1 forward primer, 5'-TATCACAACATCTTTTCCAC-3'; 29P9F 1 reverse primer, 5'-GCTCTAGCTGCTTCCTCA-3'.
The genomic DNA from M. truncatula plants was isolated using the ZenoGene40 plant DNA purification kit (Zenon Bio). BAC DNA purifications were performed according to a slightly modified method described previously (Nam et al., 1999
A. rhizogenes–Mediated Complementation of bit1-1
Sequence Analysis
Expression Analysis
Affymetrix-based expression analysis of the ERN transcript with time has been previously described (Starker et al., 2006
For microarray analysis, Affymetrix MAS 5.5 software was used to extract pixel values from scan files, and the .cel files were analyzed using DCIP 1.3 (www.dchip.org).Values were normalized across independent chips using invariant set normalization. To assess the significance of expression changes, Significance Analysis of Microarrays (SAM2.11) was used (Tusher et al., 2001
Accession Numbers
Supplemental Data
We thank A. Downie, S. Hirsch, G. Deák, G. Endre, and E. Kiss for their scientific help and advice as well as K. Karchesz and I. Szívós for skillful technical assistance. This study was supported by the Biotechnology and Biological Sciences Research Council, the Royal Society, European Grain Legumes for Food and Feed (Grant FOOD-CD-2004-506223); Hungarian national grants NKFP Medicago Genomics (Grant 4/023/2001, Ministry of Education); Medicago Biotechnology (Grant 4/031/2004); Biotechnology 2001 (Grant OMFB-00229/2002); OTKA (Hungarian Research Scientific Fund) Grants OTKA T046645 and OTKA T046819; the GVOP-3.1.1-2004-05-0101/3.0 and the U.S. Department of Energy Biosciences program.
1 These authors contributed equally to this work. 
2 Current address: Department of Plant Biology, University of Minnesota, 1445 Gortner Ave., St. Paul, MN 55108. 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.plantcell.org) is: Giles E.D. Oldroyd (giles.oldroyd{at}bbsrc.ac.uk).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.106.048264 Received October 17, 2006; Revision received January 20, 2007. accepted April 6, 2007.
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